Dietary isoflavones are biologically active in humans, but few observational data exist on the relationship between isoflavone intake and excretion in Western populations. We examined associations between self-reported soy intakes and overnight urinary isoflavone excretion in a population-based sample of western Washington State women, and we investigated the usefulness of one versus two overnight urine samples, collected 48 h apart, as a biomarker of intake. Isoflavones (genistein, daidzein, O-desmethylangolensin, and equol) were measured in two overnight urine collections from 363 women recruited from a health maintenance organization. Soy food intakes were assessed using two 1-day diet records completed on each day prior to the urine collections and a food frequency questionnaire (FFQ) that had been completed by 312 of the women with regard to their dietary habits 3.5 years (range, 2–5 years) before the urine collections. Twenty-one percent of the women consumed soy on either day of the diet recall, and 13% and 34% of the women consumed soy at least once a week or at least once a month, respectively, according to the FFQ. Women who consumed soy at either of the two diet recalls or at the FFQ (at least once a week or at least once a month) had a significantly higher urinary excretion of isoflavones than women who did not consume soy (P < 0.01). Among women who consumed soy at either of the two diet recalls or at the FFQ (soy consumed at least once a month), isoflavone intake and excretion correlated significantly (P < 0.01). Excretion of the individual isoflavones correlated significantly between the two urine samples collected 48 h apart (genistein, r = 0.41 and P < 0.001; daidzein, r = 0.30 and P < 0.001; O-desmethylangolensin, r = 0.46 and P < 0.001; equol, r = 0.60 and P < 0.001). Differences between soy consumers and nonconsumers and associations between intakes and excretion remained significant whether one or both urine collections were considered. Measuring isoflavone excretion in one overnight urine collection serves as a biomarker of recent or past isoflavone intake, even in populations whose intake of soy foods is relatively low.

Soybeans and soybean products are the richest identified sources of isoflavones, biologically active compounds that have been shown to have potential cancer-preventive and other health benefits in humans (1). Possible beneficial effects of isoflavones have been attributed to a variety of mechanisms, determined from work carried out both in vivo and in vitro. Among other effects, isoflavones have been shown to influence the levels and bioactivity of sex hormones, angiogenesis, vascular reactivity, malignant cell proliferation, lipid oxidation, and protein tyrosine kinase activity (2, 3, 4, 5, 6, 7, 8). Measuring dietary intakes of isoflavones is therefore essential if the potential health benefits of these compounds in populations are to be determined.

Typically, high concentrations of isoflavones in urine are associated with the consumption of soybeans and soybean products, such as tofu and miso; strong positive associations between self-reported dietary intakes of isoflavone-rich foods and urinary excretion of isoflavones have consistently been reported in populations that regularly consume soy (9, 10, 11). Furthermore, a linear dose-response relationship exists between dietary intake and urinary excretion of isoflavones (12). Such findings have led to the suggestion that urinary isoflavone levels could be used as a biomarker of soy consumption in epidemiological studies of diet-disease associations (11).

The majority of the studies to date, however, have focused on populations in which foods rich in isoflavones are commonly consumed, such as Chinese and Japanese populations (10, 11, 13, 14). Relatively few data from observational studies are available for populations whose intake of soy foods and therefore isoflavones is relatively low, such as Western populations. Feeding studies among individuals living in the United States have shown dose-dependent associations between soy protein intake and urinary isoflavone excretion, even at relatively low doses of soy that more closely reflect consumption patterns in the United States (15, 16, 17). In one of the few observational studies that has investigated soy intakes in relation to isoflavone excretion in a predominantly Caucasian group of men and women on a Western diet, significant correlations between intakes of soyfoods and urinary excretion of isoflavones were found when self-administered FFQs3 and 5-day food records were used to assess dietary intakes, and isoflavone excretion was measured in urine collected for 72-h (18). However, it would be impractical to implement 72-h urine collections in large-scale epidemiological studies.

Therefore, the main purpose of this study was to investigate the relationship between self-reported soy food intake and excretion of isoflavones in two overnight urine collections, collected 48 h apart, in a sample of predominantly Caucasian women who were living in the United States. Dietary intake of soy foods was assessed for each day prior to the urine collections and had also been assessed previously with regard to a time period averaging 3.5 years before the urine collections; our aim was therefore to investigate relationships between isoflavone intake and excretion using both current and past dietary data. Because isoflavones are rapidly metabolized and excreted in urine, a secondary aim was to determine whether one or two overnight urine collections would be necessary to determine a relationship between intake and excretion, using both current and past dietary data.

Participants.

Participants in this study were drawn from a larger case-control study of risk factors for uterine leiomyomata. In the case-control study, women with uterine leiomyomata newly diagnosed between September 1995 and June 1998 (cases) were identified from among the members of GHC, a large, staff-model health maintenance organization. For comparison, an age-stratified sample of women who had no history of uterine leiomyomata (controls) were identified from GHC enrollment databases. Each case and control was invited to participate in a data collection protocol that included an in-person interview and completion of a self-administered dietary assessment (see below). Of 880 eligible cases identified, 647 (73.6%) were successfully recruited. Similarly, of 861 eligible controls identified, 637 (74.0%) were successfully recruited.

Between April 1997 and October 1999, a sample of women who had participated in the case-control study were randomly selected, with an oversampling of Asian and Black women, to be recontacted for an ancillary protocol consisting of two overnight urine samples (separated by 48 h), blood samples, and dietary recall questionnaires. Women were informed that the goal of the study was to measure dietary and environmental hormonal elements in blood and urine and to investigate how these substances may affect the chance of a woman developing uterine fibroids. Of 228 cases and 241 controls selected for the additional protocol, 27 were ineligible on the basis of having used antibiotics (n = 23) or received surgery requiring anesthesia (n = 4) in the preceding 3 months. Among the remaining 218 eligible cases and 224 eligible controls, 191 cases (87.6%) and 177 controls (79.0%) participated in the ancillary data protocol. Three cases and two controls were excluded from our analyses due to incorrectly collected urine or incomplete dietary recall data, for a total of 363 participants in the ancillary study group analyses.

