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1 From the Division of Pathology (KDRS, BEW, LN-Z, and NMB), the Department of Gastroenterology and Nutrition (JEH), and the Department of Pediatrics (SJC and CH), Cincinnati Childrens Hospital Medical Center, and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH (KDRS and JEH); the Department of Gastroenterology and Hepatology, University of Perugia, Perugia, Italy (CC and DC); Sanitarium Development and Innovation, Cooranbong, Australia (SJC and CH); the Department of Physiology and Developmental Biology and The Neuroscience Center, Brigham Young University, Provo, UT (EDL); and the Department of Biomedical Sciences, Colorado State University, Fort Collins, CO (TDL and RJH)
2 These findings were presented at the 4th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, San Diego, CA, November 4-7, 2001, and at the 5th International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, Orlando, FL, September 21-24, 2003. 3 Supported by grants from the National Institutes of Health (R01CA73328) and the National Center for Research Resources (RR08084). 4 Address reprint requests to KDR Setchell, Clinical Mass Spectrometry, Cincinnati Childrens Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail: kenneth.setchell{at}cchmc.org.
ABSTRACT
Background: The discovery of equol in human urine more than 2 decades ago and the finding that it is bacterially derived from daidzin, an isoflavone abundant in soy foods, led to the current nutritional interest in soy foods. Equol, unlike the soy isoflavones daidzein or genistein, has a chiral center and therefore can occur as 2 distinct diastereoisomers.
Objective: Because it was unclear which enantiomer was present in humans, our objectives were to characterize the exact structure of equol, to examine whether the S- and R-equol enantiomers are bioavailable, and to ascertain whether the differences in their conformational structure translate to significant differences in affinity for estrogen receptors.
Design: With the use of chiral-phase HPLC and mass spectrometry, equol was isolated from human urine and plasma, and its enantiomeric structure was defined. Human fecal flora were cultured in vitro and incubated with daidzein to ascertain the stereospecificity of the bacterial production of equol. The pharmacokinetics of S- and R- equol were determined in 3 healthy adults after single-bolus oral administration of both enantiomers, and the affinity of each equol enantiomer for estrogen receptors was measured.
Results: Our studies definitively establish S-equol as the exclusive product of human intestinal bacterial synthesis from soy isoflavones and also show that both enantiomers are bioavailable. S-equol has a high affinity for estrogen receptor ß (Ki = 0.73 nmol/L), whereas R-equol is relatively inactive.
Conclusions: Humans have acquired an ability to exclusively synthesize S-equol from the precursor soy isoflavone daidzein, and it is significant that, unlike R-equol, this enantiomer has a relatively high affinity for estrogen receptor ß.
Key Words: Equol soy isoflavones humans pharmacokinetics bacteria
INTRODUCTION
Equol, [7-hydroxy-3-(4-hydroxyphenyl)-chroman], is a nonsteroidal estrogen that was first discovered in the early 1980s in the urine of adults consuming soy foods (1). It was shown to be a key metabolite of daidzin, one of the main isoflavones present in most soy foods, and to be formed after intestinal hydrolysis of the soy isoflavone glycoside (2) and subsequent colonic bacterial biotransformation (3) through an intermediate, dihydroequol (46). Equol has an infamous history, having been first identified in the urine of pregnant mares as long ago as 1932 (7) and then in the 1940s having been found to be the environmental estrogenic agent that caused a devastating reproductive disease in sheep, referred to as clover disease (8).
Equol is not of plant origin and is exclusively a product of intestinal bacterial metabolism (9), as evidenced from the finding that infants fed soy formula up to the age of 4 mo (10, 11) and germ-free rats fed soy-containing diets (12) do not make equol. When fed soy protein, a common ingredient of most commercial rodent diets (13, 14), rats and mice are prolific equol producers. In contrast, humans are unique among animals in that, for reasons that remain unclear, only 2035% of the adult population make equol after ingesting soy foods or being challenged with pure isoflavones (3, 15, 16). Several studies have suggested that those who are equol producers show more favorable responses to soy isoflavonecontaining diets (1721), which leads to the possibility that equol is a more potent isoflavone than genistein (9), which has been so extensively studied in the last decade.
