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1 This article was downloaded by: [Canadian Research Knowledge Network] On: 18 May 2011 Access details: Access Details: [subscription number ] Publisher Routledge Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Nutrition and Cancer Publication details, including instructions for authors and subscription information: Diets and Hormonal Levels in Postmenopausal Women With or Without Breast Cancer Mylène Aubertin-Leheudre a ; Esa Hämäläinen b ; Herman Adlercreutz a a Folkhälsan Research Center, Institute for Preventive Medicine, Nutrition and Cancer, and Division of Clinical Chemistry, University of Helsinki, Helsinki, Finland b Department of Obstetrics and Gynecology, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland First published on: 14 April 2011 To cite this Article Aubertin-Leheudre, Mylène, Hämäläinen, Esa and Adlercreutz, Herman(2011) 'Diets and Hormonal Levels in Postmenopausal Women With or Without Breast Cancer', Nutrition and Cancer, 63: 4, , First published on: 14 April 2011 (ifirst) To link to this Article: DOI: / URL: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Nutrition and Cancer, 63(4), Copyright C 2011, Taylor & Francis Group, LLC ISSN: print / online DOI: / Diets and Hormonal Levels in Postmenopausal Women With or Without Breast Cancer Mylène Aubertin-Leheudre Folkhälsan Research Center, Institute for Preventive Medicine, Nutrition and Cancer, and Division of Clinical Chemistry, University of Helsinki, Helsinki, Finland Esa Hämäläinen Department of Obstetrics and Gynecology, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland Herman Adlercreutz Folkhälsan Research Center, Institute for Preventive Medicine, Nutrition and Cancer, and Division of Clinical Chemistry, University of Helsinki, Helsinki, Finland The role of diet in breast cancer (BC) risk is unclear. Fiber could reduce BC risk, through the enterohepatic circulation of estrogens. We examined the relationship between diet and sex hormones in postmenopausal women with or without BC. Thirty-one postmenopausal women (10 omnivores, 11 vegetarians, and 10 BC omnivores) were recruited. Dietary records (5 days) and hormone levels (3 days) were evaluated on 4 occasions over 1 yr. Vegetarians showed a lower fat/fiber ratio, a higher intake of total and cereal fiber (g/d)/body weight (kg), a significantly lower level of plasma estrone-sulfate, estradiol, free-estradiol, free-testosterone, and ring D oxygenated estrogens, and a significantly higher level of sex-hormone-binding-globulin than BC subjects. Fiber was consumed in slightly larger amounts by omnivores than by BC subjects. Omnivores had significantly lower plasma testosterone and estrone-sulfate but higher sex-hormone-binding-globulin than BC subjects. No difference was found for the urinary 16-oxygenated estrogens. However, the 2-MeO-E1/2-OH-E1 ratio was significantly lower in omnivores than in BC group. This ratio is positively associated with the fat/fiber ratio. In conclusion, testosterone may contribute to causing alterations in the levels of catechol estrogens and 16-oxygenated estrogens. The fat/fiber ratio appears to be useful in evaluating dietary effects on estrogen metabolism. INTRODUCTION Breast cancer (BC) incidence has been significantly lower in Asian than in Western populations (1), but it is rapidly increasing Submitted 4 May 2009; accepted in final form 17 March Address correspondence to Mylène Aubertin-Leheudre, PhD, Folkhälsan Research Center, Biomedicum Helsinki, P.O. Box 63, Haartmaninkatu 8, FI University of Helsinki, Finland. Phone: Fax: mylene.aubertin-leheudre@helsinki.fi in both Japan and China. While changes in dietary habits have been suggested to reduce BC deaths by half (2), the role of diet in BC risk remains unclear. The role of dietary fat and fiber in BC risk may be important. Traditional Asian diets may be protective because they tend to be rich in soy products and low in fat (3,4). The dietary fat/fiber ratio is low in these populations (4,5). Some (6,7) but not all (8,9) studies have demonstrated a positive relationship between fat intake and BC risk. Asian women with a lower BC risk consume <20 22% fat of the total calories per day (2,4,5). These women with lower BC risk also have lower excretion of urinary estrone (E1) and estradiol (E2) and similar amounts of estriol (E3) as women in Western countries (10 12), and they have high levels of phytoestrogens (4,13). The association between diet and estrogen metabolism is further supported by vegetarians living in Western countries, showing a trend toward lower urinary E1 and E2 values and similar or slightly higher E3 values than nonvegetarians (14,15). Young premenopausal vegetarian women, specifically Asian women, have relatively low concentrations of 2-hydroxy-estrogens (2-OH-E) and relatively high concentrations of 4-hydroxy-estrogens (4-OH-E) (5). In addition to estrogen changes, Asian migrants to Hawaii have low plasma androgen levels compared with women on a Western diet (2). A Western diet with high fat and protein intake and low intake of fiber, carbohydrates, and whole-grain products is associated with high plasma sex hormone levels, low sex-hormone-binding-globulin (SHBG), and high proportion of free-testosterone and free-e2 (6). However, contrary opinions have also been presented (7,16). A meta-analysis revealed that a vegetarian diet does not necessarily protect against BC and nonvegetarian eating habits per se do not elevate BC risk in postmenopausal women in all studies (8, 18). 514

3 DIETS AND HORMONAL LEVELS IN POSTMENOPAUSAL WOMEN 515 Elevated estrogen and lower levels of SHBG have been shown to result in an excess risk of BC, specifically in obese postmenopausal women (17). Evidence from epidemiologic studies indicates that estrogen is implicated in human BC (18 20). In fact, some cohort studies have revealed a strong relationship between endogenous estrogen levels and BC risk (21,22). Moreover, the level of 16α-hydroxy-estrone (16α-OH- E1) has been demonstrated to be increased in women with BC (9). Because 2-hydroxy-estrone (2-OH-E1) is weakly estrogenic and possibly antiestrogenic (9), a high level could protect against BC risk. Furthermore, based on these and other studies, the ratio 2-OH-E1/16α-OH-E1 has been proposed to serve as a marker of BC risk (23,24). Some authors have demonstrated in pre- and postmenopausal women an association between this ratio and BC risk (23), but this has not been confirmed in other studies (25,26). To our knowledge, no study has examined separately the effect of regular omnivore and vegetarian diets in postmenopausal women comparing sex hormone status and urinary, plasma, and fecal estrogen metabolites with those in postmenopausal women with BC. The aim of this study was to evaluate the relationship between