Demographic data were collected as part of the main case-control study and were available for 360 of the 363 women with diet recall and urinary isoflavone data available from the additional protocol. The study procedures were approved by the institutional review boards of the Fred Hutchinson Cancer Research Center and GHC.

Urine Collections.

Each participating woman was asked to make two overnight urine collections 48 h apart (referred to hereafter as night 1 and night 2). The study field staff visited each woman in her home to provide her with two 3-liter brown urine sample bottles (one for each collection) that each contained 0.5 g of ascorbic acid as a preservative and to explain the urine collection protocol. For each overnight collection, the woman was instructed to empty her bladder completely before going to bed and to discard the urine. She was then told to collect all urine excreted during the night (if any) and just after rising in the morning. The urine samples were stored in a cooler with ice packs until retrieved by a member of the study staff; both samples were retrieved on the day of the second overnight urine collection. The isoflavones and their major metabolites are very stable in urine and losses are minimal with storage even at room temperature.4 When received at the laboratory, samples were refrigerated, and they were processed within 4 h of receipt. Urine volume was measured, and aliquots were frozen at −70°C until analysis.

Dietary Assessment.

In the evening on which each overnight urine collection began, each woman was asked to complete a self-administered recall of her diet during the preceding day. Thus, each participating woman completed two such dietary recalls, corresponding to each of the days prior to the overnight urine collections. The questionnaire, which had been formulated specifically for this study, consisted of a list of 82 items and included 10 foods that are known to contain appreciable quantities of isoflavones [tofu, tempeh, miso or miso soup, burgers made with soy, hot dogs made with soy, soy cheese, soy yogurt (not frozen), soy ice cream or frozen yogurt, soybean sprouts, and soymilk]. Information on the number of servings consumed during the day and serving size (small, medium, or large) was collected. In addition to the listed foods, each woman was asked to list any other dietary supplements, such as herbal products and protein, she had consumed that day.

At the time of the initial interview for the main case-control study, each woman was asked to complete a self-administered FFQ. The FFQ had been developed and validated for use in the Women’s Health Initiative (19), and each woman was asked to complete the questionnaire with regard to her dietary intake 2 years prior to her assigned reference date. Consequently, the FFQ data represented dietary intakes an average of 3.5 years (range, 2–5 years) before the urine collections were made. The FFQ consisted of three sections: (a) adjustment questions; (b) 122 foods and food groups; and (c) summary questions. Within the second section, the frequency of intake and usual serving size of “tofu and textured vegetable products” (asked as one food group) was ascertained. Soymilk consumption was ascertained using adjustment questions within section one regarding the usual type of milk consumed, in combination with questions in section two on the frequency of consumption and usual serving size of milk on cereal and in beverages. No other soy foods were listed in the main FFQ. Therefore, to ascertain soy food intakes more completely, a separate 16-item FFQ was also given to each woman at the initial interview that asked for information on frequency of intake and usual serving size of four additional soy foods [tempeh, soy cheese, soy yogurt (not frozen), and soy ice cream or frozen yogurt]. The FFQ also asked for information on consumption of vegetarian burgers and vegetarian hot dogs. If these items were consumed, two adjustment questions asked how often these items were made with soy products; vegetarian burgers or hot dogs were classed as soy-based if responses to the adjustment questions were “almost always” or “often” made with soy. FFQ data were available for 312 (86.0%) of the 363 women for whom we had both diet recall and isoflavone excretion data.

For each food item on the diet recall questionnaire and the FFQ, an indication of a medium serving size was given (for example, ½ cup of tofu), and women were asked to note whether a small, medium, or large serving size had been consumed (dietary recall) or was usually consumed (FFQ). Isoflavone levels (mg/100 g) of the soy foods were obtained from the United States Department of Agriculture data base on the genistein and daidzein contents of soy foods;5 if data on more than one type or brand of a soy food were available, a mean of the reported values was taken. From these data, isoflavone levels in a medium portion size were calculated, and this was adjusted according to the reported serving size and frequency of consumption to obtain an estimate of isoflavone intake. Data on soy protein and/or isoflavone content of specific products were obtained from the manufacturer. If data on portion size were not available for a soy-containing food noted within the “any other dietary supplements” question on the diet recall questionnaire, a medium portion size was assigned. Weekly isoflavone intakes according to the diet recall were estimated as (mean intake over the 2 days/2) × 7.

Isoflavone Analysis.

Urinary levels of total daidzein, genistein, ODMA, and equol were quantified as described previously (20), with minor modifications. Modifications included a volume of 10–12 ml of urine for analysis and combined fraction collection after quaternary aminoethyl-Sephadex chromatography. These changes were made to simplify the extraction of total isoflavones and to accommodate analysis of all target compounds in a single chromatographic run.

