One-month exposure to soy isoflavones did not induce the ability to produce equol in postmenopausal women

ORIGINAL ARTICLE

One-month exposure to soy isoflavones did not

induce the ability to produce equol in

postmenopausal women

N Ve´drine

, J Mathey

, C Morand

, M Brandolini

, M-J Davicco

, L Guy

, C Re´me´sy

, V Coxam

and C Manach

1_{CHRU Clermont-Ferrand, Service Urologie, Clermont-Ferrand, France;}2_{Unite´ des Maladies Me´taboliques et Micronutriments, INRA}

Clermont-Theix, St-Gene`s Champanelle, France and3_{Laboratory of Human Nutrition, CRNH d’Auvergne, Clermont-Ferrand, France}

Objective: As more and more postmenopausal women are taking soy isoflavone supplementation for relieving menopausal symptoms, we investigated the impact of chronic exposure on their bioavailability, with focus on achievable plasma concentrations and potential stimulation of the capacity to produce equol.

Subjects: A total of 12 Caucasian postmenopausal women.

Intervention: Volunteers ingested 100 mg isoflavones/day (aglycone equivalents, in cereal bars and yoghurts) for 1 month. Plasma concentrations of metabolites at 2, 4, 6, 8, 10, 12 and 24 h postdose, as well as urinary excretion in fractions over 36 h were compared between days 1 and 30.

Results: Similar plasma kinetic curves were obtained at day 1 and day 30 for genistein and daidzein. Maximum plasma concentrations were 1.6870.68 mmol/l on day 1 compared to 2.2770.76 mmol/l on day 30 for daidzein (P ¼ 0.056), and 3.8871.50 mmol/l on day 1 compared to 5.3072.38 mmol/l on day 30 for genistein (P ¼ 0.091). Urinary excretion of daidzein and genistein did not differ significantly between days 1 and 30. Maximum plasma concentration of equol increased significantly from 0.3170.27 to 0.9970.51 mmol/l for equol-producer volunteers (P ¼ 0.046). However, the seven volunteers who were classified as non-equol producers on day 1 did not acquire the ability to produce equol after 1-month exposure. Conclusions: Chronic exposure to isoflavones in postmenopausal women resulted in plasma concentrations as high as 2.5– 5 mmol/l of each isoflavone, but did not induce the ability to produce equol.

Sponsorship: French Ministry of Research (AQS program A01347) and Beghin-Meiji (doctoral fellowship).

European Journal of Clinical Nutrition (2006) 60, 1039–1045. doi:10.1038/sj.ejcn.1602415; published online 15 February 2006

Keywords: isoflavones; genistein; equol; pharmacokinetics; chronic exposure; dietary supplements

Introduction

Asian populations have been consuming high levels of soy isoflavones for centuries. Epidemiological data suggest that this may lead to a significant protection towards some hormone-dependant diseases such as breast or prostate cancer (Messina, 2003; Mishra et al., 2003; Magee and Rowland, 2004). Furthermore, Asian women generally

experience less troublesome symptoms of menopause than Western women (Boulet et al., 1994). As a consequence, many isoflavone-rich extracts have been developed and marketed in Western countries in the last years, with a wide commercial success. However, our understanding of the physiological impact of isoflavones is still poor and it cannot be ruled out that isoflavone-rich extracts may have detri-mental effects, possibly related to those reported for hormone replacement therapy or selective estrogen receptor modulators such as tamoxifen (Jordan, 2004). Much higher doses of isoflavones can be achieved with supplements than with foods. Most isoflavone-rich extracts are prepared from the soy germ, which has a different composition than the whole grain (Erdman et al., 2004). These extracts contains a higher proportion of glycitin and a lower proportion of

Received 19 September 2005; revised 16 December 2005; accepted 5 January 2006; published online 15 February 2006

Correspondence: Dr C Manach, Unite´ des Maladies Me´taboliques et Micronutriments, INRA Clermont-Theix, St-Gene`s Champanelle 63122, France.

E-mail: manach@clermont.inra.fr

genistein than the traditional Asian foods. As these iso-flavones may have different biological activities, effects of supplements may differ from that of traditional Asian foods. Other factors linked to the ethnic background may also differently modulate the impact of isoflavones in Asian or Western populations.

