phytoestrogens are non - steroidal plant compounds and consist of numerous classes including isoflavones , lignans , and coumestans ( 1 , 2 ) . structurally and functionally , they mimic mammalian estrogens and possess both estrogenic and antiestrogenic properties . the mechanism of their dual functions has not been completely clarified yet . some in vitro studies have suggested that the biological action of phytoestrogens may be modulated by the concentration of co - existing estrogens ; they can act estrogenically in a low - estrogen environment or antiestrogenically in a high - estrogen environment ( 3 , 4 ) . intervention studies with the supplementation of phytoestrogens have revealed inconsistent results on the clinical effectiveness of cardiovascular , menopausal , and bone health . the high intake of soy isoflavones has been shown to prevent bone loss in some studies of postmenopausal women ( 5 - 8 ) . however , these data are still controversial , and they have shown no significant effects on bone in other studies ( 9 , 10 ) . only a few studies have focused on the skeletal effect of phytoestrogens in premenopausal women . the three - year observational study revealed that soy intake had a significant effect on the maintenance of peak bone mass in women between the ages of 30 and 40 ( 11 ) , while the other intervention studies showed no effects on bone mineral density ( bmd ) in young women ( 12 , 13 ) . isoflavones are the best - studied phytoestrogens and are found in their highest amounts in soy foods . recent studies have demonstrated that the metabolism of isoflavones is highly variable in individuals , which suggests that a new paradigm considering this heterogeneity would be indispensable toward an evaluation of their clinical effects ( 14 ) . genistin and daidzin , which exist as inactive glucosides , are the two major ingredients of soy isoflavones . once ingested , they are converted to genistein ( gtn ) and daidzein ( dz ) , respectively after breakdown by intestinal microflora . gtn is partially metabolized to its non - estrogenic metabolite , para - ethylphenol , by gut microflora . some dz is also further metabolized to equol ( eql ) or o - desmethangiolensin ( o - dma ) by different metabolic pathways ( 15 - 17 ) ( fig . 1 ) . previous in vitro studies have shown that the metabolites of the o - dma pathway have weaker estrogenic properties , while eql is superior to all other daidzin metabolites , in respect to its binding affinities to estrogen receptors ( ers ) and estrogen activities ( 4 ) . because of its slower clearance , the plasma concentration of eql is normally far in excess of other daidzin metabolites ( 14 ) . however , eql is not produced in all healthy adults in response to a dietary challenge with soy or dz ; only one - third of healthy adults are capable of producing eql from dz . the inter - individual variance in the ability to produce more potent metabolites , including eql , may lead to differences in the effects of isoflavone intervention on human health . the failure to distinguish these subtypes could plausibly explain the variance in the reported data on the skeletal effects of soy . this study tested the hypothesis that soy isoflavones may compete with endogenous estrogens and thus act as estrogen antagonists , especially in premenopausal women who can produce more potent metabolites . in our randomized - controlled study , soy - induced changes of bone turnover markers were assessed in healthy , menstruating women after dividing them into subgroups according to metabolizing phenotypes . sixty healthy premenopausal female volunteers of korean ethnicity between the ages of 30 and 50 yr ( 37.24.8 yr , meansd ) were recruited to participate in this study . throughout the study , subjects had no restrictions and continued their usual activities at home and at work . informed consent was obtained from each study participant and this study was approved by the institutional review board of the cheil general hospital and women 's healthcare center . this study was designed as a randomized , double - blind , placebo - controlled study . the participants were randomly assigned to receive either isoflavone capsules ( soy group ; 120 mg / capsule , n=32 ) or capsules containing the same dose of lactose as a placebo ( n=21 ) once a day for three menstrual cycles . four of the 36 subjects in the soy group dropped out because of side effects ( breast pain and weight gain ) . three of the 24 placebo subjects dropped out for different reasons ; one subject due to pregnancy , and two subjects due to failure of follow - up . the soy extract isoflavone in 120 mg / capsules ( rexgene biotech co. , cheongwon , korea ) consisted of 3.68% daidzin , 13.97% genistin , and 18.51% glycitein . baseline blood samples and 24-hr urine collections were obtained from day 3 to day 6 of each menstrual cycle , and follow - up samples after intervention were collected on the same day of the menstrual cycle as the day when baseline samples were collected . fasting blood and 