An Observational Study of Young to Middle-Aged Women Charlotte Atkinson A, Heather E

Journal of Steroid Biochemistry & Molecular Biology 86 (2003) 71–77

Urinary equol excretion in relation to 2-hydroxyestrone and 16␣-hydroxyestrone concentrations: an observational study of young to middle-aged women Charlotte Atkinson a, Heather E. Skor a, E. Dawn Fitzgibbons b, Delia Scholes c,e, Chu Chen b,e, Kristiina Wähälä d, Stephen M. Schwartz b,e, Johanna W. Lampe a,e,∗ a Cancer Prevention Research Program, Fred Hutchinson Cancer Research Center, P.O. Box 19024, MP-900, Seattle, WA 98109-1024, USA b Program in Epidemiology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA c Center for Health Studies, Group Health Cooperative, Seattle, WA 98101, USA d Department of Chemistry, University of Helsinki, Helsinki, Finland e Department of Epidemiology, University of Washington, Seattle, WA 98195, USA Received 18 November 2002; accepted 31 March 2003

Abstract Approximately one-third to one-half of individuals harbor the colonic bacteria that are capable of metabolizing the soy isoflavone daidzein to equol. Results of prior studies suggest beneficial effects of producing equol in relation to breast cancer risk, potentially through effects on endogenous hormones. High urinary excretion of 2-hydroxyestrone (2-OH E1) relative to 16␣-hydroxyestrone (16␣-OH E1) has been associated with a reduced risk of breast cancer. In this pilot study we examined associations between urinary excretion of equol and 2-OH E1,16␣-OH E1, and their ratio, and investigated whether excretion of these estrogen metabolites differed between two samples collected 48 h apart. Isoflavones (genistein, daidzein, O-desmethylangolensin (ODMA), and equol) were measured in two overnight urines from 126 women. Excretion of 2-OH E1 and 16␣-OH E1 were measured in the first overnight urine from all 126 women and in the second overnight urine from 30 of these women; there were no significant differences between samples collected 48 h apart in excretion of 2-OH E1 or 16␣-OH E1 (P = 0.75 and 0.17, respectively). Among all women, correlations between total isoflavone excretion (sum of genistein, daidzein, ODMA, and equol) and estrogen metabolites were non-significant (P>0.05). Among women with detectable levels of equol, total isoflavone excretion was significantly positively correlated with 16␣-OH E1 (r = 0.32, P = 0.02), but was not correlated with 2-OH E1 or 2-OH E1:16␣-OH E1 ratio (r = 0.21, P = 0.14, and r =−0.05, P = 0.70, respectively). Equol excretion (adjusted for other isoflavone excretion) was significantly positively correlated with 2-OH E1:16␣-OH E1 ratio (r = 0.38, P = 0.005), but was not correlated with 2-OH E1 or 16␣-OH E1 (r = 0.15, P = 0.29, and r =−0.17, P = 0.24, respectively). The finding that equol excretion, but not total isoflavone excretion, correlated positively with the 2-OH E1:16␣-OH E1 ratio suggests that the colonic bacterial profile associated with equol production may be involved in estrogen metabolism, and may therefore possibly influence breast cancer risk. © 2003 Elsevier Ltd. All rights reserved.

Keywords: Equol; Estrogen metabolism; Isoﬂavone

1. Introduction lize daidzein to equol [4–7]. Intestinal microflora are likely involved in the conversion of daidzein to equol; young in- Soy isoflavones, such as daidzein and genistein, are bio- fants, with presumably underdeveloped gut microflora, and logically active in humans and have received considerable germ-free animals do not have the ability to produce equol attention as potential cancer-preventive compounds [1,2]. [8,9]. Furthermore, in vitro incubation of daidzein with fe- Dietary interventions with soy, one of the richest identified cal flora from an equol-producer results in the conversion sources of daidzein [3], have shown that only approximately of daidzein to equol, whereas incubation with fecal flora one-third to one-half of the population are able to metabo- from an equol non-producer does not [10,11]. Equol production is therefore likely to be a biomarker of a particu- ∗ Corresponding author. Tel.: +1-206-667-6580; fax: +1-206-667-7850. lar, although yet-to-be-defined, colonic bacterial profile in E-mail address: [email protected] (J.W. Lampe). humans.