Improving our knowledge of the isoflavone bioavailability is thus crucial, especially in postmenopausal Western women, who are the most exposed to a potential risk linked to the consumption of isoflavone supplements. Identifying the factors that control the ability to produce the microbial metabolite equol is a question of prime importance. There is a great interindividual variability in the capacity to produce equol. Only 30–40% of individuals in the Western popula-tion are able to produce this metabolite (Karr et al., 1997; Setchell et al., 2002). Equol has been reported to be more estrogenic than its precursor daidzein in in vitro studies or in experiments on animal models (Setchell et al., 2002). Furthermore, a higher impact of soy consumption has been observed for equol producers compared to nonproducers in the first clinical studies that have classified the volunteers (Lydeking-Olsen et al., 2004; Meyer et al., 2004). More favorable hormonal profiles were found in equol producers, consistently with a lower risk for breast cancer (Duncan et al., 2000). A case-controlled study on prostate cancer in Asian men also reported that the proportion of equol producers was 1.5–2-fold lower in prostate cancer patients than in control men (Akaza et al., 2004). Thus, available data suggest that the soy consumption is more highly beneficial or may be only beneficial to equol producers compared to non-producers. In these conditions, a new challenge is to find a way to stimulate the capacity to produce equol in more individuals.

Long-term exposure to isoflavones, because it could induce some bacterial activities, is one of the factors that may affect equol production. Another reason to study long-term exposure is that the systemic bioavailability of daidzein and genistein has generally been studied following a single dose of isoflavones. Pharmacokinetics data are lacking for term isoflavone exposure, despite the fact that long-term supplementation is usually proposed to postmeno-pausal women for relieving their menopostmeno-pausal symptoms.

Methods

Materials

A powdered soya isoflavone concentrate containing 46.19 g/ 100 g total isoflavone aglycone equivalents (Prevasteins

HC, Eridania Beghin-Say) was incorporated into yoghurts (Danone, Vitapole, France) and cereal bars (Nutrition Sante´, France). Each yoghurt or bar provided 25 mg of isoflavone aglycone equivalents (genistein 55–75%, daidzein 20–40% and glycitein 1–5%).

Standards of daidzein, genistein and equol were purchased from Extrasynthese (Genay, France).

Subjects

A total of 12 healthy postmenopausal women were recruited and completed the study. Subject characteristics (mean7 s.e.m.) were: weight 70.473.5 kg (range 57.5–90.0), body mass index 28.571.1 (range 24.1–34.6), age 64.872.3 (range 52–73) and menopause years 17.873.7 (range 3–37). The volunteers had stable food habits evaluated by a diet history questionnaire. Exclusion criteria included strict vegetarian and vegan, high-fiber and high-soy diets, antibiotics or hormonal replacement therapy within 3 months, menstrual bleeding within 12 months, history of chronic digestive disorders or gastrointestinal tract surgery, allergy for milk protein or soy and severe constipation.

The study protocol was approved by the regional Biome-dical Research Ethics Committee (CCPPRB, Clermont-Ferrand, France), and all participants gave their written informed consent.

Study design

During the whole experimental period and the 3 days before, the volunteers were asked to maintain their normal food intake and to avoid phytoestrogen consumption. They were given a list of authorized and prohibited food items. All soy-based foods (tofu, tempeh, miso, natto, soymilk, soybeans, soy cheese, soy-based yogurts and desserts), as well as cereal bars, breakfast cereals, foods for vegetarians, nuggets, burgers, meal substitutes and manufactured meals were prohibited. On days 1 and 30, food consumption of volunteers was assessed by a dietary interview.

Urine was collected for the last 24 h before the beginning of the kinetic study to check that isoflavones were absent from urine samples. On day 1, blood was sampled early in the morning (T0) from fasted volunteers. A catheter was installed in the forearm. Volunteers then consumed in 15 min two yoghurts and two cereal bars, providing 100 mg of isoflavones. They stayed in the experimental unit all the day. At 4 h after the isoflavone intake, ham, pasta with butter and bread were given for lunch to the volunteers. Volunteers were allowed to drink only water during the day. Another meal composed of ham, pasta with butter and bread was given for dinner 12 h after the isoflavone intake. Blood was sampled 2, 4, 6, 8, 10 and 12 h after the isoflavone intake. Urine was collected in two fractions: 0–6 and 6–12 h. After dinner, volunteers were allowed to leave the experimental unit. They continued to collect urine in fractions at home: 12–24 and 24–36 h. On day 2, they came back to the experimental unit for the 24-h blood sampling. They consumed two isoflavone-supplemented yoghurts and two experimental cereal bars at 2000 h, after the last urinary collection.