Deuterated analogues of each target compound were added as internal standards, and 4-methylumbelliferone glucuronide was added to monitor for loss during the hydrolysis step. Samples were extracted and enzymatically hydrolyzed (using Helix pomatia extract; Sigma Chemical Co., St. Louis, MO) as described previously (20) and stored in 100% methanol. Trimethylsilyl derivatives were analyzed by gas chromatography-mass spectrometry in the selected ion-monitoring mode on an Agilent Technologies (Palo Alto, CA) 5973 Mass Selective Detector (MSD) instrument with Chemstation software. The instrument configuration included a fused silica capillary column (12 m × 0.20 mm × 0.33 μm) 100% poly(dimethylsiloxane) bonded phase SBP-1 (Supelco, Bellefonte, PA). Temperature settings of the injection port, transfer line, ion source, and quadrapole were 250°C, 290°C, 230°C, and 110°C, respectively. The oven temperature was held at 100°C for 1 min and then increased to 280°C at a rate of 20°C/min, followed by a 4.5-min hold time. An injection volume of 1 μl was used for all samples and standards. Instrument detection limits for pure standards were 50–100 pg for all compounds. Method detection limits were calculated for each sample, based on urine volume analyzed and recovery of internal standards. Values ranged from 0.04–0.08 nmol/ml for all compounds. Samples with urinary isoflavone levels below the method detection limit were assigned a value of one-half the detection limit. Average 4-methylumbelliferone recovery from hydrolyzed 4-methylumbelliferone glucuronide was 80%. Each batch included 20 samples, consisting of 10 pairs (i.e., samples from nights 1 and 2) of overnight urines. Laboratory staff were blinded to all characteristics of each aliquot. Two previously unthawed aliquots of an overnight urine pool from female donors were included in each batch for quality control purposes; mean urinary daidzein, genistein, ODMA, and equol in pool samples were 4.9, 3.2, 0.6, and 0.2 nmol/ml, respectively. Based on the results for the quality control aliquots, the intra-run CV values were <5% for all compounds. Inter-run CV values for daidzein, genistein, ODMA, and equol were 12.9%, 22.4% 16.7%, and 14.8%, respectively.

Results for ODMA excretion and equol excretion on day 2 were unavailable for two women and one woman, respectively, due to interfering peaks on the chromatogram; data on the other analytes were available for these women.

Cr Analysis.

Urinary Cr concentrations were measured on aliquots from all overnight urine samples based on a kinetic modification of the Jaffe reaction using the Roche Reagent for Cr (Roche Diagnostic Systems, Nutley, NJ) on a Roche Cobas Mira Plus chemistry analyzer, after first diluting the samples 1:50 with distilled water. The assay was linear to 20 mg/dl, and the intra- and inter-assay CVs were between 1% and 2%.

Data Analyses.

Urinary isoflavone data (calculated as nmol/mg Cr) and isoflavone intake data (expressed as nmol/day or nmol/week) were skewed, and all analyses were performed on log-transformed data. For isoflavone excretion data, the natural log (ln) was taken. For isoflavone intake data, ln + 1 was computed due to a large number of zero values within the data set. In addition to performing analyses using individual isoflavones, we also used total isoflavone excretion for each woman as the sum of the excretions of the four individual isoflavones. We compared urinary excretion of isoflavones according to soy consumer status using parametric t tests. Pearson correlations were calculated to estimate the association between isoflavone excretion in the two urine samples collected 48 h apart and between isoflavone excretion and soy isoflavone intake. Geometric means and 95% CIs were calculated from the log-transformed data.

Participants in this study averaged 43.3 years of age (range, 25.0–59.0 years; Table 1) at recruitment into the main case-control study. Data on ethnicity, household income, and level of education are presented in Table 1.

Detectable levels of at least one of the four isoflavones measured were found in either overnight urine sample from 355 (97.8%) of the women. Detectable levels of genistein and daidzein were found in urine samples from 228 (62.8%) and 294 (81.0%) women, respectively, on night 1 and from 224 (61.7%) and 281 (77.4%) women, respectively, on night 2. Pearson correlations between the two measures of urinary isoflavone excretion (samples collected 48 h apart) were as follows: (a) genistein, r = 0.41 and P < 0.001; (b) daidzein, r = 0.30 and P < 0.001; (c) ODMA, r = 0.46 and P < 0.001; and (d) equol, r = 0.60 and P < 0.001. Paired t tests on log-transformed data showed no significant difference in total isoflavone excretion between the two nights when considering all women (0.76 and 0.77 nmol/mg Cr on nights 1 and 2, respectively; P = 0.82), soy consumers on either day of the diet recall (2.71 and 2.28 nmol/mg Cr on nights 1 and 2, respectively; P = 0.39), soy nonconsumers on both days of the diet recall (0.54 and 0.58 nmol/mg Cr on nights 1 and 2, respectively; P = 0.40), soy consumers at the FFQ (soy at least once a month; 1.04 and 1.14 nmol/mg Cr on nights 1 and 2, respectively; P = 0.56), and soy nonconsumers at the FFQ (0.62 and 0.64 nmol/mg Cr on nights 1 and 2, respectively; P = 0.70).

Reported intakes of genistein and daidzein according to the diet recall and the FFQ are presented in Table 2. Geometric mean weekly intakes of genistein and daidzein for all women at the FFQ were 2.0 and 1.7 nmol/week, respectively. Geometric mean excretion of isoflavones among soy consumers and soy nonconsumers is compared in Table 3. Differences in isoflavone excretion between consumers and nonconsumers of selected soy foods or any soy food according to the diet recall questionnaire were significant on both nights and when an average of the two nights was taken (Table 3). Due to low numbers of consumers on either day, separate t tests were not carried out for consumers of soy ice cream/frozen yogurt (n = 9), soy burgers (n = 7), soy cheese (n = 3), or soy hot dogs (n = 2); no women reported consuming tempeh or soy yogurt. Differences in isoflavone excretion between consumers (frequent consumers or all consumers combined) and nonconsumers of soy foods according to the FFQ were significant on both nights and when an average of the two nights was taken. There were no differences in isoflavone excretion between consumers and nonconsumers when infrequent consumers (soy once or twice a month) were considered alone.