The clinical significance of having the ability to convert of these estrogen metabolites differ between two overnight daidzein to equol remains to be established fully, but sev- urine samples collected 48 h apart. eral small studies have suggested that women who are able to produce equol may be at a reduced risk of breast cancer, potentially through effects on endogenous hormone levels 2. Experimental procedures and estrogen metabolism. In a case–control study among Australian women, there was a decreased risk of breast 2.1. Study participants and data collection cancer associated with increasing quartiles of equol excretion [12], and urinary equol excretion has been inversely Participants in this pilot study were drawn from a correlated with circulating free estradiol, and positively cor- previously described study population [31]. Briefly, 363 related with sex hormone binding globulin (SHBG) [13]. women, aged 25–59 years, who had participated in a larger Furthermore, within specific menstrual cycle phases in a case–control study of risk factors for uterine leiomyomata soy feeding study, women with the ability to produce equol had also completed an ancillary protocol consisting of had statistically significantly lower plasma concentrations two overnight urine collections (made 48 h apart). Ascor- of estrone, estrone-sulfate, testosterone, androstenedione, bic acid had been added to all samples as a preservative. dehydroepiandrosterone (DHEA), DHEA-sulfate, and cor- As part of the case–control study, all urine samples were tisol, and statistically significantly higher concentrations analyzed for isoflavones (daidzein, genistein, equol, and of sex hormone binding globulin than women without the O-desmethylangolensin (ODMA)) and creatinine concen- ability to produce equol. These differences were found trations. Key features of the urine collection, isoflavone at all soy doses given, and it was suggested that the assay, and creatinine assay have been described previously ability to produce equol might represent colonic bacte- [31]. There were no significant differences between cases rial enzyme activity that increases fecal steroid excretion and controls in urinary excretion of isoflavones (P>0.05; [14]. data not shown). For the present pilot study, 126 women In the 1970s, it was proposed that estrogen metabolites (63 cases and 63 controls) who had taken part in the an- might be important in breast cancer development [15].Two cillary study were selected as follows: total isoflavone ex- major estradiol metabolites, 2-hydroxyestrone (2-OH E1) cretion (the sum of daidzein, genistein, equol, and ODMA and 16␣-hydroxyestrone (16␣-OH E1) [16,17], are produced excretions) per 24 h was estimated by multiplying data by competing pathways and have markedly different prop- expressed as nmol/h by 24, and women were placed into erties. 2-OH E1 is weakly estrogenic, and increased 2-OH one of three groups: low (0–1000 nmol/24 h; n = 206), E1 has been associated with a reduction in breast cancer risk intermediate (>1000–2000 nmol/24 h; n = 83), and high (reviewed in [17]). In contrast, 16␣-OH E1 is estrogenic, has (>2000 nmol/24 h; n = 74) excretors. We selected 40, 40, been shown to form covalent bonds with estrogen receptors, and 46 women at random from the low-, intermediate-, and may be genotoxic [18–20]. Therefore, it has been sug- and high-excretion groups, respectively. Women were gested that urinary excretion of these estrogen metabolites over-sampled from the high excretion group in order to can be used as a risk marker for breast cancer [21,22].In test the hypothesis that high excretion of isoflavones is support of this, several retrospective and prospective studies associated with a higher 2-OH E1:16␣-OH E1 ratio. have reported an increased risk of breast cancer associated Demographic data were collected as part of the main with low urinary excretion of 2-OH E1 relative to 16␣-OH case–control study and also as part of the ancillary study E1 [23–26]. Some evidence exists to suggest that dietary soy at the time of the urine collections. The study procedures can modulate estrogen metabolite profiles; two intervention were approved by the Institutional Review Boards of the studies in premenopausal women reported an increase in the Fred Hutchinson Cancer Research Center and Group Health 2-OH E1:16␣-OH E1 ratio with soy consumption [27,28], Cooperative, and all study participants provided written in- and in postmenopausal women, there was a trend towards formed consent. increased 2-hydroxylation of estrogens with soy consump- α tion [29]. Furthermore, urinary 2-OH E1 excretion increased 2.2. 2-Hydroxyestrone and 16 -hydroxyestrone analysis in mice fed either a fermented soybean extract, daidzein, or genistein [30]. Urine samples from the first overnight urine collection Because intestinal bacteria are involved in the metabolism from each of the 126 selected women were analyzed for of estrogens, inter-individual differences in host bacte- 2-OH E1 and 16␣-OH E1. To determine if excretion of 2-OH rial populations may result in differences in estrogen E1 and 16␣-OH E1 differed between two overnight urine metabolism, and therefore potentially breast cancer risk. samples collected 48 h apart, urine samples from the second Our main objective was to investigate the relationship overnight collection were also assayed for a subset of these between equol excretion and estrogen metabolism, specif- women (n = 30). The 30 pairs were randomly selected, ically the urinary concentrations of 2-OH E1,16␣-OH E1 distributed evenly among the tertiles of total isoflavone ex- and their ratio, in the context of an observational study. A cretion from which women were selected for the study (see secondary aim was to determine if urinary concentrations above), and balanced by case and control status. C. Atkinson et al. / Journal of Steroid Biochemistry & Molecular Biology 86 (2003) 71–77 73