type of diet and sex hormone levels in postmenopausal Finnish women. METHODS Subjects Three groups of postmenopausal women (10 healthy omnivores, 11 healthy vegetarians, and 10 BC omnivores) living in the Helsinki, Finland area were recruited. Recruitment of BC women and sample collection started 3 6 mo after tumor operation. Women included in the BC group had a tumor diameter 2 cm (Stage 1 = 66.7%; Stage 2 = 33.3%) and no detectable metastases or nodules prior to study involvement. No further treatment was given after the operation or during the collection period. At the time of this study, no further regular medical treatment was given to patients with operated small tumors. No tumors, metastases, or nodules had developed after the operation or during the collection period. All subjects agreed to consume the same diet as before the recruitment or before the operation for the BC group. To obtain a broad range of food intake, we included vegetarians in the study. Omnivorous women consumed all major types of foods, and vegetarians were vegans [consuming no animal products; n = 2 (18%)], lacto-vegetarians [consuming milk products; n = 6 (55%)], or lacto-ovo-vegetarians [consuming milk products and eggs; n = 3 (27%)]. To be considered as a regular vegetarian, women were required to have eaten this diet for at least 2 yr (mean = 14 yr). Some women enrolled in the study were physically active; others were not. This aspect was controlled by questionnaire, which included the frequency and type of physical activity practiced by women. Subjects with a history of cancer (except for the BC group) or any major diseases or using drugs, such as oral contraceptives, hormonal replacement therapy, or antibiotics, were excluded. All subjects gave their informed consent and were initially interviewed by a doctor who disclosed the study protocol. The ethics committee of the Helsinki University Central Hospital approved the research program. Collection of Samples We studied the subjects over 5 consecutive days on 4 occasions (1 visit each season, 20 days total). Each of these visits included a 72-h urine collection, a 3-day blood collection, and a 5-day food record starting 2 days prior to the urine collection. The 3-day blood and urine collections were pooled to reduce the number of analyses from 12 to 4. Hormones Plasma samples were collected from fasting subjects. To the heparin plasma samples, we added 0.1% sodium azide and 0.1% ascorbic acid, and the samples were then frozen at -20 C until analyzed. As previously described, plasma E1 and E2 were determined by radioimmunoassay (RIA) after chromatographic separation on an LH-20 column (27,28). Plasma testosterone (T) and androstenedione (A-dione) were determined as previously described by Kuoppasalmi et al. (29). Sex-hormone-bindingglobulin was determined as described by Rosner (30), with slight modifications (31). Free-E2 [in percentages (%) and in pmol/l] was calculated from total E2 and SHBG values, and free-testosterone (in% and in pmol/l) was calculated from T and SHBG values according to Bergink et al. (32). Estrone-sulfate (E1-s) was determined by RIA after separation of estrone-3- glucuroide from E1-S and enzymatic hydrolysis and extracton. Dehydroepiandrosterone-sulfate (DHEA-s) was measured by RIA with a commercial kit (Wien Laboratories, NJ). Growth hormone (GH) and insulin were also measured by RIA with commercial kits (CIS International, France). Urinary estrogen metabolites were measured by isotope dilution gas chromatography/mass spectrophotometer (GC/MS) in the selected ion monitoring mode (33,34). Urinary phytoestrogens [enterodiol, enterolactone, daidzein, O-desmethylangolensin (O-Dma), and Equol] were measured by isotope dilution GC/MS in the selected ion monitoring mode (33,34). Urinary genistein and plasma phytoestrogens (enterolactone, genistein, and daidzein) were measured by timeresolved fluoroimmunoassay (TR-FIA) (35 37). All assays with the exception of E1-S and phytoestrogens were carried out within 1 yr of the last collection, and all samples of 1 individual were analyzed simultaneously. Dietary Intake Subjects were instructed to maintain a habitual diet throughout the study. Each subject was provided with a letter balance and was instructed on how to complete the dietary records. The validity of a 3-day dietary record to estimate dietary intake in adults without cognitive impairments has been confirmed (38). We collected dietary records during 5 days in all 4 seasons including at least 1 weekend day. The urinary 72-h collection was

4 516 M. AUBERTIN-LEHEUDRE ET AL. initiated on the third day. Dietary analyses were completed by a nutritionist using the 1983 version of a coding system from the Department of Nutrition (University of Helsinki) for total, lipid, carbohydrate, protein, cholesterol, monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA), starch, saccharose, lactose, calcium, and alcohol intake. For fiber data, we used Southgate tables (39) and for some typical Finnish foods (e.g., rye products), we used the data given by manufacturers. Fiber values are presented separately for cereal, vegetables, berries and fruits, legumes, and total. Vegetable fiber values include legume fiber values. Physical Activity Level The level of physical activity was evaluated with a frequency questionnaire (how long and how many sessions per wk). We categorized women as physically inactive if they engaged in physical activity 3 or fewer times or 3 hr or less per wk. The level of physical activity was considered low or moderate when the intensity was between 0 and 8 kcal/min or less than 1,000 kcal/wk. Statistical Analyses Results are presented as means (95% CIs). Normality of distribution was determined using the Kurtosis test. Data were logtransformed if nonnormally distributed. Homogeneity of variances was tested using the Levene test. Because of the skewed distributions and the small number of subjects in each group, omnivores, vegetarians, and BC omnivores were compared using the nonparametric Kruskal-Wallis test for all variables. Pairwise comparisons of the groups were made using the nonparametric Mann-Whitney test for all variables. A general linear model, with Bonferroni post-doc test and using SHBG as a covariable was performed. Partial correlation was used to estimate the degree of association between urinary estrogen metabolites and significant variables, with SHBG, age, and body mass index (BMI) as covariables. We also performed a stepwise linear regression for urinary estrogen metabolites that included all significant variables in the model. P values 0.05 were considered statistically significant. Analyses were performed using SPSS 15.0 software (Chicago, IL). RESULTS No difference between groups for anthropometric characteristics, other than age (P = 0.002), was found. We also