A significantly greater proportion of Asian women reported consuming soy on either of the diet recalls compared with non-Asian women (37.5% versus 18.4%, respectively; χ2 = 9.3; P < 0.01). Similarly, 75.0% of Asian women reported consuming soy on the FFQ, compared with 27.6% of non-Asian women (χ2 = 35.1; P < 0.01). Irrespective of consumer status, geometric mean total isoflavone excretion was significantly higher among Asian women than non-Asian women on day 2 (1.31 and 0.72 nmol/mg Cr for Asian and non-Asian women, respectively; P = 0.04) and when a mean of the 2 days was taken (1.62 and 0.88 nmol/mg Cr for Asian and non-Asian women, respectively; P = 0.02). The difference was not significant on day 1 (1.08 and 0.71 nmol/mg Cr for Asian and non-Asian women, respectively; P = 0.10). When considering Asian women and non-Asian women separately, differences in isoflavone excretion between consumers and nonconsumers according to the two diet recalls were significant on both nights and when an average of the two nights was taken for Asian women and also for non-Asian women (Table 3). Differences in isoflavone excretion between consumers and nonconsumers according to the FFQ were significant for non-Asian women, but not for Asian women (Table 3).

Forty (38.1%) women who reported consuming soy products on the FFQ (soy at least once a month) were also classified as soy consumers according to the two diet recall questionnaires, and 182 (87.9%) women who were nonconsumers of soy at the FFQ (soy never or less than once a month) were also classified as nonconsumers according to the diet recall questionnaires. Geometric mean total isoflavone excretion for these women was 4.3 nmol/mg Cr (range, 0.4–50.8 nmol/mg Cr) and 0.7 nmol/mg Cr (range, 0.1–9.4 nmol/mg Cr), respectively. Geometric mean total isoflavone excretion for women who were soy consumers according to the diet recalls but not the FFQ (n = 25) and for women who were soy consumers according to the FFQ but not the diet recalls (n = 65) was 2.4 nmol/mg Cr (range, 0.2–11.2 nmol/mg Cr) and 0.7 nmol/mg Cr (range, 0.2–41.3 nmol/mg Cr), respectively.

We examined associations between isoflavone intake and isoflavone excretion; using data on isoflavone intake (nmol/day) determined from the diet recall, we found significant positive correlations on both days and for the average of the 2 days (Table 4 and Fig. 1,A). The correlation between mean intake (sum of genistein and daidzein) and mean excretion (sum of daidzein, genistein, ODMA, and equol) over the 2 days for women who consumed soy on both days of the diet recall (n = 22) was 0.39 (P = 0.07). Using intake data determined from the FFQ, correlations between intake (nmol/week) and mean excretion over the 2 days are presented in Table 4 and Fig. 1 B. Correlations between intake and excretion were not significant when considering the frequent and infrequent consumers separately but were significant when the two groups were combined. Correlations between total intake (sum of genistein and daidzein) and total excretion (sum of daidzein, genistein, ODMA, and equol) for frequent and infrequent consumers combined (n = 105) remained significant when considering excretion on days 1 and 2 separately (day 1, r = 0.22 and P = 0.02; day 2, r = 0.28 and P < 0.01).

We report the results of one of the few observational studies to examine self-reported soy food intake in relation to overnight urinary isoflavone excretion in a population-based sample of women living in the United States. We observed that women who reported consuming soy foods had a significantly higher urinary isoflavone excretion than women with no reported soy intake when intakes were determined using diet recall questionnaires completed at the time of the urine collections or using an FFQ that ascertained intake 3.5 years prior to the urine collections. In addition, we observed significant correlations between intakes, which were determined using either the diet recall or the FFQ (women who reported consuming soy at least once a month), and excretion of isoflavones. These findings were of a similar magnitude if one or both urine collections were considered, which suggests that one overnight urine collection may be useful for reflecting current and/or usual consumption of soy foods in this predominantly Caucasian population living within the greater Seattle area. Urinary excretion of the individual isoflavones correlated significantly between the two nights, and there were no significant differences in isoflavone excretion between the two nights for soy consumers, nonconsumers, or all women combined. This further supports our finding that one overnight urine collection captures information about both recent or historical isoflavone intakes.

Our findings of significant associations between isoflavone intakes and urinary isoflavone excretion are consistent with those of studies carried out in predominantly Asian populations (9, 10). However, in comparison with those studies, associations between intakes and excretion among the soy consumers in our study (using diet recall or FFQ data) were slightly weaker. For example, the correlation between isoflavone intake (previous 24 h) and overnight urinary isoflavone excretion among women in a multiethnic population was 0.62 (P < 0.001; Ref. 9), and the correlation between isoflavone intake (previous 5 years) and overnight urinary isoflavone excretion in a Chinese population was 0.54 (P < 0.001; Ref. 10).

Relatively few women in our study population consumed soy foods, despite the increased availability and heightened consumer awareness in the United States of the potential health benefits of consuming soy. Only 21% of the women in our study reported consuming soy on either day of the diet recall, and fewer than 13% reported consuming soy at least once a week according to the FFQ. In comparison, Chen et al.(10) reported that in a Chinese population, almost all (97%) of their study participants consumed soy foods at least once a week. The prevalence of soy food consumption among individuals residing in the United States has been assessed in two previous studies (18, 21), but both were based on highly selected populations (high/low fruit and vegetable consumers and the faculty, staff, and students of a naturopathic university, respectively) and were relatively small in size (n = 98 and n = 51, respectively). Our data therefore provide an indication of soy food consumption patterns from a larger, more representative population of United States residents than has been previously studied. However, our study population was restricted to females ages 25–59 years who resided in a metropolitan area of western Washington State and who were enrollees in a health maintenance organization; thus, the current soy food consumption patterns in other United States populations remain to be determined.