Urine samples were assayed using the commercially avail- isoflavone data (nmol/mg Cr) and estrogen metabolite data able EstrametTM 2/16 enzyme immunoassay (EIA) kits (Im- (ng/mg Cr) were skewed and all analyses were performed munacare Corporation, Bethlehem, PA). All kits were from on log-transformed (natural log) data. Data from women the same kit lot and, upon receipt, all components were whose urinary excretion of 2-OH E1 and 16␣-OH E1 had stored as recommended by the manufacturer. The assay was been measured on both nights (n = 28) were used to exam- conducted as described elsewhere [32], with minor modifi- ine the within-woman, short-term reliability of the metabo- cations. Briefly, urine samples that had not previously been lite measures. For the measurement of urinary isoflavone thawed were diluted one part urine to one part deionized (DI) excretion, method detection limits were calculated for each water. The in-house pool sample was diluted one part urine sample based on urine volume analyzed, and recovery of in- to three parts DI water. These dilutions were established ternal standards. Values ranged from 0.04 to 0.08 nmol/ml based on the results of an initial test run of urine samples for all compounds. For samples with urinary isoflavone lev- from the study population and pooled sample, and DI water els below the method detection limit, a value of one-half the was used in place of the sample diluent supplied with the kit detection limit for that sample was assigned. In addition to to allow for larger volumes of urine and diluent to be used. performing analyses using equol excretion, we also used to- We tested the effect of using DI water versus sample diluent tal isoflavone excretion for each woman as the sum of the ex- on the concentrations of estrogen metabolites in six urine cretions of the four individual isoflavones (equol, daidzein, samples from the study population with a range of metabo- genistein, and ODMA). For all analyses regarding isoflavone lite concentrations. There were no significant differences for excretion, the mean excretion over both nights was used for either metabolite; mean 2-OH E1 concentrations for sam- a more representative measure of usual isoflavone excre- ples diluted with DI water and sample diluent were 8.21 and tion. We compared urinary excretion of estrogen metabolites 8.00 ng/ml, respectively, P = 0.91, and mean 16␣-OH E1 according to equol excretion using parametric t-tests. Ge- concentrations for samples diluted with DI water and sample ometric means were calculated from log-transformed data. diluent were 6.43 and 6.01 ng/ml, respectively, P = 0.77. Pearson correlations were calculated to estimate associa- All urine samples, standards, and controls were assayed tions between equol excretion and estrogen metabolite ex- in triplicate (three wells per sample). An in-house control cretion, total isoflavone excretion and estrogen metabolite sample (two per plate) and positive control sample (supplied excretion, and estrogen metabolite excretion in urine sam- with kit; one per plate) were included. A coefficient of vari- ples collected 48 h apart. Partial correlations were carried ation (CV) was calculated for each sample using data from out to assess the potential impact of ‘other’ isoflavone ex- the triplicate measurements. Samples with a CV greater than cretion (sum of daidzein, genistein, and ODMA), age, body 15% (primarily due to the sample being too concentrated or mass index (BMI), case–control status, smoking status, and too dilute and therefore falling within non-linear parts of the time since last menstrual period on the relationship between curve) were re-assayed using the appropriate dilution (n = equol excretion and estrogen metabolite excretion. When en- 64), or the mean of two wells was used (n = 16), contingent tered separately into a model, ‘other’ isoflavone excretion, upon the CV of the two wells being less than 15%. Four BMI, and days since last menstrual period appeared to alter samples (three from night 1 and one from night 2) were ex- the relationship, therefore associations between equol excre- cluded from all analyses due to being either too dilute or too tion and estrogen metabolite excretion were calculated with concentrated, or having unacceptable CVs that remained so and without adjustment for these three variables. even when duplicates or re-assays were considered. Thus, estrogen metabolite data were available for 123 of the 126 women on night 1, and for 28 of these women on night 2. 3. Results Mean concentrations of 2-OH E1 and 16␣-OH E1 in the in-house control urine sample were 22.2 and 10.0 ng/ml, Demographic data, and geometric mean isoflavone excre- respectively, and mean intra-plate CVs were 4.7% (range tion and estrogen metabolite data are presented in Table 1, 0.8–9.2%) and 7.3% (range 0.2–19.8%), respectively. for the 123 women for whom estrogen metabolite data were Between-plate CVs for 2-OH E1 and 16␣-OH E1 were 9.2 available. and 13.2%, respectively, for the in-house control sample, Urinary excretion of 2-OH E1 and 16␣-OH E1, and and 10.3 and 19.5%, respectively, for the positive control their ratio were highly correlated between the two samples supplied with the kit. collected 48 h apart (Fig. 1); similar results were obtained when untransformed data were used (data not shown). 2.3. Data analysis Paired t-tests showed no statistically significant difference between nights for 2-OH E1 (geometric mean = 7.85 and Statistical analyses were performed using the SAS statisti- 8.05 ng/mg Cr on nights 1 and 2, respectively, P = 0.75), cal package version 6.12 (SAS Institute, Cary, NC, USA) un- 16␣-OH E1 (geometric mean = 5.64 and 5.25 ng/mg Cr der the Windows operating system. Urinary isoflavone data on nights 1 and 2, respectively, P = 0.17), or the 2-OH and estrogen metabolite data were expressed per mg creati- E1:16␣-OH E1 ratio (geometric mean = 1.39 and 1.53 on nine (Cr) to adjust for variability in urinary output. Urinary nights 1 and 2, respectively, P = 0.21). 74 C. Atkinson et al. / Journal of Steroid Biochemistry & Molecular Biology 86 (2003) 71–77