Equol, unlike its precursor daidzein or genistein, is unique in having a chiral carbon atom at position C-3 of the furan ring (Figure 1). It therefore can occur as 2 distinct enantiomeric forms, S-equol and R-equol, which differ significantly in their conformational structure. We recently showed that S-equol is unique in that it not only possesses estrogenic properties but also is a potent antagonist of dihydrotestosterone in vivo, which has significant implications for the prevention of prostate cancer and other androgen-related conditions (22). We can find no other example of a molecule that is both a selective estrogen and an androgen antagonist. Establishing the diastereoisomerism of equol production is therefore important both in view of the possible differences in biological actions of the enantiomers and in aid of the future development of strategies either to use equol pharmacologically or to manipulate equol production in humans to enhance the effectiveness of soy diets.
FIGURE 1.. Comparison of the chemical structures of the diastereoisomers of equol, showing the site position of the chiral carbon center.
When equol was originally isolated, it was found to be optically active (7), and subsequently its enantiomeric assignment as R-equol was questioned and redefined (2326). The few studies of equol to date have been exclusively performed on the racemic [(±)] mixture because, when equol is chemically synthesized, it is the (±)equol that is usually obtained. Little is known about the enantiomers of equol. For example, it is unclear which form of equol circulates in human blood or is excreted in human urine, because all of the analytic methods for measuring equol in these fluids fail to distinguish the diastereoisomers. To our knowledge, before the current studies, little was known about the pharmacologic or biological activities of the enantiomers. In this report, we focus on several key questions related to equol in humans. First, are the R-, S-, or both enantiomeric forms of equol found in human urine and blood? Second, are intestinal bacteria stereoselective in their synthesis of equol? Third, are both enantiomers absorbed and bioavailable? And fourth, are there differences in the biological activity of the equols enantiomers, specifically with regard to their binding affinity for estrogen receptors?
SUBJECTS AND METHODS
Human studies
Identity of the enantiomeric form of equol in human urine and plasma
The characterization of equols diastereoisomerism was determined in plasma and urine samples (n = 10 each) selected from study subjects in previous studies (NIH grant no. R01CA73328) of the pharmacokinetics of soy isoflavones in healthy adults consuming soy foods (18). In addition, plasma and urine samples taken from a group of Seventh-Day Adventist vegetarians (n = 10) after they had consumed soymilk (2 x 240 mL/d for 4 d) were analyzed. These samples were collected by staff members of the Pathology Department of the Sydney Adventist Hospital after informed consent was obtained. Characterization of the enantiomeric form of equol was accomplished by chiral-phase HPLC coupled to electrospray ionizationmass spectrometry (ESI-MS) after enzymic hydrolysis with a mixed ß-glucuronidase and sulfatase preparation (Helix pomatia), and solid-phase extraction of equol using a Bond Elut C18 cartridge (Varian, Harbor City, CA). The methanolic extracts of urine and plasma were taken for direct analysis by ESI-MS, or, where gas chromatographymass spectrometry (GC-MS) was used, a volatile tert-butyldimethylsilyl (t-BDMS) ether derivative was prepared as described previously (18, 27, 28).
Written informed consent was obtained from all subjects. The studies were approved by the Cincinnati Childrens Hospital Medical Center Institutional Review Board.