Despite the low frequency of soy consumption in our study, we detected isoflavones in the urine of almost all women on at least one of the two nights. Furthermore, isoflavone excretion varied considerably among both the consumers and nonconsumers of soy foods. These findings suggest widespread exposure to isoflavones in our population. It is now well established that a wide variety of processed foods contain appreciable quantities of isoflavones (22), which could account for variations in urinary levels of isoflavones among individuals with no reported soy intakes. To determine isoflavone intakes accurately using questionnaires, a comprehensive data base of the isoflavone content of many foods available in specific populations, in addition to traditional soy-based foods, will therefore be necessary (22, 23, 24). Urinary isoflavone excretion can also be influenced by a number of factors other than dietary intakes, including bacterial populations in the gut, intestinal transit time, and alterations in the redox level in the large intestine (25), sex (26, 27), and length of time of soy consumption (16), which also may have contributed to the variation in isoflavone excretion seen in this study among both consumers and nonconsumers of soy foods.

The half-lives of plasma isoflavones are approximately 6–8 h (28). Urinary isoflavone concentrations will therefore most likely reflect recent soy exposure, and, as expected, we observed a significantly higher excretion of isoflavones in women who had consumed soy immediately before the urine collections than in women who had not done so. However, we also observed a significant difference in isoflavone excretion between consumers and nonconsumers identified using the FFQ, which initially suggested that the soy intake patterns of the women in this study did not change appreciably in the approximately 3.5 years between the two assessment periods. Interestingly, only 38% of the women who were soy consumers at the time of the FFQ were also soy consumers at the diet recall, suggesting that persistent soy intake does not explain the associations. Although we saw a significant correlation between isoflavone intake and excretion among all consumers according to the FFQ, we did not observe this relationship among frequent or infrequent consumers alone. This is presumably due to the small sample sizes within the subsets and insufficient statistical power to detect a relationship.

Estimated intakes of isoflavones among the women in our study were considerably lower than those reported among Asian populations (9, 11, 29). However, when considering the soy consumers alone in our study, daily intakes according to the diet recall began to approach levels reported among some Asian populations (9, 29). Similarly, overnight urinary excretion of isoflavones only began to approach levels similar to those reported in overnight urines from a Chinese population (10) when soy consumers alone were considered.

Asian and black women were oversampled for this ancillary study, which led to a higher proportion of Asians and blacks and a lower proportion of Caucasians in our study population than those reported in census data for the King County area of Seattle (30). Asian populations have traditionally consumed soy foods more frequently than non-Asian populations, and in accordance with this, we found that the proportion of Asian women who were classified as soy consumers was significantly higher than the proportion of non-Asian women classed as soy consumers according to both the diet recall and the FFQ. Furthermore, and in agreement with Maskarinec et al.(9), who examined isoflavone excretion in a multiethnic population, we observed that Asian women had a significantly higher urinary excretion of isoflavones than non-Asian women. When our data were analyzed separately for Asians and non-Asians, differences in isoflavone excretion between consumers and nonconsumers according to the diet recall remained significant when considering either Asians or non-Asians alone. The number of Americans who perceive soy and soy products to be healthy has shown an increasing trend since 1997. Furthermore, the percentage of Americans who use soy or soy protein products almost doubled from 15% in 1998 to 27% in 2000.6 Ethnic differences that have typically been observed may therefore become less apparent with time.

In conclusion, our data support the use of urinary isoflavone excretion as a biomarker of both recent and past soy food intake in young-to-middle-aged women living in western Washington State, who have a relatively low and infrequent intake of soy foods. Furthermore, we have shown that isoflavone measurement in one overnight urine collection can provide information on current and/or usual consumption of soy foods in such populations and can distinguish, on average, between soy consumers and nonconsumers.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by National Institute of Environmental Health Sciences Grant ES 08305, National Institute of Child Health and Human Development Grant HD 29829, and the Fred Hutchinson Cancer Research Center.

3

The abbreviations used are: FFQ, food frequency questionnaire; ODMA, O-desmethylangolensin; GHC, Group Health Cooperative of Puget Sound; CV, coefficient of variation; CI, confidence interval; Cr, creatinine.

4

Unpublished data.

5

United States Department of Agriculture-Iowa State University Database on the Isoflavone Content of Foods, Release 1.2, 2000. www.nal.usda.gov/fnic/foodcomp/Data/isoflav/isoflav.html.

6

Consumer Attitudes about Nutrition, United Soybean Board National Report, 2001. www.talksoy.com.

Fig. 1.

Correlation between intake and excretion of isoflavones. A shows intakes measured by diet recall [r (consumers either day) = 0.52; P < 0.001]; B shows intakes measured by FFQ [r (all consumers) = 0.29; P < 0.01]. Isoflavone intake = sum of genistein and daidzein; isoflavone excretion = sum of genistein, daidzein, ODMA, and equol (mean of days 1 and 2).

Fig. 1.

Correlation between intake and excretion of isoflavones. A shows intakes measured by diet recall [r (consumers either day) = 0.52; P < 0.001]; B shows intakes measured by FFQ [r (all consumers) = 0.29; P < 0.01]. Isoflavone intake = sum of genistein and daidzein; isoflavone excretion = sum of genistein, daidzein, ODMA, and equol (mean of days 1 and 2).