Table 1 4 Participant characteristics 3.5 Mean age 41.9 years (range 25–56) 3 Mean BMIa 27.0 (range 17.2–48.4) 2.5 Case–control status [n (%)] Cases (with uterine fibroids) 62 (50.4) 2 Controls (without uterine fibroids) 61 (49.6) 1.5 r = 0.85 Smoking status [n (%)]b 1 Log 2-OH E1 night 2 p < 0.001 Current smokers 7 (5.8) 0.5 Non-smokers 114 (94.2) 0 Race [n (%)]c 0124 3 White 90 (73.2) Asian 20 (16.3) (a) Log 2-OH E1 night 1 Black 13 (10.6) 3.5 Isoflavone excretion (mean of nights 1 and 2)d Daidzein (nmol/mg Cr) 0.77 (0.03–18.18) 3 Genistein (nmol/mg Cr) 0.28 (0.02–5.02) 2.5 Equol (nmol/mg Cr) 0.07 (0.02–6.06) O-Desmethylangolensin (nmol/mg Cr) 0.15 (0.02–5.48) 2 d Estrogen metabolites 1.5 2-OH E (ng/mg Cr) 10.58 (1.64–80.83) 1 r = 0.93 16␣-OH E1 (ng/mg Cr) 7.27 (1.75–34.26) 1 2-OH E :16␣-OH E ratio 1.45 (0.37–5.55) 1 1 0.5 p < 0.001 Log 16alpha-OH E1 night 2 a 2 Weight (kg)/height (m) (unavailable for one woman). 0 b Unknown for two women. 02134 c Adds to 100.1% due to rounding up of significant figures. d Geometric means and range of untransformed data presented. Log 16alpha-OH E1 night 1