24-hr urine samples were also collected , and the serum was prepared immediately and frozen at -70 until it was assayed . serum osteocalcin ( oc ) levels were determined by radio immunoassay ( ria ) using an osteocalcin 125i ria kit ( incstar corp . , stillwater , mn , u.s.a . ; intra- and interassay cv , 7.8% and 9.2% , respectively ) . urine deoxypyridinoline ( dpd ) was assayed by the enzyme - linked immuno - sorbent assay ( elisa ) method ( osteomark , ostex , seattle , wa , u.s.a . ; intra- and interassay cv , 5.7% and 3.5% , respectively ) and corrected for creatinine . serum 17-estradiol ( e2 ) was assayed via ria ( diagnostic system laboratories , webstar , tx , u.s.a . ; cv=11% ) . serum lutenizing hormone ( lh ) and follicular stimulating hormone ( fsh ) levels were measured by immunoradiometric assays ( diagnostic products corp . ; twenty - four hour urine samples were analyzed for phytoestrogens using solid - phase extraction ( spe ) followed by hplc and tandem mass spectrometry . conjugated analytes in urine were hydrolyzed by the addition of -glucuronidase / sulfatase ( helix pomatia , h-1 , sigma chemical , st . louis , mo , u.s.a . ) and incubated overnight at 37. deconjugated samples were spiked with an internal standard ( flavone , sigma chemical ) and then extracted with oasis hydrophilic - lipophilic balance ( hlb ) solid - phase extraction ( spe ) ( 30 mg hlb , 1 ml , waters corporation , milford , ma , u.s.a . ) . the phytoestrogens were separated by a reversed - phase high - performance liquid chromatography ( hplc ) ( 1501.0 mm alltima column , alltech , deerfield , il , usa ) and a flow rate of 50 l / min ( 0.1% formic acid , 25% meoh , 25% acetonitrile ) . all analytes were measured by tandem mass spectrometry ( api 2000 , mds / sciex , concord , ontario , canada ) using electrospray ionization ( esi ) in the positive - ion multiple - reaction monitoring mode . statistical analyses were performed with the statistical package for the social sciences ( spss , inc . , data were evaluated using a one - way analysis of variance ( anova ) and a paired two - tailed t test . to assess interrelationships between variables , spearman 's correlation coefficient analyses were also used . in the case of significant differences , a non - parametric test was used to further specify these differences . p values below 0.05 ( paired two - tailed t test ) were considered significant . three of the 24 placebo subjects and four of the 36 subjects in the soy group dropped out because of difficulties complying with the study protocol . therefore , we included 21 women in the placebo group and 32 women in the soy group for the final data analyses . at baseline , there were no significant differences in age , body mass index ( bmi ) , food intake , bone markers , or hormone status between the soy and placebo groups . no significant differences could be found in the baseline excretion concentrations between the two groups ( fig . the daily urinary excretion of all measured isoflavone metabolites was significantly increased in the soy group , while significant changes were not observed in the placebo group except for the excretion of dihydrogenistein ( dgtn ) , which showed only a slight but statistically significant incremental increase at the follow - up ( fig . the marked inter - individual variation was observed in the urinary excretion of isoflavone metabolites after soy intake ( fig . 3 ) . individual variations of dz and gtn after a challenge exceeded normal levels by 100-fold and 400-fold , respectively . eql also had a high individual difference in its excretion , exceeding normal levels by 400-fold . urinary excretion of dz was highly correlated with gtn , dihydrodaidaein ( ddz ) and o - dma , but not with eql . the urinary concentration of eql did not show any significant correlations with any other metabolites ( fig . significant changes of circulating hormones , including lh , fsh , and estradiol ( e2 ) , were not found after a challenge in both groups ( data not shown ) . body weight and bmi did not change significantly after follow - up in both groups ( data not shown ) . however , the serum concentrations of oc ( the bone formation marker ) increased significantly after a challenge in the soy group but not in the placebo group ( fig . 