observed a significant difference for BMI (P = 0.026) and body weight (BW; P = 0.024) between the omnivore and vegetarian groups (Table 1). No difference between groups for dietary intake, except alcohol (P = 0.018), total fiber/kgbw (P = 0.004), and cereal fiber/kgbw (P = 0.030), was observed. Vegetarians had a significantly higher intake of total fiber (P = 0.036), legume fiber (P = 0.027), vegetable fiber (P = 0.015), total fiber/kgbw (P = 0.002), and cereal fiber/kgbw (P = 0.021), and a lower cholesterol intake (P = 0.046) and lower total fat/total fiber ratio (P = 0.036) than the BC group (Table 2). Moreover, we observed a significant difference between groups for plasma SHBG (P = 0.002), plasma E2 (P = 0.024), plasma E1-S (P = 0.043), and plasma free-e2 (pmol/l; P = 0.020), and a significant difference between vegetarian and BC groups for plasma E1-S (P = 0.023), free-e2 (pmol/l; P = 0.009), free-t (%;P = 0.043), and free-t (pmol/l; P = 0.043). Vegetarians showed a lower level of plasma E1-S, E2, free-e2, and free-t and a higher level of SHBG than omnivores or BC subjects (Table 3). We found significant differences between groups for urinary 16α-OH-E1 (P = 0.009), 16β-OH-E1 (P = 0.014), 16-keto-E2 (P = 0.009), 17-epi-estriol (P = 0.025), and 2-MeO-E1/2OH-E1 ratio (P = 0.042). This ratio was significantly higher in the BC group than in omnivores and tended to be significantly higher in BC subjects than in vegetarians. We also showed a significant difference between BC and vegetarian groups for 15α-OH-E1 (P = 0.021) and 16-epi-estriol (P = 0.035) and between omnivore and vegetarian groups for urinary E3 (P = 0.036). No difference was observed for any other urinary estrogen metabolites (Table 4). Significant differences emerged between groups for plasma genistein (P = 0.004), plasma daidzein (P = 0.014), urinary enterolactone (P = 0.014), urinary enterodiol (P = 0.010), urinary genistein (P = 0.047), and urinary daidzein (P = 0.012) (Table 5). In addition, a significant difference was present between omnivore and vegetarian groups for plasma genistein (P = 0.001), plasma daidzein (P = 0.003), O-Dma (P = 0.020), urinary genistein (P = 0.009), urinary daidzein (P = 0.002), and urinary enterolactone (P = 0.005) (Table 5). We also found a significant difference between omnivore and BC groups for urinary enterodiol (P = 0.034). Finally, a significant difference was seen between vegetarian and BC groups for plasma genistein (P = 0.016), urinary enterodiol (P = 0.009), urinary enterolactone (P = 0.033), and urinary daidzein (P = 0.048). Based on these results, we used a general linear model with SHBG as a covariable because this protein is known to play an important role in estrogen metabolism. Interestingly, no difference was subsequently found between groups for any variable (data not shown), except E1-S (between groups: P = 0.007; between vegetarians and BC group: P = 0.003; between omnivores and BC group: P = 0.010). We also carried out partial correlation calculations between estrogen metabolites and significant dietary variables, controlling for SHBG, age, and BMI. We observed that enterodiol, enterolactone, total fiber intake, cholesterol intake, fat intake, total fat/total fiber ratio, total fat/cereal fiber ratio, cereal fiber/kgbw, and total fiber/kgbw were correlated significantly with some estrogen metabolites (Table 6). We observed a significant correlation between 2-OH-E1 ans E1-sulfate (r 2 = 0.659; P < 0.001; Fig. 1). To further evaluate these results, we carried out a stepwise linear regression analysis with all significant variables found with Mann-Whitney and

5 DIETS AND HORMONAL LEVELS IN POSTMENOPAUSAL WOMEN 517 TABLE 1 Participants characteristics Characteristic Omnivores (n = 10) Vegetarians (n = 11) BC (n = 10) P value Age (yr) 57 (53 60) 60 (57 63) 66 (61 71) Height (cm) 162 ( ) 161 ± 5 ( ) 161 ± 5 ( ) NS Body weight (kg) 69.1 ( ) 57.6( ) 65.1( ) NS Body mass index (kg/m 2 ) 26.3 ( ) 22.3( ) 25.3( ) NS Age at menarche (yr) 13 (12 14) 14 (13 15) 13 (12 14) NS Age at menopause (yr) 52 (42 55) 51 (47 52) 50 (38 56) NS Age at first pregnancy (yr) 25 (22 29) 27 (24 30) 30 (23 38) NS Number of children 2.78 ( ) 2.80 ( ) 1.83 ( ) NS Smoker (%) NS Physically active (%) NS Means (95% CIs). P value is a significant difference between groups obtained using the nonparametric Kruskal-Wallis test. P < 0.05, significantly different from vegetarian group, obtained using the nonparametric Mann-Whitney test. P < 0.05, significantly different from BC group, obtained using the nonparametric Mann-Whitney test. Data derived using crosstabs and chi-square tests. TABLE 2 Dietary intake Kruskal-Wallis tests in the model and using all significant estrogen metabolites as dependent variables (Table 7). Enterodiol and fat/fiber ratio were both independent predictors of 2-OH- E1/16α-OH-E1 ratio, explaining 66% of the variance (adjusted r 2 = 0.66). Free-T and fat/fiber ratio were both independent predictors of 2-OH-E1, explaining 40% of the variance (adjusted r 2 = 0.40). Testosterone and legume fibers were both independent predictors of 4-OH-E1, explaining 91% of the variance (adjusted r 2 = 0.91). Fat/fiber ratio, T, and total fiber/kgbw were both independent predictors of 2-MeO-E1/2-OH-E1 ratio, explaining 47% of the variance (adjusted r 2 = 0.47). Testosterone and total fiber were both independent predictors of 2-MeO-E1, explaining 39% of the variance (adjusted r 2 = 0.39). Testosterone and fat/fiber ratio were both independent predictors of Intake Omnivores (n = 9) Vegetarians (n = 10) BC (n = 10) P value Energy (kcal/day) 1748 ( ) 1838 ( ) 1751 ( ) NS Total fat (g/day) 72 (54 89) 71 (62 81) 75 (61 89) NS Protein (g/day) 63 (49 77) 64 (55 74) 64 (56 72) NS Carbohydrates (g/day) 210 ( ) 243 ( ) 208 ( ) NS SFA 23 (11 38) 25 (17 55) 21 (16 37) NS MUFA (g/day) 39 (29 49) 34 (23 44) 39 (32 46) NS PUFA (g/day) 7 (6 9) 9 (7 12) 9 (5 13) NS Cholesterol (mg/day) 373 ( ) 238 ( ) 373 ( ) NS Total fiber (g/day) ( ) ( ) ( ) NS Legume fiber (g/d) 0.22 ( ) 0.87 ( ) 0.14 ( ) NS Cereal fiber (g/day) 9.52 ( ) ( ) 9.32 ( ) NS Vegetable fiber (g/day) 3.32 ( ) 5.33 ( ) 2.84 ( ) NS Berry fiber (g/day) 2.82 ( ) 6.43 ( ) 2.35 ( ) NS Total fat (g/day)/total fiber (g/day) 4.33 ( ) 3.32 ( ) 4.99 ( ) NS Total fiber (g/day)/body weight (kg) 0.25 ( ) 0.44 ( ) 0.23 ( ) Cereal fiber (g/day)/body weight (kg) 0.14 ( ) 0.19 ( ) 0.13 ( ) Total fat (g/day)/cereal fiber (g/day) 8.01 ( ) 7.27 ( ) 8.45 ( ) NS Means (95% CIs). MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid. P value is a significant difference between groups obtained using the nonparametric Kruskal-Wallis test. P < 0.05, significantly different from vegetarian group, obtained using the nonparametric Mann-Whitney test. P < 0.05, significantly different from BC group, obtained using the nonparametric Mann-Whitney test.