(b) Fifty-seven (46.3%) of the 123 women had detectable 2 levels of equol in at least one of their two urine collections. 1.5 There were no statistically significant differences between women with (n = 57) and without (n = 66) detectable lev- 1 els of equol for 2-OH E1 excretion (geometric mean = 10.0 r = 0.70 and 11.1 ng/mg Cr, respectively, P = 0.43), 16␣-OH E1 0.5 p < 0.001 excretion (geometric mean = 7.1 and 7.4 ng/mg Cr, re- 0 spectively, P = 0.63), and the 2-OH E1:16␣-OH E1 ratio = . P = . Log 2:16 ratio night 2 -1.5 -1 -0.5 0 0.5 1 1.5 2 (geometric mean 1 4 and 1.5, respectively, 0 54). -0.5 Correlations between isoflavone excretion and 2-OH E1, 16␣-OH E1, and 2-OH E1:16␣-OH E1 ratio are shown in -1 Table 2. Among all women, the positive correlation be- (c) Log 2:16 ratio night 1 tween equol excretion and the 2-OH E1:16␣-OH E1 ratio was of borderline statistical significance. When analyses Fig. 1. Relationship between urinary excretion of estrogen metabolites ␣ ␣ were restricted to women with detectable levels of equol (a) 2-OH E1; (b) 16 -OH E1; (c) 2-OH E1:16 -OH E1 ratio, in two overnight urine samples collected 48 h apart. there was a statistically significant positive correlation be- ␣ tween total isoflavone excretion and 16 -OH E1, and a the 2-OH E1:16␣-OH E1 ratio, and adjustment for urinary statistically significant positive correlation between equol excretion of the other isoflavones strengthened this associa- excretion (adjusted for ‘other’ isoflavone excretion) and the tion. In addition, we observed a non-significant inverse cor- ␣ 2-OH E1:16 -OH E1 ratio. All other correlations were not relation between equol excretion (adjusted for excretion of P> . statistically significant ( 0 05) (Table 2). other isoflavones) and 16␣-OH E1. In contrast, there was a significant positive correlation between total isoflavone excretion and 16␣-OH E1, which is potentially less desirable, 4. Discussion although this was apparent only when considering women with detectable levels of equol and not all women. The main aim of this pilot study was to assess the relation- A secondary aim of this pilot study was to determine if ship between equol excretion and urinary excretion of two concentrations of the estrogen metabolites, 2-OH E1 and major estrogen metabolites, 2-OH E1 and 16␣-OH E1.We 16␣-OH E1, differed between two overnight urine samples observed a positive correlation between equol excretion and collected 48 h apart. In agreement with Chen et al. [33],we C. Atkinson et al. / Journal of Steroid Biochemistry & Molecular Biology 86 (2003) 71–77 75

did not ﬁnd a statistically signiﬁcant difference in the 2-OH

0.70) ␣ 0.005) 0.07) E1:16 -OH E1 ratio between nights, nor did we ﬁnd a statis- = = = P tically signiﬁcant difference between nights for the individ- P P ual metabolites. These data suggest that one overnight urine 0.05 ( 0.38 ( 0.26 ( sample is useful for examining potential determinants of the = =− = ratio, however long-term variability in overnight urinary ex- 2:16 ratio r r r cretion of the estrogen metabolites was not investigated in this study.