From day 3 to 28, subjects consumed one yoghurt and one cereal bar twice a day in addition to their usual diet. Supplements were taken in the morning and in the after-noon, out of the meals. On day 29, volunteers were asked to

take their two yoghurts and the two cereal bars in the morning.

On day 30, the same experimental design as that on day 1 was used. Blood was sampled 0, 2, 4, 6, 8, 10, 12 and 24 h following isoflavone ingestion and urine was collected over 36 h in four fractions.

Isoflavone analysis in plasma and urine samples

Blood was collected using vacuum tubes containing heparin. Blood was immediately centrifugated (10 min at 3000 g) and plasma was acidified with 10 mmol/l acetic acid.

Urine was collected in plastic bottles containing 1 g ascorbic acid. At the end of each collection period, urine volume was measured and sodium azide (1 g/l) was added. Aliquots of plasma and urine were stored at 201C.

Isoflavones were quantified in biological samples after enzymatic hydrolysis of their glucuronidated and sulphated forms. Plasma and urine samples (180 ml) were acidified with acetic acid to pH 4.9, and incubated for 18 h at 371C in the presence of 1000 units of b-glucuronidase and 45 units of sulfatase (from Helix pomatia, Sigma G0876). Incubation time and conditions for hydrolysis were optimized in a prior kinetics study (data not shown). Samples were then mixed with 4 volumes of methanol/HCl (200 mmol/l) and centri-fuged for 5 min at 12 500 g. The supernatant was analysed by HPLC with Coularray detection.

Calibration curves have been prepared in human plasma and urine by spiking control pools with known concentra-tions of isoflavones: 0, 0.025, 0.25, 0.5, 1 and 2 mmol/l for plasma and 0, 1, 2, 5, 15 and 30 mmol/l for urine. These standards were treated exactly in the same way as the samples (i.e., they contained the same ingredients as samples and were subjected to the same hydrolysis and extraction procedure). Quality control samples (duplicates of two different concentrations) were added in each batch of analyses to control the accuracy of the quantification. The stock solutions of isoflavones used to prepare these quality control samples were different from that used to prepare the calibration curve samples. The accuracy was good, with differences between the measured value and the actual value alwayso7%. The limit of quantification was estimated to be 25 and 50 nmol/l in plasma and urine, respectively.

HPLC analysis was performed using a system consisting of two pumps (Model 580, ESA, Chelmsford, MA, USA) for high-pressure gradient, a temperature-controlled autosam-pler (Gilson, Villiers-le-Bel, France), a 15  2.1 mm Symme-tryShield RP18-5m column (Waters) and an eight-channel CoulArray detector (model 5600, Eurosep, Cergy, France). Mobile phases consisted of 30 mmol/l NaH3PO4buffer (pH 3) containing 20% acetonitrile (A) and 40% acetonitrile (B). Separation was achieved using a gradient elution (flow ¼ 0.5 ml/min): 0–15 min: linear gradient from 100%A to 100%B, 15–19 min: 100%B, 19.01–25 min: 100%A. Poten-tials were set at 200; 280; 450; 550; 600; 650; 700; 750 mV (Pd as reference).

Statistics

Values are given as means_{7s.d. The significance of} differ-ences was determined using paired t-test (INSTAT, Graphpad Software, San Diego). A P–value o0.05 was considered significant.

Results

The 12 healthy postmenopausal women consumed 100 mg isoflavones per day for 1 month. Dietary interviews on days 1 and 30 and absence of isoflavone metabolites in urine samples collected prior to the beginning of isoflavone supplementation showed good compliance to the protocol. Five volunteers were classified as equol producers on the basis of isoflavone metabolite concentration in plasma (40.04 mmol/l on day 1) and urine (410 mmol excreted/ 36 h).

Plasma

Maximum plasma concentrations of isoflavones were slightly higher at day 30 than at day 1: 2.27_{70.76 compared} to 1.6870.68 mmol/l for daidzein (P ¼ 0.056), 5.3072.38 compared to 3.8871.50 mmol/l for genistein (P ¼ 0.091) (Figure 1). Yet, two of the 12 volunteers had lower or equal plasma concentrations of genistein and daidzein at day 30 compared to day 1.