6 518 M. AUBERTIN-LEHEUDRE ET AL. TABLE 3 Profile of plasma steroid hormones Hormone Omnivores (n 10) Vegetarians (n = 11) BC (n = 10) P value Estrone (pmol/l) 135 ( ) 137 ( ) 155 ( ) NS Estradiol (pmol/l) 82 (59 105) 59 (48 71) 86 (57 115) Estrone-sulfate (nmol/l) 2.16 ( ) 2.00 ( ) 3.37 ( ) Testosterone (nmol/l) 1.37 ( ) 1.45 ( ) 1.81 ( ) NS SHBG (nmol/l) 52.9 (18 87) 75.8 (64 87) 38.6 (28 49) Free-estradiol (%) 2.11 ( ) 2.09 ( ) 2.14 ( ) NS Free-estradiol (pmol/l) 1.73 ( ) 1.21 ( ) 1.85 ( ) Free-testosterone (%) 1.01 ( ) 0.85 ( ) 1.05 ( ) NS Free-testosterone (pmol/l) ( ) ( ) ( ) NS Androstenedione (nmol/l) 2.86 ( ) 3.36 ( ) 3.73 ( ) NS DHEA-sulfate (µmol/l) 3.11 ( ) 1.84 ( ) 3.61 ( ) NS Testosterone + A-dione (nmol/l) 4.23 ( ) 4.81 ( ) 5.54 ( ) NS Means (95% CIs). DHEA = Dehydroepiandrosterone. P value is a significant difference between groups obtained using the nonparametric Kruskal-Wallis test. P < 0.05, significantly different from vegetarian group, obtained using the nonparametric Mann-Whitney test. P < 0.05, significantly different from BC group, obtained using the nonparametric Mann-Whitney test. 2-OH-E1/E1 ratio, explaining 65% of the variance (adjusted r 2 = 0.65). Finally, legume fiber and total fat/cereal fiber ratio were both independent predictors of 4-OH-E1/E1 ratio, explaining 89% of the variance (adjusted r 2 = 0.89). DISCUSSION Because estrogen is thought to be important in the etiology of BC, the aim of this study was to carry out a very careful evaluation of dietary intake and estrogen metabolism in postmenopausal women with or without BC. The main limitation of our study is the low number of participants in each group. This is partly compensated by the collection of food intake data over 5 consecutive days and the urine and blood collections over 3 days on 4 occasions. We determined a great number of urinary estrogen and phytoestrogen metabolites as well as plasma estrogens and phytoestrogens. To the best of our knowledge, no study has taken this approach with regard to the problem of diet, estrogen, and BC. An advantage for the calculation of correlations between dietary intake and hormone values is the wide range of food intake due to the inclusion of vegetarian and omnivore controls. We confirmed our previous study results and we suggest that the primary cause of increased estrogen and androgen is cortical stromal hyperplasia, despite not having studied it as it would need a biopsy of the ovaries. The difference between vegetarians and the 2 other groups is more likely to be caused by dietary differences. Cereal fiber intake/kg body weight is clearly higher in the vegetarians. The high SHBG is typical for vegetarians, leading to lower free estradiol and testosterone, and the lower fat/cereal fiber ratio in the vegetarians leads to lower plasma estrogens because lower amounts of fat and more cereal fiber increases the fecal excretion of estrogen (5,27,40). Omnivores had a similar intake of total fiber and cereal fiber/kg body weight as the BC subjects, but significantly lower than the vegetarians. Most fiber types were consumed in slightly larger amounts by the omnivores, but the differences were not significant. The omnivores had significantly lower plasma testosterone and E1S but higher SHBG than the BC subjects. The urinary 16-oxygenated estrogens did not differ between the omnivores and the BC subjects. However, the 2-MeO-E1/2-OH- E1 ratio in the omnivores was significantly lower than in the BC group. This ratio was found to be positively associated with fat/fiber ratio. The age of subjects varied from 53 to 71 yr and none had menstruated for 3 yr. The range of ages was taken into account during the statistical analyses, but we have not found in the literature that hormone values change in healthy women after 3 yr in menopause. We know that estrogen excretion in urine (41) and plasma testosterone (42) does not change with age in postmenopausal women. There