b Intervention studies in both pre- and post-menopausal 55) 0.24) women have reported increases in the 2-OH E1:16␣-OH E1 0.02) 0.74) = = n = = ratio with dietary soy supplementation, resulting from either P 1 P P an increase in the excretion of 2-OH E1 [28,29] or a decrease

0.17 ( in the excretion of 16␣-OH E1 [27]. Our data suggest that, 0.32 ( 0.05 ( -OH E ␣ =− = = independent of total isoflavone excretion, equol excretion 16 r r r is positively associated with the 2-OH E1:16␣-OH E1 ratio, which is broadly consistent with the findings of Duncan et al. [14]; in a sample of 14 premenopausal women, they 0.29) 0.14) 0.12) found that, compared to non-producers, equol producers had = = = plasma hormone profiles consistent with the hypothesis of a P P P

1 reduced risk of breast cancer associated with reduced circu- 0.15 ( 0.21 ( 0.22 ( lating levels of estrogens [34]. However, they did not report = = = the effects of equol production on the 2-OH E1:16␣-OH E1 Women with detectable levels of equol ( r r r ratio. Gut bacteria are involved in the metabolism of both steroid hormones and isoflavones, and perturbations in 0.05) 0.69) 0.07) colonic microflora such as that seen with antibiotics, can re- = = = P P P sult in alterations in estrogen metabolism [35–39]. Our find- ratio

1 ing of an association between equol excretion and estrogen 0.18 ( 0.04 ( 0.17 ( metabolism, in combination with the likelihood that intesti- = = = -OH E 2:16 ratio 2-OH E r r r ␣ nal bacteria are responsible for equol production [10,11], :16 avone data were log transformed.

1 suggests that equol production may be a biomarker of gut ﬂ bacteria associated with pathways of estrogen metabolism that could inﬂuence risk of breast and other hormone de- 0.77) 0.13) 0.77)

= pendent cancers. Alternatively, equol itself may inﬂuence and 2-OH E = = P

1 the activity of enzymes involved in estrogen metabolism. 1 P P

ve women. Lu et al. [28] suggested that the increase in 2-OH E1 ex- ﬁ 0.03 ( -OH E 0.14 ( 0.03 (

-OH E cretion seen with soy consumption may have been due to ␣ ␣ = = =− alterations in enzymes involved in the formation of 2-OH 16 r r r ,16 1 E1, including the cytochromes P-450, and in vitro, equol

a has been shown to inhibit the activity of aromatase, an

118) enzyme involved in estrogen synthesis [40]. 0.14) 0.15) 0.29) = Although several hypotheses have been put forward to n = = =

P P P explain inter-individual differences in equol production, it

1 remains unclear why some people have the ability to pro-

0.14 ( 0.14 ( 0.10 ( duce equol while others do not. Composition of the intesti- = = =

All women ( 2-OH E r r r nal microﬂora, intestinal transit time, and variability in the redox potential of the colon might contribute to variation in equol production in humans [11]. Diet has also been asso- avone excretion and urinary 2-OH E c ciated with the equol-producer phenotype; however, results ﬂ d

, are conﬂicting and sample sizes are relatively small [41,42]. c In a cross-sectional study, we observed that equol-producing avone excretion is the sum of daidzein, genistein, and ODMA. ﬂ c

avones women had, on average, a higher intake of dietary ﬁber com- iso ﬂ

’ pared to non-producers [5]. However, in a feeding study, avone excretion iso ﬂ ’ we were unable to induce equol production by supplement- Other ‘ Data on BMI or days since last menstrual period unavailable for two women. Data on BMI or days since last menstrual period unavailable for Adjusted for BMI and days since last menstrual period; estrogen metabolite and iso a b c d