Close modal
Table 1

Demographic characteristics of participants

CharacteristicNo. of women (%)
Age (yrs)  
 25–29 12 (3.3) 
 30–34 26 (7.2) 
 35–39 51 (14.0) 
 40–44 109 (30.0) 
 45–49 106 (29.2) 
 50–54 48 (13.2) 
 55–59 11 (3.0) 
Race/ethnicity  
 Caucasian 275 (76.4) 
 Black 36 (10.0) 
 Chinese 14 (3.9) 
 Japanese 14 (3.9) 
 Other Asian 20 (5.6) 
 Native American 1 (0.3) 
Income level (U.S.a dollars)  
 <15,000 or 15,000–24,999 37 (10.3) 
 25,000–34,999 45 (12.5) 
 35,000–49,999 79 (21.9) 
 50,000–69,999 89 (24.7) 
 70,000–89,999 55 (15.3) 
 ≥90,000 49 (13.6) 
 Refused or not known 6 (1.7) 
Level of education  
 High school only 50 (13.9) 
 More than high school but no college graduation 97 (26.9) 
 College graduation or more 213 (59.2) 
CharacteristicNo. of women (%)
Age (yrs)  
 25–29 12 (3.3) 
 30–34 26 (7.2) 
 35–39 51 (14.0) 
 40–44 109 (30.0) 
 45–49 106 (29.2) 
 50–54 48 (13.2) 
 55–59 11 (3.0) 
Race/ethnicity  
 Caucasian 275 (76.4) 
 Black 36 (10.0) 
 Chinese 14 (3.9) 
 Japanese 14 (3.9) 
 Other Asian 20 (5.6) 
 Native American 1 (0.3) 
Income level (U.S.a dollars)  
 <15,000 or 15,000–24,999 37 (10.3) 
 25,000–34,999 45 (12.5) 
 35,000–49,999 79 (21.9) 
 50,000–69,999 89 (24.7) 
 70,000–89,999 55 (15.3) 
 ≥90,000 49 (13.6) 
 Refused or not known 6 (1.7) 
Level of education  
 High school only 50 (13.9) 
 More than high school but no college graduation 97 (26.9) 
 College graduation or more 213 (59.2) 
a

U.S., United States.

Table 2

Geometric mean daily and weekly genistein and daidzein intakes according to the diet recall and FFQ

RecallFFQ
Day 1 (52; 363)aDay 2 (46; 363)Mean daily intake (76; 363)Estimated weekly intake (76; 363)Once per week consumers (39)Once or twice a month consumers (66)All soy consumers (105)
Mean [95% CI] Rangeb (nmol/day)cMean [95% CI] Range (nmol/day)Mean [95% CI] Range (nmol/day)Mean [95% CI] Range (nmol/week)Mean [95% CI] Range (nmol/week)Mean [95% CI] Range (nmol/week)Mean [95% CI] Range (nmol/week)
Soy consumers        
 Genistein 61.1 [55.1–67.7] 42.6 [37.9–47.8] 30.6 [27.0–34.5] 104.6 [92.3–118.4] 81.3 [70.2–94.1] 11.9 [10.5–13.4] 24.7 [19.4–31.3] 
 5.8–1075.5 4.0–430.4 2.0–752.9 7.0–2635.2 9.0–1014.7 0.9–64.4 0.9–1014.7 
 Daidzein 45.3 [40.8–50.3] 32.1 [28.7–35.9] 23.0 [20.4–26.0] 78.2 [69.0–88.5] 60.4 [52.4–69.7] 8.4 [7.4–9.4] 17.8 [14.0–22.7] 
 3.5–794.8 2.6–319.1 1.3–556.9 4.6–1949.2 6.8–755.3 0.8–37.1 0.8–755.3 
All women        
 Genistein 0.8 [0.5–1.1] 0.6 [0.4–0.8] 1.1 [0.8–1.4] 1.7 [1.2–2.3]    
 0–1075.5 0–430.4 0–752.9 0–2635.2    
 Daidzein 0.7 [0.5–1.0] 0.6 [0.4–0.8] 0.9 [0.7–1.2] 1.5 [1.1–2.0]    
 0–794.8 0–319.1 0–556.9 0–1949.2    
RecallFFQ
Day 1 (52; 363)aDay 2 (46; 363)Mean daily intake (76; 363)Estimated weekly intake (76; 363)Once per week consumers (39)Once or twice a month consumers (66)All soy consumers (105)
Mean [95% CI] Rangeb (nmol/day)cMean [95% CI] Range (nmol/day)Mean [95% CI] Range (nmol/day)Mean [95% CI] Range (nmol/week)Mean [95% CI] Range (nmol/week)Mean [95% CI] Range (nmol/week)Mean [95% CI] Range (nmol/week)
Soy consumers        
 Genistein 61.1 [55.1–67.7] 42.6 [37.9–47.8] 30.6 [27.0–34.5] 104.6 [92.3–118.4] 81.3 [70.2–94.1] 11.9 [10.5–13.4] 24.7 [19.4–31.3] 
 5.8–1075.5 4.0–430.4 2.0–752.9 7.0–2635.2 9.0–1014.7 0.9–64.4 0.9–1014.7 
 Daidzein 45.3 [40.8–50.3] 32.1 [28.7–35.9] 23.0 [20.4–26.0] 78.2 [69.0–88.5] 60.4 [52.4–69.7] 8.4 [7.4–9.4] 17.8 [14.0–22.7] 
 3.5–794.8 2.6–319.1 1.3–556.9 4.6–1949.2 6.8–755.3 0.8–37.1 0.8–755.3 
All women        
 Genistein 0.8 [0.5–1.1] 0.6 [0.4–0.8] 1.1 [0.8–1.4] 1.7 [1.2–2.3]    
 0–1075.5 0–430.4 0–752.9 0–2635.2    
 Daidzein 0.7 [0.5–1.0] 0.6 [0.4–0.8] 0.9 [0.7–1.2] 1.5 [1.1–2.0]    
 0–794.8 0–319.1 0–556.9 0–1949.2    
a

Numbers in parentheses represent (number of soy consumers; number of all women).

b

Range of original (untransformed) data.

c

1 nmol genistein = 0.270 μg, 1 nmol daidzein = 0.254 μg.