is one study indicating a decrease of plasma estrogen during menopause from 1 yr to 8 yr (43), but in our study we found that a year after menopause there may be higher values for estrogen and, therefore, our participants were included in the study first 3 yr after menopause. SHBG may increase during menopause (44) and in obesity, but there was no correlation between SHBG and weight in our material. The low SHBG values in the BC group must, therefore, depend on factors other than weight. The mean age at first pregnancy in the BC group was highest, albeit not significantly. The mean number of children was lowest (but not significantly) in the BC group. Higher weight (17) and high age at first birth are known risk factors for BC in menopause. A smaller number of children may be related to higher age at first birth. The lack of significant differences is

7 DIETS AND HORMONAL LEVELS IN POSTMENOPAUSAL WOMEN 519 TABLE 4 Profile of urinary estrogen metabolite Metabolite Omnivores (n = 10) Vegetarians (n = 11) BC (n = 10) P value 2-OH-E1 (nmol/24h) 4.76 ( ) 4.35 ( ) 5.20 ( ) NS 4-OH-E1 (nmol/24h) 0.40 ( ) 0.58 ( ) 0.66 ( ) NS 2-OH-E2 (nmol/24h) 1.58 ( ) 1.39 ( ) 2.13 ( ) NS 2-MeO-E1 (nmol/24h) 1.37 ( ) 1.69 ( ) 2.11 ( ) NS Estrone (E1) (nmol/24h) 9.74 ( ) 8.09 ( ) 8.74 ( ) NS Estradiol (E2) (nmol/24h) 4.60 ( ) 4.05 ( ) 3.78 ( ) NS Estriol (E3) (nmol/24h) ( ) 8.90 ( ) ( ) NS 16α-OH-E1(nmol/24h) 2.28 ( ) 1.29 ( ) 2.71 ( ) β-OH-E1 (nmol/24h) 0.85 ( ) 0.52 ( ) 0.86 ( ) keto-E2 (nmol/24h) 1.18 ( ) 0.79 ( ) 1.34 ( ) α-OH-E1 (nmol/24h) 0.28 ( ) 0.19 ( ) 0.32 ( ) NS 16-epi-estriol (nmol/24h) 1.06 ( ) 0.66 ( ) 1.33 ( ) NS 17-epi-estriol (nmol/24h) 1.21 ( ) 0.41 ( ) 0.88 ( ) OH-E1/4OH-E1 (nmol/24h) ( ) 9.49 ( ) ( ) NS 2-OH-E1/E1 (nmol/24h) 0.45 ( ) 0.45 ( ) 0.53 ( ) NS 16α-OH-E1/E1 (nmol/24h) 0.30 ( ) 0.18 ( ) 0.37 ( ) NS 4-OH-E1/E1 (nmol/24h) 0.05 ( ) 0.07 ( ) 0.07 ( ) NS 2-MeO-E1/2OH-E1 (nmol/24h) 0.29 ( ) 0.34 ( ) 0.45 ( ) OH-E1/16α-OH-E1 (nmol/24h) 2.56 ( ) 4.34 ( ) 3.38 ( ) NS Total estrogens 42 (33 51) 33 (15 51) 39 (30 50) NS Means (95% CIs). 2 OH/16α-OH = (2OHE1 + 2OHE2)/(16α-OHE1 + E3). P value is a significant difference between groups obtained using the nonparametric Kruskal-Wallis test. P < 0.05, significantly different from vegetarian group, obtained using the nonparametric Mann-Whitney test. P < 0.05, significantly different from BC group, obtained using the nonparametric Mann-Whitney test. TABLE 5 Plasma and urinary phytoestrogens probably due to the small number of subjects in each group (Table 1). Fat intake (6) and fat/fiber ratio (45) have been demonstrated to be positively related to BC risk, and fiber intake to be protective (40,46,47). Our results confirmed those in the literature, with vegetarians having a significantly higher intake of total and cereal fiber/kgbw than omnivores or BC subjects (Table 2). The reason for calculating fiber intake/kgbw is based on the view Phytoestrogens Omnivores (n = 10) Vegetarians (n = 11) BC (n = 10) P value P-Enterolactone (nmol/l) 27 (16 37) 81 (12 149) 43 (12 72) NS P-Genistein (nmol/l) 4 ( ) 45 ( ) 7.2 ( ) P-Daidzein (nmol/l 3.8 ( ) 15 ( ) 8.5( ) U-Equol (nmol/72h) 53 (36 70) 260 (59 580) 45 (30 60) NS U-O-Dma (nmol/72h) 16 ( ) 81 ( ) 63 ( ) NS U-Enterolactone (µmol/72h) 2.8 ( ) 30 ( ) 3.2 ( ) U-Enterodiol (nmol/72h) 297 ( ) 1436 (0 3, 299) 486 ( 303 1, 276) U-Daidzein (nmol/72h) 71 (36 105) 373 ( ) 133 ( ) U-Genistein (µmol/72h) 96 (63 129) 516 ( ) 288 ( 431 1, 007) Means (95% CIs). U = urine; P = plasma; O-Dma = O-desmethylangolensin. Plasma values and urinary genistein values were obtained by time-resolved fluorimmuno assay. P value is a significant difference between groups obtained using the nonparametric Kruskal-Wallis test. P < 0.05, significantly different from vegetarian group, obtained using the nonparametric Mann-Whitney test. P < 0.05, significantly different from BC group, obtained using the nonparametric Mann-Whitney test.