4 ) . of the subjects in the soy group , eight women ( 25% ) could be categorized as eql high - excretors ( > 1,000 nm/24 hr ) ( 17 ) . we recategorized subjects according to the excretion of gtn , because gtn also has the strongest er - binding affinities and estrogenic activities among all the metabolites ( 4 , 29 ) . when the gtn high - excretors were defined arbitrarily as women whose urinary excretion of gtn was greater than 2,000 nm / day , 15 women were included in the gtn high - excretor group . all dz metabolites after intervention were significantly higher in gtn high - excretors compared with the placebo group and the gtn low - excretors ( data not shown ) . daily excretion of dz was over 2,500 nm / day in all gtn high - excretors . seven of the eight women who were categorized as eql excretors could be also categorized as gtn high - excretors . basal characteristics including age , weight , bmi , and sex hormone levels were not different among these subgroups . serum oc concentrations in the gtn high - excretors increased significantly after the challenge but did not increase in either the placebo group or the gtn low - excretors ( fig . the bone - resorption marker , urine dpd , was decreased after a challenge in both the placebo and soy groups ( fig . however , was not observed in either the eql excretors or the gtn high - excretors ( data not shown ) . when we subdivided women by the excretion levels of o - dma , the weak metabolite of dz , any tendency for soy - induced increments in bone remodeling could not be found in the o - dma high - excretors ( data not shown ) . our results indicate that the high ingestion of isoflavones increases bone turnover in the early follicular phase of menstruation . this antiestrogenic action is observed prominently in women who can produce and absorb the metabolites with relatively strong binding affinities to ers . we also noted a marked inter - individual variation after a soy challenge in the excretion of isoflavone metabolites including the intermediate metabolites of gtn and dz . the personal variation of daily urinary excretion of metabolites exceeds several hundred - fold after the soy challenge . the difference between minimum and maximum was over 2,000-fold in the excretory levels of some intermediate metabolites such as ddz and dgtn . these -glucosides , including daidzin and genistin , are biologically inactive and can be absorbed only after being hydrolyzed into the aglycones , dz and gtn , respectively ( 1 , 2 , 15 - 17 ) . hydrolysis is known to be an extremely efficient process , occurring along the entire length of the intestine by the action of both the brush border membrane and the bacterial -glucosidase . in our study , a broad range of metabolites could be detected in urine after a challenge , which is in agreement with previous studies ( 15 - 17 ) . a proportion of dz and gtn appeared to escape further metabolism within the intestine , being absorbed as aglycones just after hydrolysis by the glucosidase and largely cleared in the urine . the remnants underwent more intestinal fermentation via the bacterial reaction that takes place distally and presumably in the colon , yielding various intermediate and end products , which are also readily absorbed and then largely cleared in urine . interestingly , in our study the tested metabolites of dz were highly correlated with each other , except for eql . gtn also showed a significant correlation with its metabolite dgtn and even with other dz metabolites except for eql . this finding suggests that dz and gtn undergo a similar metabolism using the same metabolizing enzymes to produce o - dma or dgtn , respectively . eql , however , seems to have a distinct metabolic pathway using different enzymes from different intestinal bacteria strains . it has been demonstrated that the formation of eql is exclusively dependent on intestinal microflora . the lack of eql in germ - free animals and infants fed infant formula revealed the need for an active microflora for its formation ( 18 - 20 ) . the repeated administration of isoflavones to the same adults showed consistently that those who are " eql excretors " seem to remain " eql excretors " over time ( 21 ) . three strains of bacteria responsible for eql formation have been identified in japanese subjects by in vitro culturing of fecal flora ( 14 ) . in approximately one - fourth of our study subjects , all of the tested isoflavones were excreted in concentrations of less than 500 nm / day even though the participants were challenged daily with a large amount of soy extract . the reasons why some people do not have an ability to excrete enough isoflavonoids after a challenge are unclear . some studies have shown that some dietary components could alter the intestinal environment and affect the metabolism of isoflavones , but the data are still controversial ( 14 ) . our study shows that the high consumption of isoflavones can affect physiological functions of bone remodeling in premenopausal women . to our knowledge , only two small studies have been reported to evaluate the effects of isoflavones on bone turnover during menstrual cycles . in a randomized , cross - over study with 14 young , menstruating women , the daily consumption of 65 mg and 130 mg of soy isoflavones increased the bone - resorption marker , urine dpd , at the early follicular phase after three menstrual cycles ( 22 ) . in another study of a randomized , cross - over design , after daily ingestion of 52 mg isoflavones for more than one menstrual cycle , 14 premenopausal women showed a significant increase in tartrate - resistant acid phosphatase ( trap ) compared with the placebo group , but not in another bone - resorption marker , c - telopeptide ( ctx ) ( 23 ) . however , no significant