Table 3

Geometric mean isoflavone excretion (nmol/mg Cr)a comparing soy consumers versus nonconsumers based on intakes of individual soy foods and any soy foods at the dietary recall and on frequencies of consumption of soy foods at the FFQ

Geometric mean isoflavone excretion (nmol/mg Cr)a comparing soy consumers versus nonconsumers based on intakes of individual soy foods and any soy foods at the dietary recall and on frequencies of consumption of soy foods at the FFQ
Geometric mean isoflavone excretion (nmol/mg Cr)a comparing soy consumers versus nonconsumers based on intakes of individual soy foods and any soy foods at the dietary recall and on frequencies of consumption of soy foods at the FFQ
a

Sum of genistein, daidzein, ODMA, and equol.

b

In parentheses, (number of nonconsumers at the diet recall; number of nonconsumers at the FFQ with isoflavone data available).

c

Statistical analyses performed on log-transformed variables; back-transformation of mean (geometric mean) and 95% CI are presented.

d

Range of untransformed data.

Table 4

Correlations (r) between isoflavone intake and isoflavone excretion among soy consumers, by instrument used for assessing soy intakea

Intake (nmol)bExcretion (nmol/mg Cr)Soy consumer as determined by
Dietary recallFFQ
Day 1 (n = 52)Day 2 (n = 46)Either day (n = 76)Frequent (n = 39)cInfrequent (n = 66)dAll (n = 105)e
rrrrrr
Genistein Genistein 0.36 0.56 0.43 0.06 0.25 0.31 
  [P < 0.01]f [P < 0.001] [P < 0.001] [P = 0.74] [P = 0.05] [P < 0.01] 
Daidzein Daidzein 0.39 0.46 0.49 0.03 0.12 0.24 
  [P < 0.01] [P < 0.01] [P < 0.001] [P = 0.88] [P = 0.35] [P = 0.01] 
Daidzein Sum (daidzein)g 0.45 0.51 0.55 0.07 0.13 0.28 
  [P < 0.001] [P < 0.001] [P < 0.001] [P = 0.65] [P = 0.30] [P < 0.01] 
Daidzein + genistein Sum (isoflavones)h 0.45 0.56 0.53 0.07 0.16 0.29 
  [P < 0.001] [P < 0.001] [P < 0.001] [P = 0.66] [P = 0.20] [P < 0.01] 
Intake (nmol)bExcretion (nmol/mg Cr)Soy consumer as determined by
Dietary recallFFQ
Day 1 (n = 52)Day 2 (n = 46)Either day (n = 76)Frequent (n = 39)cInfrequent (n = 66)dAll (n = 105)e
rrrrrr
Genistein Genistein 0.36 0.56 0.43 0.06 0.25 0.31 
  [P < 0.01]f [P < 0.001] [P < 0.001] [P = 0.74] [P = 0.05] [P < 0.01] 
Daidzein Daidzein 0.39 0.46 0.49 0.03 0.12 0.24 
  [P < 0.01] [P < 0.01] [P < 0.001] [P = 0.88] [P = 0.35] [P = 0.01] 
Daidzein Sum (daidzein)g 0.45 0.51 0.55 0.07 0.13 0.28 
  [P < 0.001] [P < 0.001] [P < 0.001] [P = 0.65] [P = 0.30] [P < 0.01] 
Daidzein + genistein Sum (isoflavones)h 0.45 0.56 0.53 0.07 0.16 0.29 
  [P < 0.001] [P < 0.001] [P < 0.001] [P = 0.66] [P = 0.20] [P < 0.01] 
a

FFQ correlations are based on mean isoflavone excretion over the 2 days.

b

nmol/day for intake data derived from the diet recall, nmol/week for intake data derived from the FFQ.

c

Women who consumed soy once a week or more.

d

Women who consumed soy once or twice a month.

e

Women who consumed soy at least once a month.

f

In brackets, P for correlation coefficient.

g

Sum (daidzein) = daidzein plus its metabolites ODMA and equol.

h

Sum (isoflavones) = daidzein + genistein + ODMA + equol.

We thank Joia Hicks for recruiting women into the urine collection protocol, Dick Jacke for data management and computer programming, Jean Jue and Vicky Tran for data entry, and Noemi Epstein for specimen repository management.