Studies of S- and R-equol bioavailability
Purified samples of S-equol and R-equol (20 mg) obtained by semipreparative chiral-phase HPLC chromatographic separation of a racemic mixture were prepared in capsules and randomly administered orally to 3 healthy adults (1 female and 2 male) on different occasions separated by a washout period of 1 wk. This study was performed by the ethical standards of the Department of Gastroenterology and Hepatology, University of Perugia, Italy, in accordance with the Helsinki Declaration of 1975, as revised in 1983. Each equol enantiomer was taken with a glass of water after an overnight fast and before eating breakfast. Blood samples (10 mL) were obtained via an indwelling catheter placed in the antecubital vein at timed intervals immediately before and then 1, 2, 3, 4, 8, 12, and 24 h after the administration of equol. These samples were centrifuged for 10 min at 2200 x g and room temperature, and the plasma was removed for the measurement of equol concentrations by stable-isotope dilution GC-MS with selected ion monitoring, as detailed below. The plasma equol concentrationtime profiles for the 3 persons were ascertained by using a noncompartmental approach. The WINNONLIN computer program (version 3.0; Pharsight Corporation, Cary, NC) was used for this analysis. The total area under the plasma concentrationtime curve (AUC 0 ; AUCinf) was computed by using the following equation:
RESULTS
Characterization of S-equol in humans and rats by chiral-phase HPLC and mass spectrometry
A typical separation of S- and R-equol enantiomers when a racemic mixture was subjected to chiral-phase chromatography and the isoflavones were detected by their ultraviolet absorbance at 260 nm is shown in Figure 2. S-equol eluted from the chiral-phase column with a shorter retention time than that of R-equol, and baseline resolution was achieved. The identity of both enantiomers was confirmed by isolation of both peaks and measurement of optical dichroism. Also shown in Figure 2 is a typical HPLC-ESI-MS mass chromatogram of the negative ion m/z 241 corresponding to the pseudomolecular ion ([M-H]) of equol obtained from the analysis of hydrolyzed extracts of human urine. All samples of human urine analyzed were found to contain a single equol enantiomer with a retention time (6.75 min) that exactly corresponded to the retention time of the pure standard of S-equol (ie, 6.77 min). There was no evidence for the presence of R-equol (which elutes from the HPLC column with a significantly longer retention time of 7.51 min) in any sample of human urine when analyzed under the same chromatographic conditions. Likewise, a sample of urine from a rat, a species that is exclusively an equol producer (12, 14), was found, on the basis of its retention index, to have exclusively the S-equol enantiomer (Figure 2). Similar findings were obtained from the analysis of a selection of samples (n = 10) of human plasma collected from equol producers who had consumed 2 x 240 mL soymilk. Consistent with the urinary analysis, the ESI-MS profiles of plasma showed one major peak in all samples analyzed, and this had a retention time corresponding to that of the S-equol standard.
FIGURE 2.. Chiral-phase HPLC separation with ultraviolet detection (260 nm) showing resolution of a standard mixture of S- and R-equol (left). These profiles are compared with the superimposed ESI-MS mass chromatograms of mass-to-charge ratio (m/z) 241 ([MH] ion) obtained from the analysis of hydrolyzed extracts of human and rat urine collected after ingestion of soy foods (right), which established S-equol as the only enantiomer excreted in human urine. ESI-MS, electrospray ionizationmass spectrometry.
Pharmacokinetics of S- and R-equol enantiomers
Mean (±SEM) appearance and disappearance concentration curves for equol in plasma after single-bolus oral administration of R-equol and S-equol to 3 healthy adults are shown in Figure 3. The administration of both enantiomers yielded similar plasma pharmacokinetic profiles, which confirmed that the 2 diastereoisomers are similarly bioavailable. Equol rapidly appeared in plasma and disappeared with a terminal elimination half-life of 4.9 ± 1.6 and 6.2 ± 0.2 h, respectively, for the S- and R-enantiomers, and there was no obvious difference between these values. A comparison of the computed pharmacokinetics of the diastereoisomers is shown in Table 1. There were no statistical differences in maximum plasma concentration, time to reach maximum plasma concentration, terminal elimination half-life, AUCinf, apparent volume of distribution, and systemic clearance between R- and S-equol, and no difference in the absorption rates of the 2 enantiomers.
FIGURE 3.. Mean (±SEM) plasma concentrations of S- and R-equol in 3 healthy adults given a single-bolus oral 20-mg dose of each enantiomer on separate occasions. Data are expressed as linear-linear (left) and log-linear (right) plots. There were no significant differences.
View this table:
TABLE 1. Computed plasma pharmacokinetics for S- and R-equol administered to 3 healthy adults in a single-bolus oral dose of 20 mg of each diastereoisomer in a randomized crossover design with a 1-wk washout interval1
Because GC-MS was used to quantify equol in these plasma samples, and thus this technique cannot resolve the individual enantiomers as their t-BDMS ether derivatives, it was essential to definitively confirm the identity of the R-equol enantiomer in plasma to exclude the possibility of racemization to S-equol occurring during or after its absorption. Confirmation that the administered R-equol remained unaltered during absorption or uptake was established by taking the 2-h plasma sample extract and subjecting it to direct chiral-phase HPLC analysis with ESI-MS used as the detection system. The mass chromatograms for m/z 241 corresponding to the [M-H] ion of equol for the plasma extract collected 2 h after administration of the R-equol enantiomer are shown in Figure 4. For comparison, the mass chromatograms of a pure mixture of S- and R- equol are also shown, and, on the basis of the retention time, only R-equol was found in the plasma. After administration of S-equol, ESI-MS confirmed that S-equol appeared unchanged in the 2-h plasma sample (data not shown). These data provided evidence that both enantiomeric forms of equol were absorbed without change and that no racemization occurred during or after intestinal uptake. Finding only the S-equol enantiomer in human urine and plasma suggested that this must be the result of the exclusive bacterial production of S-equol in the intestine.