For both isoflavones, plasma concentration/time curves exhibit two peaks: one peak 2 h after the isoflavone intake and the highest peak at 6 h. This biphasic shape has already been observed in other bioavailability studies in humans. It probably reflects the enterohepatic recycling as well as absorption occurring successively in the small intestine and in the colon. The shape of the curves was not strongly modified by long-term supplementation with isoflavones, either for daidzein and genistein, except that the concentra-tions decreased more rapidly after the maximum peak at day 30 than at day 1. However, timepoints were not numerous enough to calculate elimination half-lives. The shapes of the kinetic curves varied between individuals (especially regard-ing the relative height of the two peaks). But, similar curves were obtained at day 30 compared to day 1 for each individual (data not shown).

The maximum plasma concentration of equol increased significantly from 0.3170.27 mmol/l at day 1 to 0.9970.51 mmol/l at day 30 for equol-producer volunteers (Figure 1). In contrast to genistein and daidzein, the peak of equol concentration was reached earlier at day 30 than at day 1 (mean Tmax¼ 6 vs 10 h). This may be due to an adaptation of the bacteria species responsible for the production of this metabolite.

For the three compounds, plasma concentrations were not returned to baseline level 24 h after the isoflavone intake, which is consistent with the slight increase of plasma concentrations after long-term daily supplementation.

Five volunteers among the 12 were found to be equol producers at day 1. Equol was only recovered in the plasma of these five volunteers at day 30, which indicates that the other seven volunteers did not acquire the ability to produce equol.

Urine

At days 1 and 30, the maximum urinary excretion of genistein and daidzein occurred between 6 and 12 h

(Figure 2). Urinary excretion of daidzein and genistein did not differ significantly between days 1 and 30, except that a higher proportion was excreted in the 0–6 h fraction at day 30.

Urinary excretion (36 h) of isoflavone metabolites at day 1 was 66.2_{715.9 mmol for daidzein and 48.9719.1 mmol for} genistein. The daily intakes were estimated to be 118 and Day 30 Day 1 3.0 2.5 2.0 1.5 1.0 0.5 0.0
mol/l
mol/l
mol/l 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0 4 8 12 16 20 24 Time (h) 0 4 8 12 16 20 24 Time (h) Day 30 Day 1 Day 30 Day 1 0 4 8 12 16 20 24 Time (h) 1.6 1.4 1.2 1.0 0.5 0.6 0.4 0.2 0.0 -0.2

a

b

c

Figure 1 Plasma concentrations of daidzein (a), genistein (b) and

equol (c) after a single (day 1) or a 1-month supplementation with 100 mg isoflavones (day 30). Values are means7s.d. (n ¼ 12). Means of equol concentrations were calculated from concentrations of the five equol producer volunteers.

Day 1 Day 30 Day 1 Day 30 Day 1 Day 30 0-6h 6-12h 12-24h 24-36h 0-36h 0-6h 6-12h 12-24h 24-36h 0-36h 0-6h 6-12h 12-24h 24-36h 0-36h NS NS NS NS NS _NS NS NS NS NS ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ _∗ 120 100 80 60 40 20 0 120 100 80 60 40 20 0 120 100 80 60 40 20 0
mol excreted
mol excreted
mol excreted

a

b

c

Figure 2 Urinary excretion of daidzein (a), genistein (b) and equol

(c) after a single (day 1) or a 1-month supplementation with 100 mg isoflavones (day 30). Values are means7s.d. (n ¼ 12). Means of equol excretion were calculated from urinary excretion data for the equol producer volunteers (n ¼ 5). Differences between days 1 and 30 were analyzed using a paired t-test with ***Po0.0005, **Po0.005, *Po0.05, NS: Po0.05.

241 mmol aglycone equivalent/day, respectively. Urinary excretion at day 1 thus represented about 56 and 20% of the ingested dose for daidzein and genistein, respectively. Equivalent percentages were not calculated at day 30 because isoflavone excretion was spread over more than 24 h, so that a part of the isoflavone metabolites excreted at day 30 comes from the isoflavone intake on day 29.