1
Adlercreutz H., Mazur W. Phyto-oestrogens and Western diseases.
Ann. Med.
,
29
:
95
-120,  
1997
.
2
Lu L. J., Anderson K. E., Grady J. J., Nagamani M. Effects of soya consumption for one month on steroid hormones in premenopausal women: implications for breast cancer risk reduction.
Cancer Epidemiol. Biomark. Prev.
,
5
:
63
-70,  
1996
.
3
Fotsis T., Pepper M., Adlercreutz H., Fleischmann G., Hase T., Montesano R., Schweigerer L. Genistein, a dietary-derived inhibitor of in vitro angiogenesis.
Proc. Natl. Acad. Sci. USA
,
90
:
2690
-2694,  
1993
.
4
Honore E. K., Williams J. K., Anthony M. S., Clarkson T. B. Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques.
Fertil. Steril.
,
67
:
148
-154,  
1997
.
5
Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shibuya M., Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases.
J. Biol. Chem.
,
262
:
5592
-5595,  
1987
.
6
Adlercreutz H. Diet, breast cancer, and sex hormone metabolism.
Ann. N. Y. Acad. Sci.
,
595
:
281
-290,  
1990
.
7
Tikkanen M. J., Wähälä K., Ojala S., Vihma V., Adlercreutz H. Effect of soybean phytoestrogen intake on low density lipoprotein oxidation resistance.
Proc. Natl. Acad. Sci. USA
,
95
:
3106
-3110,  
1998
.
8
Pagliacci M. C., Smacchia M., Migliorati G., Grignani F., Riccardi C., Nicoletti I. Growth-inhibitory effects of the natural phyto-oestrogen genistein in MCF-7 human breast cancer cells.
Eur. J. Cancer
,
30A
:
1675
-1682,  
1994
.
9
Maskarinec G., Singh S., Meng L., Franke A. A. Dietary soy intake and urinary isoflavone excretion among women from a multiethnic population.
Cancer Epidemiol. Biomark. Prev.
,
7
:
613
-619,  
1998
.
10
Chen Z., Zheng W., Custer L. J., Dai Q., Shu X. O., Jin F., Franke A. A. Usual dietary consumption of soy foods and its correlation with the excretion rate of isoflavonoids in overnight urine samples among Chinese women in Shanghai.
Nutr. Cancer
,
33
:
82
-87,  
1999
.
11
Seow A., Shi C. Y., Franke A. A., Hankin J. H., Lee H. P., Yu M. C. Isoflavonoid levels in spot urine are associated with frequency of dietary soy intake in a population-based sample of middle-aged and older Chinese in Singapore.
Cancer Epidemiol. Biomark. Prev.
,
7
:
135
-140,  
1998
.
12
Slavin J. L., Karr S. C., Hutchins A. M., Lampe J. W. Influence of soybean processing, habitual diet, and soy dose on urinary isoflavonoid excretion.
Am. J. Clin. Nutr.
,
68
:
1492S
-1495S,  
1998
.
13
Adlercreutz H., Honjo H., Higashi A., Fotsis T., Hamalainen E., Hasegawa T., Okada H. Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet.
Am. J. Clin. Nutr.
,
54
:
1093
-1100,  
1991
.
14
Arai Y., Uehara M., Sato Y., Kimira M., Eboshida A., Adlercreutz H., Watanabe S. Comparison of isoflavones among dietary intake, plasma concentration, and urinary excretion for accurate estimation of phytoestrogen intake.
J. Epidemiol.
,
10
:
127
-135,  
2000
.
15
Karr S. C., Lampe J. W., Hutchins A. M., Slavin J. L. Urinary isoflavonoid excretion in humans is dose dependent at low to moderate levels of soy-protein consumption.
Am. J. Clin. Nutr.
,
66
:
46
-51,  
1997
.
16
Xu X., Duncan A. M., Merz B., Kurzer M. S. Effects of soy isoflavones on estrogen and phytoestrogen metabolism in premenopausal women.
Cancer Epidemiol. Biomark. Prev.
,
7
:
1101
-1108,  
1998
.
17
Xu X., Harris K. S., Wang H-J., Murphy P., Hendrich S. Bioavailability of soybean isoflavones depends upon gut microflora in women.
J. Nutr.
,
125
:
2307
-2315,  
1995
.
18
Lampe J. W., Gustafson D. R., Hutchins A. M., Martini M. C., Li S., Wähälä K., Grandits G. A., Potter J. D., Slavin J. L. Urinary isoflavonoid and lignan excretion on a Western diet: relation to soy, vegetable, and fruit intake.
Cancer Epidemiol. Biomark. Prev.
,
8
:
699
-707,  
1999
.
19
Patterson R. E., Kristal A. R., Tinker L. F., Carter R. A., Bolton M. P., Agurs-Collins T. Measurement characteristics of the Women’s Health Initiative food frequency questionnaire.
Ann. Epidemiol.
,
9
:
178
-187,  
1999
.
20
Lampe J. W., Skor H. E., Li S., Wähälä K., Howald W. N., Chen C. Wheat bran and soy protein feeding do not alter urinary excretion of the isoflavan equol in premenopausal women.
J. Nutr.
,
131
:
740
-744,  
2001
.
21
Kirk P., Patterson R. E., Lampe J. Development of a soy food frequency questionnaire to estimate isoflavone consumption in U.S.
adults. J. Am. Diet. Assoc.
,
99
:
558
-563,  
1999
.
22
Horn-Ross P. L., Barnes S., Lee M., Coward L., Mandel J. E., Koo J., John E. M., Smith M. Assessing phytoestrogen exposure in epidemiologic studies: development of a database (United States).
Cancer Causes Control
,
11
:
289
-298,  
2000
.
23
Liggins J., Bluck L. J., Runswick S., Atkinson C., Coward W. A., Bingham S. A. Daidzein and genistein content of fruits and nuts.
J. Nutr. Biochem.
,
11
:
326
-331,  
2000
.
24
Liggins J., Bluck L. J., Runswick S., Atkinson C., Coward W. A., Bingham S. A. Daidzein and genistein contents of vegetables.
Br. J. Nutr.
,
84
:
717
-725,  
2000
.
25
Setchell K. D. R., Borriello S. P., Hulme P., Kirk D. N., Axelson M. Nonsteroidal estrogens of dietary origin: possible roles in hormone-dependent disease.
Am. J. Clin. Nutr.
,
40
:
569
-578,  
1984
.
26
Kirkman L. M., Lampe J. W., Campbell D. R., Martini M. C., Slavin J. L. Urinary lignan and isoflavonoid excretion in men and women consuming vegetable and soy diets.
Nutr. Cancer
,
24
:
1
-12,  
1995
.
27
Lu L-J. W., Anderson K. E. Sex and long-term soy diets affect the metabolism and excretion of soy isoflavones in humans.
Am. J. Clin. Nutr.
,
68
:
1500S
-1504S,  
1998
.
28
Watanabe S., Yamaguchi M., Sobue T., Takahashi T., Miura T., Arai Y., Mazur W., Wähälä K., Adlercreutz H. Pharmacokinetics of soybean isoflavones in plasma, urine, and feces of men after ingestion of 60 g baked soybean powder (kinako).
J. Nutr.
,
128
:
1710
-1715,  
1998
.
29
Wakai K., Egami I., Kato K., Kawamura T., Tamakoshi A., Lin Y., Nakayama T., Wada M., Ohno Y. Dietary intake and sources of isoflavones among Japanese.
Nutr. Cancer
,
33
:
139
-145,  
1999
.
30
1990 Census of Population and Housing: Population and Housing Characteristics for Census Tracts and Block Numbering Areas; Seattle-Tacoma, WA CMSA (Part), Seattle, WA PMSA. Section 1 of 2: Bureau of the Census, Economics and Statistics Administration, U.S. Department of Commerce, 1993.