8 520 M. AUBERTIN-LEHEUDRE ET AL. TABLE 6 Partial correlation between urinary estrogen metabolites and significant variables using sex-hormone-binding-globulin, age, and body mass index as covariables Variables in Variable nmol/24 h r 2 Enterodiol (nmol/72 2-OH-E1/16α-OH-E h) Enterolactone 2-OH-E1/16α-OH-E (nmol/72h) Total fiber (g/d) 2-OH-E1/16α-OH-E OH/16α-OH Total fat/total 2-OH-E fiber ratio (g/d) 4-OH-E OH-E1/16α-OH-E OH-E α-OH-E MeO-E1/2OH-E E OH/ 16α-OH Fat intake (g/d) 2-OH-E OH-E α-OH-E E OH/ 16α-OH Cholesterol intake E (g/d) Cereal fiber (g/d)/ 15α-OH-E body weight (kg) Total fiber (g/d)/body 2-OH/ 16α-OH weight (kg) 4-OH-E OH-E1/16α-OH-E OH-E1/E Total fat/cereal fiber 2-OH-E ratio (g/d) 2-OH-E1/4-OH-E MeO-E1/2-OH-E P P OH/16α-OH = (2OHE1 + 2OHE2)/(16α-OHE1 + E3). that body weight is related to body size. A tall woman has a higher weight and a larger gut. Height is a risk factor for BC, and this must be corrected for (5). In animals eating mainly meat, the volume of the large intestine, where the estrogens are reabsorbed, is related to body size (48). We have not found any literature regarding this for human subjects, but in the above study, most of the animals were primates. In addition, as expected, cholesterol intake was lowest in the vegetarian group. No significant differences emerged for total fat, MUFA, and PUFA intakes between groups. This is surprising because we anticipated that vegetarians would consume more unsaturated fats. However, at the time of our study collection, there was little knowledge of the benefit of unsaturated fat in the diet that FIG. 1. Correlation between E1-sulfate and 2-OHE1. may explain the similar intake in the 3 groups. We observed the fat/fiber ratio to be significantly lower in vegetarians than in BC subjects. An advantage of using the fat/fiber or fat/cereal fiber ratio for the evaluation of the relationship between diet and hormone levels is that this ratio is completely independent of body size and better reflects the situation in the intestine with regard to proportions of fat and fiber, factors influencing absorption and the enterohepatic circulation of estrogens (40). In a recent study (40) as well as earlier studies, we have discussed how diet influences enterohepatic circulation of estrogens and particularly the effect of fat and fiber. The most interesting results are the low SHBG, high testosterone, high estrone sulfate, and tendency to high estrogens (NS) in the BC subjects. The opposite was the case for the vegetarians, but this could at least to some extent be explained by the high fiber low fat diet. However, the results for the BC survivors and omnivores were found despite absence of differences in food habits. The pattern of steroid hormones was very similar to what we found in American postmenopausal women in 1989 (49) and with regard to estrone sulfate identical to what was found by Hankinson et al. (50). In the United States, women s androstenedione was significantly higher, but in our material androstenedione (and also DHEA-sulfate) did not reach significance, but they were highest in the BC group (NS). Our results confirm numerous studies showing that androgens are elevated in BC (22,51 55). The strength of our study compared to other studies is that, despite the low numbers of participants, we studied the subjects during a whole year with multiple sampling. There are considerable variations in sex hormones and also food intake during the year, and methodological variations are frequent. In our study, all food was weighed with a balance provided to the participants. It is important that all samples from 1 subject are analyzed in the same series. We suggest that the high testosterone and estrogen levels may be the primary factor affecting BC risk in this age group. The

9 DIETS AND HORMONAL LEVELS IN POSTMENOPAUSAL WOMEN 521 TABLE 7 Linear stepwise regression model Dependent Variable R 2 β (nmol/24 h) Model adjusted VIF (standardized) 2-OH/16α-OH Fat/fiber ratio (g/d) OH-E1/16α-OH-E1 Enterodiol (nmol/72h) Fat/fiber ratio (g/d) OH-E1 Fat/fiber ratio (g/d) Free-testosterone (pmol/l) OH-E1 Legumes fiber (g/d) Testosterone (nmol/l) β-OH-E1 Total fiber (g/d)/body weight (kg) keto-E2 Vegetable fiber (g/d) epi-estriol Free-testosterone (%) MeO-E1/2OH-E1 Fat/fiber ratio (g/d) Testosterone (nmol/l) Total fiber (g/d)/body weight (kg) OH-E2 E1-S (nmol/l) MeO-E1 Testosterone (nmol/l) Total fiber (g/d) OH-E1/4-OH-E1 E1-S (nmol/l) OH-E1/E1 Testosterone (nmol/l) Fat/fiber ratio (g/d) α-OH-E1/E1 Fat/fiber ratio (g/d) OHE1/E1 Legumes fiber (g/d) Total fat/cereal fiber ratio (g/d) Included all significant variables [age, weight, body mass index, estradiol, estrone-sulfate, testosterone, free-testosterone (%), free-testosterone (pmol/l), SHBG, free-estradiol (pmol/l), enterodiol, enterolactone, O-Dma, daidzein, cholesterol intake, total fiber intake, legumes fiber intake, vegetable fiber intake, fat/fiber ratio, cereal fiber intake, total fiber intake/body weight, cereal fiber intake/body weight, alcohol intake] on the models. 2 OH/16α-OH = (2OHE1 + 2OHE2)/(16α-OHE1 + E3). No model found for 16α-OH-E1, 15α-OH-E1, and 17-epi-estriol. androgens are converted to estrogens in peripheral tissues, in this case mainly estrone sulfate, but also the other estrogens are highest in the BC group. But what causes the high testosterone and other androgens and the low SHBG increasing free estradiol and testosterone? We know that ovarian tumors can cause high androgen levels, but it is unlikely that all have ovarian tumors. We also know that the adrenals produce androgens, which are converted to testosterone. This production is increased in some women but can hardly be the reason for high testosterone in our study. Previously, this was suggested to be the reason for high estrogen found in postmenopausal women (17). We also know that high endogenous testosterone or testosterone treatment may reduce circulating SHBG. Low SHBG may be developing not only if testosterone production is high but also in subjects having hypothyroidism or polycystic ovaries (PCO). In our subjects, the thyroid function was normal studied by thyroid function tests, but we did not exclude PCO. The number of children influences SHBG because women with many children have higher SHBG. Parous women have higher SHBG than nonparous, but there is no trend (56). In our material, the difference between the BC subjects and the healthy omnivores was small (1 child) and cannot explain the low SHBG. The BC survivors were significantly older than the omnivores and vegetarians, but we know that urinary estrogen excretion does not change in aging postmenopausal women (41). However, within a 10-yr period it was found that plasma testosterone increases with age and estradiol decreases (57), which could have influenced the results. If testosterone increases, estrogens must also increase. A further possibility is ovarian cortical stromal hyperplasia, which usually leads to moderate increases in androgens and estrogens in the blood [(58 61); but sometimes to higher values due to, e.g., accompanying Leydig celle or other tumors (62)]. Ovarian cortical hyperplasia is not uncommon but is seldom diagnosed as a disease if not associated with tumors causing hirsutism or virilism or endometrial cancer. We suggest that mild ovarian cortical hyperplasia could be the cause of increased androgen and estrogen production with resultant SHBG-lowering independent of weight in some of our BC subjects, resulting in decrease of SHBG and increased free steroids. The high androgen levels in BC have, to our knowledge, not been well explained. But