Analysis of urine samples from day 30 confirmed that, after 1-month exposure to isoflavones, equol was not produced by the seven women who were classified on day 1 as non-producers. In equol producers, urinary excretion of equol was significantly higher at day 30 than at day 1.

Discussion

The dose and the mode of exposure (twice a day, out of the meals) were chosen to maximize the plasma concentrations of isoflavones. The dose was about twice the mean intake in Asian populations, and the absence of co-ingested foods probably increases the intestinal absorption of isoflavones by improving their bioaccessibility throughout the gut. The urinary excretion rates previously reported in the literature range from 16 to 66% of the ingested dose for daidzein and from 10 to 24% for genistein (Manach et al., 2005). Similarly, peak plasma concentrations vary in the literature from 1.6 to 2.6 mmol/l for each isoflavone after an intake of 50 mg aglycone equivalent (Manach et al., 2005). Data measured in the present study were consistent with the reported data but high in the range, as expected.

A high interindividual variability was found in the present study like in most available studies, dealing with the bioavailability of isoflavones. Many factors such as the ethnic background, the age and the gender, the microflora composition of individuals and some genetic polymor-phisms for metabolic enzymes may contribute to this varia-bility. The impact and the relative weight of each factor have been poorly studied. However, interindividual variability in isoflavone bioavailability should be taken into account for safety concerns, because some individuals, independently of potential variability in isoflavone activities, absorb these phytoestrogens much more efficiently than the average population.

Plasma concentrations at T0 on day 30 were 0.7070.11 mmol/l for daidzein, 2.2170.37 mmol/l for genis-tein and 0.6870.16 mmol/l for equol, which is approximately equivalent to the increase of concentrations between days 1 and 30, and also to the concentrations measured 24 h postdose. On the other hand, 36-hurinary excretions of daidzein and genistein were similar at days 1 and 30. Thus, slightly higher plasma concentrations of isoflavones ob-served at day 30 compared to day 1 must reflect the steady-state level achieved with daily supplementation. It is unlikely that further increase occurs with longer exposure. The same finding can be drawn from the few studies that used long-term exposure of isoflavones (Miltyk et al., 2003;

Wiseman et al., 2004), although our study is the only one that compared the plasma kinetic curves of isoflavones after a single dose or a 1-month exposure to isoflavones. Accordingly, Lampe et al. (2000) found no significant differences in the 24-h urinary excretion of daidzein and genistein after 4 days or 30 days of soy protein supplementa-tion.

Future studies evaluating the beneficial or detrimental effects of isoflavones should thus consider that plasma concentrations as high as 2.5–5 mmol/l of each isoflavone can be achieved in women after chronic intake of isoflavone supplements. This is quite higher than the concentrations reported for other flavonoids, generally lower than 1 mmol/l for ingested doses of 100 mg (Manach et al., 2005). Such information may provide preliminary insights into the mechanisms of action likely to be involved in the effect of isoflavones. Activities requiring isoflavone concentrations higher than 10 mmol/l have typically little chance to occur in vivo.

In a study investigating the potential genotoxic effects of isoflavones in patients with prostate cancer, peak plasma concentration as high as 27 mmol/l genistein was reported for one volunteer, whereas the mean peak concentration was 8 mmol/l for the group of volunteers (Miltyk et al., 2003). The administered dose far exceeded nutritional levels in this study: volunteers were challenged with 300 mg genistein/day for 28 days and then 600 mg/day for another 56 days. These results confirm that long-term exposure does not induce continuous increase of plasma concentrations and that the high interindividual variability must be taken into account. The results also suggest a dose/response effect, with very high doses being less efficiently absorbed than normal nutritional doses. The conclusion is that plasma concentra-tion higher than 10 mmol/l can only be achieved in a low proportion of individuals and after intake of pharmacologi-cal doses at least six-fold higher than nutritional doses.

We observed a three-fold increase in the peak plasma concentration and urinary excretion of equol in equol-producer women after the 1-month exposure to isoflavones. Long-term exposure to isoflavone has already been shown to increase the level of equol production in equol producers, probably because of metabolic adaptation of the microflora (Lu et al., 1996). Another known way to stimulate equol production in equol producers is to provide isoflavones as glycosides rather than as aglycones (Setchell et al., 2001; Zubik and Meydani, 2003). However, inducing the ability to produce equol in non-equol producers is a much more difficult challenge. We found that long-term exposure to a quite high dose of isoflavones was not efficient on this purpose. The same findings were reported in three other studies that tested 4–10 weeks exposure to isoflavone-enriched diets (Lu et al., 1996; Lampe et al., 2001; Wiseman et al., 2004). Furthermore, Bowey et al. showed that one-month exposure to isoflavones of germ-free rats colonized with feacal flora from a non-equol producer did not result in induction of equol production.