FIGURE 4.. Chiral-phase HPLC-electrospray ionizationmass spectrometry (ESI-MS) analysis of the plasma collected 2 h after administration of R-equol to a healthy adult (left), which confirmed its presence as unchanged R-equol. For comparison, the ESI-MS mass chromatograms for the ion mass-to-charge ratio (m/z) 241 obtained for S- and R-equol standards are superimposed (right).
Evidence for enantiomer-specific synthesis of S-equol by human intestinal bacteria
In vitro studies were performed on cultured human fecal flora collected from healthy adults who were challenged for 4 d with soy foods and who were determined from plasma and urinary isoflavone analysis to be either equol producers or equol nonproducers as defined previously (9). Daidzein (20 µg), the precursor isoflavone of equol, was then incubated with cultured fecal flora at 37 °C for 72 h; after extraction of the supernatant fluid by solid-phase chromatography, the extract was analyzed by direct ESI-MS with chiral-phase HPLC separation. Superimposed mass chromatograms of the negative ion recordings for m/z 241 ([M-H] ion) that were specific for a pure standard of S-equol and for the equol isolated from the 72-h supernatant fluid from one of the equol producers and from one equol nonproducer are shown in Figure 5. The cultured fecal flora from the equol producers showed a peak corresponding to equol that had a retention time identical to that of the authentic pure standard of S-equol. By contrast, the supernatant fluid from an equol nonproducer showed negligible production of S-equol. These results establish conclusively that fecal bacteria are selective in producing only S-equol as the principal metabolite of the soy isoflavone, daidzein.
FIGURE 5.. Definitive evidence for the enantiomer-specific synthesis of S-equol by cultured human fecal flora. Mass chromatograms obtained by using chiral-phase HPLC-mass spectrometric analysis of a pure standard of S-equol (bottom trace) are compared with extracts from in vitro bacterial metabolism of daidzein by cultured fecal flora from a known equol producer (top trace) and an equol nonproducer (middle trace). ESI-MS, electrospray ionizationmass spectrometry.
Estrogenic activity of equol enantiomers
Competitive binding studies were used to assess the estrogenic properties of R- and S-equol. On the basis of the ability of R- and S-equol to compete with [3H]E2 in ER binding, their affinities for ERs translated in vitro were shown to be very different. S-equol showed the greatest affinity for ERß (Ki = 0.73 ± 0.2 nmol/L), whereas its affinity for ER (Ki = 6.41 ± 1 nmol/L) was relatively poor. In contrast, R-equol possessed only 4.8% and 25.0% as much relative binding affinity, respectively, for ERß (Ki = 15.4 ± 1.3 nmol/L) and for ER (Ki = 27.38 ± 3.8 nmol/L) as did S-equol. For comparison, 17ß-estradiol binds ER with a Kd of 0.13 nmol and ERß with a Kd of 0.15 nmol. S-equol thus shows ER selectivity with a high affinity for ERß, whereas R-equol can, at best, be classified as a weak estrogen.