effects of soy - intervention were observed for the bone - formation marker oc in both studies . the discrepancy between our study and the previous data on the bone - formation marker is not clear . the failure of oc to show a significant , soy - induced change in previous studies may be due to the heterogeneity of study subjects , many of whom might be low excretors of additional estrogenic isoflavones , such as eql or gtn . after soy - induction , we could not find any significant changes of circulating hormones including serum e2 , lh , and fsh . however , some intervention studies have shown decreased estrogen levels and changes in menstrual - cycle length after increased soy intake ( 24 , 25 ) . in the intervention study from california , the soy - induced estrogen - lowering effect was restricted to asian women ( 26 ) . other intervention studies have failed to observe any significant effect of isoflavones on sex hormones and menstrual patterns ( 27 ) . in a previous study , five premenopausal eql excretors had lower concentrations of estrone and estron - sulfate and higher sex hormone - binding globulin than nine eql non - excretors in certain phases of the menstrual cycle including the early follicular phase . serum e2 , however , did not show any significant differences between the subgroups ( 28 ) . most of the reported data are not at the proper scale to adjust for a large number of unmeasured factors that may influence the clinical effects of soy . therefore , the mechanism of a soy - induced antiestrogenic effect on premenopausal bone remodeling is still unclear ; it may be an indirect effect caused by the changes of circulating sex hormones or a direct effect on bone . isoflavones can bind both types of er , especially er , and exhibit weak estrogenic activity . the antiestrogenic activity may be partially explained by its competition with endogenous e2 for ers and then attenuating estrogenic action by their lower intrinsic activity . gtn has been shown to be similar or even stronger than eql in er - binding affinities and estrogenic activities ( 4 , 29 ) . in our study , a significant increment of bone turnover could be observed more prominently in women whose urinary gtn excretion was more than 2,000 nm / day after soy - intervention , rather than in eql excretors . since most women who were categorized as gtn high - excretors could also excrete high concentrations of dz and its metabolites including even eql , the gtn effect in our study may reflect a sum effect including other isoflavone metabolites . a recently published , large - scale clinical trial , however , has shown a gtn - induced positive effect on bmd in postmenopausal women ; gtn can independently increase bmd at both the lumbar and femoral neck areas by means of a two - year treatment of daily doses of 54 mg of gtn ( 7 ) . although most reported studies are small - scale and do not contain enough participants to confirm the skeletal effect of isoflavones especially on premenopausal bone remodeling , the previous data are consistent with our results ; isoflavones can modulate the bone turnover rate and may act as estrogen antagonists in certain phases of the menstrual cycle . furthermore , our results identified the importance of the awareness that a large degree of individual variation exists in the metabolism and biological functions of phytoestrogens . since the magnitude of the effect of isoflavones on bone turnover is quite small despite relatively high doses of isoflavones , their clinical importance should be clarified by further , large - scale studies investigating the long - term effects on bmd according to the metabolic phenotype .
soy - isoflavones may act as estrogenic agonists or antagonists depending on the endogenous hormone status . 
 these clinical effects can be exerted variably in individuals by the metabolic ability to produce a more potent metabolite than precursors . 
 the objective of this randomized , double - blind , placebo - controlled study was to investigate the skeletal effect of isoflavones according to their metabolic variability in premenopausal women . 
 volunteers were randomly assigned to receive either soy - extract isoflavones ( n=32 ) or lactose ( n=21 ) once a day for three menstrual cycles . 
 after intervention , the urinary excretions of isoflavones and their metabolites were significantly higher in the soy group than in the placebo group and showed a large inter - individual variation . 
 women in the soy group were divided into subgroups according to their ability to excrete more potent metabolites . 
 serum osteocalcin and urine deoxypyridinoline showed a tendency to increase after a challenge in equol high - excretors . 
 serum osteocalcin concentration in the genistein high - excretors increased significantly after a challenge ( p=0.04 ) but did not increase in either the placebo or genistein low - excretors . 
 an estrogenic antagonistic effect of isoflavones on bone turnover was observed in premenopausal women who are able to produce more potent metabolites .