10 522 M. AUBERTIN-LEHEUDRE ET AL. our results do not indicate that diet has a major influence on the androgen production and low SHBG. However, the different hormone pattern in vegetarians with reduced estrogens and androgens and high SHBG is probably caused by a diet low in fat and high in fiber (40). The mechanisms have been discussed [see (40)]. The ring D oxygenated estrogens (Table 4) of BC subjects were highly significantly elevated compared with the low values in vegetarians. Catechol estrogens and estrone were also slightly higher in the BC group. We obtained a similar pattern in a recent study of postmenopausal women living in the United States (40), in which we evaluated the effect of the dietary fat/fiber ratio on hormone patterns. We found that those with the highest fat/fiber ratio have significantly higher reabsorption of biliary estrogens in the gut, leading to a urinary estrogen profile pattern with high 16-oxygenated estrogens. This could be demonstrated by measuring a specific estriol conjugate, estriol 3-glucuronide, in urine. This estriol conjugate is formed exclusively in the mucosal cells of the large bowel and is thereafter excreted unchanged in urine (40,63,64). In addition, we found that high urinary estrogens were associated with an increased 2- OH-E1/16α-OH-E1 ratio, as proposed earlier. This means that this ratio, when high, is a biomarker of BC risk because of the high estrogen level. We previously demonstrated that young Asian immigrant women in Hawaii had very low catechol estrogen excretion but also lower 16-oxygenated estrogens than Finnish women (5). The 2-OH-E1/16α-OH-E1 ratio was very low in the immigrants with low BC risk. Interestingly, plasma E1-S, the most abundant plasma estrogen, correlates highly and significantly with urinary 2-OH-E1 excretion (Fig. 1) (65,66). 2-hydroxy-estrone is, therefore, a biomarker of estrogen levels in the body. These results are in opposition to the view that a high 2-OH-E1/16α-OH-E1 ratio is associated with low BC risk. Urinary and plasma phytoestrogens (Table 5) showed, as expected, that vegetarians excreted phytoestrogens, particularly urinary lignans, in abundance. In addition, their equol production was relatively high, in accordance with the literature (67). These results confirm that vegetarians, like Asians, may have a lower BC risk because of their dietary habits (4). Table 6 shows that enterodiol, enterolactone, and total fiber (g/d)/body weight (kg) correlate significantly with the 2-OH-E1/16α-OH-E1 ratio. Fat intake is negatively associated with catechol estrogens and positively with 16 α-oh-e1. The two lignans (enterodiol and enterolactone) are positively associated with the ratio that Bradlow thinks is protective. Enterolactone also seems to be protective (68), but this is still controversial. A high dietary fat/fiber ratio was associated with high 16α-hydroxyestrone and low cathecol estrogens. This result is in agreement with our recent study on the effect of fat/fiber ratio on 16α-hydroxylation in women in Boston, Massachusetts (40). High urinary excretion of lignans was in the present study associated with high 2-OH- E1/16a-OHE1 ratio. Increased catechol estrogen formation with increasing phytoestrogen excretion could be due to competition of the phenolic compounds for estrogen-metabolizing enzymes (45). In addition, total fiber intake (g/kgbw) correlated with 4-OH-E1 and 4-OH-E1/2-OH-E1. The fiber complex contains numerous phenolic compounds, so the mechanism may be the same as suggested above. In subjects with low BC risk, the concentrations of 2-OH-E and 16α-OH-E are low (5). Interestingly, fat or cholesterol intake is negatively associated with E1 (Table 6). High fat intake is also associated with lower catechol estrogens. The finding of an increase of 4-hydroxylated estrogens related to fiber intake was unexpected, as well as the effect of fat intake on catechol estrogens. Most results in Table 6 are not in accordance with our previous work. Finally, linear stepwise regression (Table 7) shows that the predictors of the level of 4-OH-E1 are legume fiber and testosterone, together explaining more than 90% of the concentration. Legume fiber combined with dietary total fat/cereal fiber ratio explains 89% of the level of the 4-OH-E1/E1 ratio, a measure of 4-hydroxylation. While 4-OH-E2 is known to be associated with high cancer risk (69), it could not be measured in the present study. Fat/fiber ratio combined with free-t predicted 40% of the level of 2-OH-E1, and fat/fiber ratio combined with T explained 65% of the 2-OH-E1/E1 ratio. Up to 47% of the 2-MeO-E1/2- OH-E1 ratio was predicted by dietary fat/fiber ratio, plasma testosterone, and intake of total fiber/kg body weight. This ratio was highest in the BC group and significantly different from the omnivores. The level of 2-MeO-E1 was also predicted by plasma testosterone and total fiber intake up to 39%. Plasma testosterone and fiber intake were consequently involved in the sequence from E1 to 2-OH-E1 and further to 2-MeO-E1 (Table 7). Both T and estrogens increase SHBG production in liver cells (70). This pathway starting from E2 is probably an important factor in the production of SHBG. Catechol O-methyltransferase (COMT) blocks in vitro the SHBG production caused by E2, showing that methylation of 2-OH-E1 is necessary for SHBG production (70). The effect of intake of fiber and its lignans on SHBG may be of physiological importance. We have previously shown that fiber intake increases plasma SHBG (71) and have suggested that this is due to the weakly estrogenic enterolactone. These results show the importance of (cereal) fiber intake in estrogen metabolism because of the content of lignans. In conclusion, dietary impact on estrogen metabolism is much more complicated than believed 20 yr ago. The effect of fiber on enterohepatic circulation, discussed in a previous study (40), is only one side of the story. Fiber contains lignans and legumes contain isoflavonoids and other phenolic compounds that compete with estrogens for metabolic enzymes, causing changes in estrogen metabolism and, consequently, biological activities. The estrogens also compete with each other, and the pattern varies based on the level of estrogens in the body. Increased estrogen production increases both 2- hydroxylated and 16-oxygenated estrogens but relatively more the 2-hydroxylated (5). Fiber increases 2-hydroxylation, and fat increases 16α-hydroxylation. In addition, other hormones may participate in causing alterations to the levels of catechol