DISCUSSION
In 1932, Marrian and Haslewood, working at the University College London, first isolated and elucidated the chemical structure of the isoflavone metabolite equol (7). It was found as a minor metabolite of the urine of pregnant mares and shown to be optically active, although in subsequent years there was some confusion as to its enantiomeric assignment; it was first assigned the R-configuration, and only later was the absolute configuration defined as the S-enantiomer (25). The importance of defining the nature of the stereoisomerism in humans relates to the marked differences in the conformational structure of the diastereoisomers and the expectation that there would be differences in the biological activity, primarily related to binding to the ER. When equol was first isolated, it was reported by Marrian and Haslewood to have no estrus-producing activity when injected into ovariectomized mice in doses as large as 0.86 mg/animal (7). This observation was inconsistent with the later finding that equol was the estrogenic agent responsible for the devastating reproductive abnormalities, referred to as clover disease, observed in sheep in the mid-1940s (8, 33, 34). Later, in a period that predated knowledge of specific ER subtypes (35, 36), equol was shown in vitro to bind to uterine cytosolic ERs (37, 38). Given the predominance of ER in the uterus, it is almost certain that these early reports of binding affinities refer to equols binding to ER, rather than to ERß. Using preparations of recombinant steroid receptors, we have shown that only the S enantiomer of equol binds to ERs with sufficient affinity to be of physiologic relevance based on circulating equol concentrations typically found in humans (9). Furthermore, almost 50% of equol circulates unbound to serum protein (39), whereas endogenous estrogen is >95% protein bound. This protein-binding status of equol is likely to enhance its biological potency because it is only the "free" or unbound fraction that is available for receptor occupancy. The relative binding affinity of the R- and S-equol enantiomers for ER were 0.47% and 2.0% with that of 17ß-estradiol. However, S-equol is largely ERß selective and has a relatively high affinity for this receptor subtype. S-equol binds ERß 20% with as much affinity as does 17ß-estradiol (equol: Ki = 0.7 nmol/L; 17ß-estradiol: Kd = 0.15 nmol/L), whereas the R enantiomer bound at 1% of the affinity. These findings are corroborated by several studies (4042) that also show equol to have selective affinity for ERß, and, therefore, equol can be defined as a type of selective ER modulator.
As a potent antagonist of dihydrotestosterone (DHT) in vivo, equol is also unique, in that we can find no other example of a compound that has selective estrogen action and yet also has the ability to be an antagonist of androgen action (22). It is interesting that the mechanism of its anti-androgen action differs from that of the anti-androgen drugs used in clinical practice to block the effects of DHT. For example, equol has no affinity for the androgen receptor (22) and therefore does not function as an androgen receptor blocker. It also does not appear to alter the synthesis of DHT in the way that 5-reductase inhibitors do, but, rather, it appears to bind directly to DHT (22), and this effect is seen with both R- and S-equol (TD Lund, RJ Handa, ED Lephart, KDR Setchell, unpublished data, 2003).
Given the distinctly different biological actions of the diastereoisomers of equol, particularly with regard to their affinity toward ERs, it is relevant to define the stereoisomerism of equol production in humans. Using ESI-MS with selected ion monitoring, we analyzed urine and plasma samples from healthy adults, and by our ability to separate the 2 diastereoisomers with the use of chiral-phase column chromatography, we have shown for the first time that S-equol is the only enantiomer circulating in human blood and excreted in urine (Figures 2 and 3). This is also true in the rat, a species that is predominantly an equol producer (14). The logical explanation for the finding of a single enantiomer in plasma and urine is that intestinal bacteria are stereoselective in their synthesis, but the possibility that both enantiomers would be made in the intestine, but only one, the S enantiomer, would be absorbed required addressing. Furthermore, racemization of R-equol to S-equol during the formers absorption was an alternative possibility that was feasible and required investigation.
The separate oral administration of pure S- and R-equol to 3 healthy adults clearly showed that both enantiomers, when present in the intestine, are efficiently absorbed and appear rapidly in plasma. There were no differences in the pharmacokinetics of the 2 enantiomers. The bioavailability of equol as measured by the dose-adjusted AUCinf is relatively high when compared with the bioavailability of genistein and daidzein reported in previous studies (18, 27, 28). The clearance rate of equol was also much slower than that of the soy isoflavones, which contributes to the maintenance of high circulating equol concentrations observed in rodents (14). ESI-MS established that, after its administration, R-equol appeared in plasma unchanged, and therefore the possibility of bacterial production of R-equol in the intestine with racemization to S-equol during absorption can be confidently excluded. Thus, these data taken together are indicative of the enantiomer-specific production of S-equol by intestinal bacteria. This is now confirmed by in vitro experiments in which human fecal flora from equol producers were cultured under anerobic conditions and incubated with daidzein or soy isoflavones. After 72 h in culture, S-equol was the sole enantiomer identified in the supernatant. Thus, given that humans, rats, and sheep all produce S-equoland it is likely that macaque monkeys (43), chimpanzees (44), dogs (45), domestic fowl (46), cows (47), and mice (14) also do soit is evident that the bacteria responsible for equol production are all highly selective in performing an asymmetric synthesis with production of the one enantiomer that shows the highest ligand affinity for ERß.