other ing the diets of non-producers with high-fiber cereals [43]. ‘ Table 2 Correlations between iso Total iso Equol excretion Equol excretion adjusted for There do not appear to be sex or ethnic/racial differences 76 C. Atkinson et al. / Journal of Steroid Biochemistry & Molecular Biology 86 (2003) 71–77 in the prevalence of equol producers [5,41], and, unless a [2] M. Messina, V. Persky, K.D.R. Setchell, S. Barnes, Soy intake and person is on chronic antibiotic therapy, the capacity to pro- cancer risk: a review of the in vitro and in vivo data, Nutr. Cancer duce equol remains relatively stable [43]. The stability of 21 (1994) 113–131. [3] K. Reinli, G. Block, Phytoestrogen content of foods—a compendium the equol-producer phenotype raises the possibility that the of literature values, Nutr. Cancer 26 (1996) 123–148. populations of equol-producing bacteria in the colon may [4] G.E. Kelly, C. Nelson, M.A. Waring, G.E. Joannou, A.Y. Reeder, be determined by host genetics; however, there are no data Metabolites of dietary (soya) isoflavones in human urine, Clin. Chim. as yet to support this. Acta 223 (1993) 9–22. This observational study in a well-characterized study [5] J.W. Lampe, S.C. Karr, A.M. Hutchins, J.L. Slavin, Urinary equol excretion with a soy challenge: influence of habitual diet, Proc. Soc. population adds further support to the data on potential as- Exp. Biol. Med. 217 (1998) 335–339. sociations between the equol-producer phenotype and hor- [6] A.M. Hutchins, J.L. Slavin, J.W. Lampe, Urinary isoflavonoid mones [14]. However, a major limitation of the study is that phytoestrogen and lignan excretion after consumption of fermented we did not use a soy challenge to determine equol-producer and unfermented soy products, J. Am. Diet. Assoc. 95 (1995) 545– status, which may have resulted in misclassification. Some 551. [7] K.D. Setchell, N.M. Brown, E. Lydeking-Olsen, The clinical women who did not have detectable levels of equol might importance of the metabolite equol—a clue to the effectiveness of be classified as equol producers following a sufficient soy soy and its isoflavones, J. Nutr. 132 (2002) 3577–3584. dose. Conversely, some people with very low, but detectable [8] M.L.A. Cruz, W.W. Wong, F. Mimouni, D.L. Hachey, K.D.R. levels of equol in our study would still only excrete small Setchell, P.D. Klein, R.C. Tsang, Effects of infant nutrition on amounts of equol following a soy challenge; the excretion cholesterol synthesis rates, Pediatr. Res. 35 (1994) 135–140. [9] I. Rowland, H. Wiseman, T. Sanders, H. Adlercreutz, E. Bowey, of low levels of equol by equol non-producers may be due Metabolism of oestrogens and phytoestrogens: role of the gut to dietary sources [44]. Nevertheless, despite this potential microflora, Biochem. Soc. Trans. 27 (1999) 304–308. limitation we still observed a relationship between equol ex- [10] C. Atkinson, O. Humbert, H.E. Skor, J.W. Lampe, Soy isoflavone cretion and the 2-OH E1:16␣-OH E1 ratio, which was more metabolism: investigating a biomarker of colonic environment, Proc. pronounced among women with detectable levels of equol Am. Assoc. Cancer Res. 42 (2001) 766 (abstract). [11] K.D.R. Setchell, S.P. Borriello, P. Hulme, D.N. Kirk, M. than among all women. This suggests that if equol-producer Axelson, Nonsteroidal estrogens of dietary origin: possible roles in status had been more clearly defined, relationships between hormone-dependent disease, Am. J. Clin. Nutr. 40 (1984) 569–578. equol-producer status and the estrogen metabolites may have [12] D. Ingram, K. Sanders, M. Kolybaba, D. Lopez, Case–control study been even stronger. of phyto-oestrogens and breast cancer, Lancet 350 (1997) 990–994. In conclusion, we have provided evidence that equol ex- [13] H. Adlercreutz, K. Hockerstedt, C. Bannwart, S. Bloigu, E. ␣ Hamalainen, T. Fotsis, A. 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