In contrast, Lu et al. (1995) reported that three among five non-equol producer women acquired the ability to produce equol after 2 weeks soymilk consumption. One hypothesis for these discrepancies is that the bacteria species involved in the production of equol were different among individuals of the various studies. Indeed, recent studies showed that a combination of several bacterial species is necessary to produce equol from daidzin or daidzein. Some strains have been identified, but emerging hypothesis is that the combination of bacteria involved may differ among indivi-duals (Hur et al., 2000; Ueno and Uchiyama, 2001; Atkinson et al., 2004; Decroos et al., 2005; Wang et al., 2005). Between-subject differences in the effects of antibiotics on daidzein conversion into equol were shown by Atkinson et al. (2004) in cultures of fecal microflora from seven equol producers. We also observed interindividual differences in kinetics of equol appearance in plasma at day 1, with either an appearance between 6 and 8 h with a peak concentration at 10 h or a progressive increase beginning 10 h postdose. This could reflect different microflora compositions between equol producer volunteers.

A more comprehensive knowledge of the factors that may influence equol production is essential because equol producers may gain more benefits from soya consumption than non-producers (Setchell et al., 2002).

Whereas 30–40% of individuals from Western populations have consistently been found to be equol producers in numerous studies, a higher percentage has been reported for Asian populations in two studies. Morton et al. (2002) observed 58% equol producers in Japanese men and Akaza et al. (2004) found 46% equol producers in Japanese men and 59% in Korean men with prostate cancer. If confirmed, these data indicate an influence of the ethnic background, which can cover genetic factors, microflora composition, and dietary habits. The gender and age seem to have a poor impact on equol production. The same percentage of equol producers has been found in men and women (Setchell et al., 1984; Kelly et al., 1993; Lampe et al., 1998; Hedlund et al., 2005). No major differences were reported depending on the age, except for old adults and neonates probably due to age-related changes in intestinal microflora (Setchell et al., 2002; Frankenfeld et al., 2004).

Conflicting results have been found regarding the impact of dietary habits on equol production. Fat, meat and dietary fibers were positively or negatively associated with the ability to produce equol (Adlercreutz et al., 1991; Lampe et al., 1998; Rowland et al., 2000; Hedlund et al., 2005). A high proportion of carbohydrates in the diet (Lampe et al., 1998; Rowland et al., 2000) and intake of green tea (Miyanaga et al., 2003) have been shown to be associated with a higher proportion of equol producers. Some diet components known to stimulate the activity of the micro-flora, such as dietary fibers and probiotics, have been tested in intervention studies, but no change in the proportion of equol producers was observed with wheat bran fibers (Lampe et al., 2001) or probiotic supplements (Bonorden et al., 2004;

Nettleton et al., 2004). In mice, equol production was increased by addition of fructo-oligosaccharides in the diet, whereas inulin decreased equol production in rats (Ohta et al., 2002; Zafar et al., 2004). These results needs to be confirmed in humans because of interspecies differences, demonstrated by the fact that all rodents are constitutive equol producers.

In conclusion, inability to produce equol seems to result from the lack of specific bacteria in the colon, which suggests that specific dietary intervention has poor impact. Further research on the combination of bacterial species involved in equol production in various populations will be crucial to find efficient ways of intervention to stimulate equol production capacity. Plasma concentrations achieved with long-term exposure to isoflavones were about 2.5–5 mmol/l for daidzein and genistein, with high interindividual variability in absorption and metabolism. Such key informa-tion will be used to establish guidelines for optimal isoflavone intakes, especially towards postmenopausal women.

Acknowledgements

This work was supported in part by the French Ministry of Research (AQS program A01347) and Beghin-Meiji (doctoral fellowship). We are grateful to Eridania Beghin-Say and Beghin-Meiji for kindly supplying the powdered isoflavone concentrate (PrevasteinsHC).

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