The formation of equol from its precursor daidzein proceeds through an intermediate, dihydrodaidzein. Our pharmacokinetic studies show that equol is rapidly absorbed from the intestine, but its formation after the initial intake of daidzein or of soy foods containing daidzin or daidzein is a time-dependent process. It generally takes >12 h for equol to appear in the plasma, and, in some adults, it may not appear for 36 h, which indicates that the colon is the site of its formation (18, 28, 48). Identification of the bacteria responsible for equol production has thus far been elusive. It is apparent that there is more than one bacterium involved because we have observed cases in which dihydrodaidzein is present in urine in the absence of equol, which is consistent with partial conversion of daidzein to equol (KDR Setchell, unpublished observations, 1995). Furthermore, in vitro incubation of fecal homogenates from some adults was shown to produce dihydrodaidzein and O-desmethylangolensin but not equol, whereas recently it was shown that some antibiotics, such as rifampicin and kanamycin, may inhibit the production of equol but not of dihydrodaidzein (6). In contrast, kanamycin virtually eliminated equol production in the plasma of cynomologus monkeys (49), which highlights the complexity of the bacterial production of equol. Attempts to identify the species of bacteria involved in equol production have yielded some information regarding strains that are capable of hydrolyzing the ß-glucoside of daidzin (50, 51) or of converting daidzein to dihydrodaidzein (52), and one report claimed that Streptococcus intermedius spp, Ruminococcus productus spp, and Bacteroides ovatus in vitro perform the required conversion (53).
In view of the apparent advantages of being an equol producer (9, 17, 1921, 54), the question of whether it is possible to manipulate the intestinal milieu in favor of equol production when soy foods are given is relevant. Early studies by Setchell and Cassidy (55) using an in vitro model of human colonic fermentation showed that, with a background of a high nonstarch polysaccharide environment, which affords increased colonic fermentation, the conversion of daidzein to equol is complete, but no conversion occurs under low-carbohydrate conditions. This in vitro observation is supported by data from a study of 24 healthy adults showing that good equol producers were associated with a diet that was lower in saturated fat and higher in total carbohydrate (16). The role of carbohydrate in equol production, also reported by Lampe et al (15), suggests that there are important prebiotic or probiotic factors that may influence equol formation in adults. The addition of fructooligosaccharides to the diet of mice has been shown to yield higher equol concentrations (56), as did feeding potato starch (57), and, in the former study, higher equol concentrations were associated with a greater effect on bone density in this animal model. This has also been shown to be the case in a 2-y clinical study of the effectiveness of soy foods in preventing bone loss in postmenopausal women (20), in which equol producers showed a significant increase in lumbar spine bone mineral density. Whether probiotic or prebiotic diets can influence the metabolism of soy isoflavones to enhance equol production in humans is unclear (5860), but, if not, the potential benefits of equol, with its selective ER modulator properties and its antiandrogen actions, could be optimized by the use of pure enantiomeric forms of equol as a supplement or nutraceutical.
In conclusion, our studies described here definitively establish S-equol as the only enantiomer found in human plasma and urine, and we show for the first time that this exclusivity is not due to differences in the bioavailability or metabolism of S- and R-equol but rather to the fact that intestinal microflora show enantiomeric specificity in their production of equol. These findings are of immense clinical relevance because we also found that S-equol, but not R-equol, has a relatively high affinity for ERß and is in fact a more potent estrogen than is estradiol; these findings have been corroborated by others (4042). The significance of these findings is that humans have acquired intestinal microflora that perform an asymmetric synthesis of the only diastereoisomer of equol that has affinity for the ER and thus has the greatest potential for physiologic effects.
ACKNOWLEDGMENTS
KDRS was the principal investigator; conceived, designed, and directed these studies; and prepared the manuscript. CC and DC were responsible for conducting the pharmacokinetic studies of R- and S-equol in healthy subjects. SC and CH performed the studies on human fecal flora and collected blood and urine samples from healthy vegetarian Seventh Day Adventist volunteers in Sydney. EDL, TDL, and RJH conducted the estrogen-binding studies of equol. NMB was the clinical coordinator for studies conducted on healthy subjects in Cincinnati, and JEH was the physician responsible for the monitoring of these studies conducted on the General Clinical Research Center. BEW and LN-Z, the research assistants, performed the laboratory analysis of equol by mass spectrometry. All authors provided input to the manuscript, and none of the authors had any conflict of interest.
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