11 DIETS AND HORMONAL LEVELS IN POSTMENOPAUSAL WOMEN 523 estrogens and 16-oxygenated estrogens. The hormone influencing the levels of certain estrogen metabolites seems to be testosterone. Each estrogen also has its own effect on peripheral tissues, and thus estrogens should not be treated as a group. Two special findings were made. The first is the significant association between legume intake and 4-OH-E1. The biological consequences of these metabolites are still unknown, but 4-OHE2 has been found to be carcinogenic. The second is an association between total fat/fiber ratio and 2-MeO-E1/2-OH-E1 ratio. A high ratio should be beneficial because of the stimulation of SHBG production, but the fact that total fat/fiber ratio increases this ratio is somewhat confusing. Both these two special observations warrant further studies. The dietary fat/fiber ratio determines the environment for the metabolic enzymes in the gut and also for absorption. This ratio seems useful for the evaluation of dietary effects on estrogen metabolism, as it is independent of the weight and height of subjects. We conclude that the typical BC subject has high urinary 16-oxygenated estrogens, high plasma estrogens, low SHBG, and high testosterone. Vegetarians, in most aspects, show the opposite pattern. The primary event leading to increased BC risk may be increased testosterone of ovarian (or adrenal) origin, resulting in low SHBG and higher estrogens because of conversion in peripheral tissues and higher 16-oxygenated estrogens. Because of these, 16a-OH-E1 is known to be highly estrogenic. ACKNOWLEDGMENTS This work was supported by the Sigrid Jusélius Foundation and the Folkhälsan Research Center, Helsinki, Finland. Mylène Aubertin-Leheudre was supported by the Canadian Institutes of Health Research. We thank Anja Koskela, Inga Wiik, and Adile Samaletdin for excellent technical assistance, Dr. K. Höckerstedt and Dr. T. Fotsis for support during the study, and all participants. Finally, this manuscript has been edited by Ann-Caroll Pelly. No conflicts of interest occurred. REFERENCES 1. Parkin DM: Cancers of the breast, endometrium and ovary: geographic correlations. Eur J Cancer Clin Oncol 25, , Willett WC: Diet, nutrition, and avoidable cancer. Environ Health Perspect 103(Suppl 8), , Barnes S: Evolution of the health benefits of soy isoflavones. Proc Soc Exp Biol Med 217, , Adlercreutz H, Honjo H, Higashi A, Fotsis T, Hamalainen E, et al.: Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. Am J Clin Nutr 54, , Adlercreutz H, Gorbach SL, Goldin BR, Woods MN, Dwyer JT, et al.: Estrogen metabolism and excretion in Oriental and Caucasian women. J Natl Cancer Inst 86, , Boyd NF, Martin LJ, Noffel M, Lockwood GA, and Trichler DL: A metaanalysis of studies of dietary fat and breast cancer risk. Br J Cancer 68, , Armstrong B and Doll R: Environmental factors and cancer incidence and mortality in different countries, with special reference to dietary practices. Int J Cancer 15, , Hunter DJ, Spiegelman D, Adami HO, Beeson L, Van Den Brandt PA, et al.: Cohort studies of fat intake and the risk of breast cancer a pooled analysis. NEnglJMed334, , Smith-Warner SA, Spiegelman D, Adami HO, Beeson WL, Van Den Brandt PA, et al.: Types of dietary fat and breast cancer: a pooled analysis of cohort studies. Int J Cancer 92, , Goldin BR, Adlercreutz H, Gorbach SL, Woods MN, Dwyer JT, et al.: The relationship between estrogen levels and diets of Caucasian American and Oriental immigrant women. Am J Clin Nutr 44, , Macmahon B, Cole P, Brown JB, Aoki K, Lin TM, et al.: Urine oestrogen profiles of Asian and North American women. IntJCancer14, , Trichopoulos D, Yen S, Brown J, Cole P, and Macmahon B: The effect of Westernization on urine estrogens, frequency of ovulation, and breast cancer risk. A study of ethnic Chinese women in the Orient and the USA. Cancer 53, , Morton MS, Arisaka O, Miyake N, Morgan LD, and Evans BA: Phytoestrogen concentrations in serum from Japanese men and women over forty years of age. JNutr132, , Shultz TD and Leklem JE: Nutrient intake and hormonal status of premenopausal vegetarian Seventh-Day Adventists and premenopausal nonvegetarians. Nutr Cancer 4, , Gray GE, Williams P, Gerkins V, Brown JB, Armstrong B, et al.: Diet and hormone levels in Seventh-Day Adventist teenage girls. Prev Med 11, , Fung TT, Hu FB, Holmes MD, Rosner BA, Hunter DJ, et al.: Dietary patterns and the risk of postmenopausal breast cancer. Int J Cancer 116, , Key TJ, Appleby PN, Reeves GK, Roddam A, Dorgan JF, et al.: Body mass index, serum sex hormones, and breast cancer risk in postmenopausal women. JNatlCancerInst95, , Russo J and Russo IH: Genotoxicity of steroidal estrogens. Trends Endocrinol Metab 15, , Yue W, Santen RJ, Wang JP, Li Y, Verderame MF, et al.: Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis. J Steroid Biochem Mol Biol 86, , Yager JD and Davidson NE: Estrogen carcinogenesis in breast cancer. N EnglJMed354, , Toniolo PG, Levitz M, Zeleniuch-Jacquotte A, Banerjee S, Koenig KL, et al.: A prospective study of endogenous estrogens and breast cancer in postmenopausal women. JNatlCancerInst87, , Berrino F, Muti P, Micheli A, Bolelli G, Krogh V, et al.: Serum sex hormone levels after menopause and subsequent breast cancer. JNatlCancerInst 88, , Muti P, Bradlow HL, Micheli A, Krogh V, Freudenheim JL, et al.: Estrogen metabolism and risk of breast cancer: a prospective study of the 2:16alphahydroxyestrone ratio in premenopausal and postmenopausal women. Epidemiology 11, , Pasagian-Macaulay A, Meilahn EN, Bradlow HL, Sepkovic DW, Buhari AM, et al.: Urinary markers of estrogen metabolism 2- and 16 alphahydroxylation in premenopausal women. Steroids 61, , Longcope C, Gorbach S, Goldin B, Woods M, Dwyer J, et al.: The effect of a lowfat diet on estrogen metabolism. J Clin Endocrinol Metab 64, , Wellejus A, Olsen A, Tjonneland A, Thomsen BL, Overvad K, et al.: Urinary hydroxyestrogens and breast cancer risk among postmenopausal women: a prospective study. Cancer Epidemiol Biomarkers Prev 14, , Goldin BR, Adlercreutz H, Gorbach SL, Warram JH, Dwyer JT, et al.: Estrogen excretion patterns and plasma levels in vegetarian and omnivorous women. NEnglJMed307, , Adlercreutz H, Fotsis T, Heikkinen R: Current state of the art in the analysis of estrogens. In Advances in Steroid Analyses. Görög S. ed. Budapest, Ahadémia Kiado, 1982, pp