1 Division of Geriatrics, School of Medicine, University of California, Los Angeles, Los Angeles, CA.
2 New England Research Institute, Watertown, MA.
3 Division of Research, Kaiser Permanente, Oakland, CA.
4 Department of Epidemiology and Preventive Medicine, School of Medicine, University of California, Davis, Davis, CA.
5 National Institute on Aging, National Institutes of Health, Bethesda, MD.
6 Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
bone density; cohort studies; diet; genistein; isoflavones; menopause; soybeans; women
Abbreviations: BMD, bone mineral density; FFQ, food frequency questionnaire; SWAN, Study of Women's Health Across the Nation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The phytoestrogen components of soybeans and soybean products may account for a bone protective effect of these foods. Phytoestrogens, heterocyclic phenols structurally analogous to human estrogens, bind to estrogen receptors alpha and beta and stimulate transcriptional activity (57
). Phytoestrogens include coumestans, lignans, and isoflavones; genistein and daidzein are the main compounds within the isoflavone class of phytoestrogens (8
). Genistein and daidzein are found largely in soybeans and soy foods such as tempeh, miso, and tofu and have been the focus of most in vitro and in vivo experimental studies of the physiologic effects of isoflavones (3
, 4
, 8
10
).
Although ecologic inference and cellular biology suggest that soy isoflavones may benefit bone, epidemiologic studies of dietary isoflavone intake and bone health are scant, hampered by insensitive dietary assessment methods and by limited databases to estimate the isoflavone content of foods (11, 12
). One Japanese study of almost 1,000 women aged 4049 years reported a positive association between metacarpal bone mineral density (BMD) and soybean intake assessed by a food frequency questionnaire (FFQ) (13
). In contrast, among 50 Japanese women aged 3268 years, isoflavone intake calculated from a 3-day food record was unrelated to BMD (14
). Interventional studies of isolated soy isoflavones and BMD in humans are also few and have yielded discordant results to date, possibly because of differences in the dosages of isoflavones administered as well as small sample sizes (15
, 16
).
The Study of Women's Health Across the Nation (SWAN) is a US-based, multisite, multiethnic, longitudinal cohort study with over 3,000 participants aged 4252 years at baseline. A major focus of SWAN is to study the determinants of bone health in pre-, peri-, and postmenopausal women. At baseline, we measured participants' dietary isoflavone consumption and BMD at five of the seven SWAN clinical sites. We used these SWAN data to address the following questions: What is the relation between dietary isoflavone intake and BMD? Does this relation vary by race/ethnicity or by menopausal status?
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cohort recruitment and enrollment have been described in detail previously (17). In brief, participants were enrolled at seven clinical sites in the following geographic areas of the United States: Boston, Massachusetts; Chicago, Illinois; Detroit, Michigan; Los Angeles, California; Newark, New Jersey; Oakland, California; and Pittsburgh, Pennsylvania. All seven SWAN clinical sites enrolled Caucasians; the Boston, Chicago, Detroit, and Pittsburgh sites enrolled African Americans; and the remaining three sites enrolled Japanese, Hispanic, and Chinese women, respectively. The Chicago and Newark sites did not perform BMD testing, leaving a potential maximum of 2,413 participants for BMD analyses.
Of the women at the five SWAN BMD testing sites, 82 did not have a baseline measurement of lumbar spine BMD and 54 had technically unsatisfactory baseline scans of spine BMD, resulting in 2,277 participants for whom lumbar spine BMD data were usable (94.4 percent of participants at the five BMD sites). A baseline hip BMD measurement was lacking for 81 women, and three had technically unsatisfactory baseline hip BMD measurements, resulting in 2,329 women at BMD sites with usable data on hip BMD (96.5 percent of women at the BMD sites). Because a few women had some health conditions that affect BMD, we excluded those with self-reported anorexia or bulimia (n = 30 (spine) and n = 32 (hip)), hypercalcemia (n = 13 in each sample), tamoxifen use (n = 4 in each sample), medroxyprogesterone acetate use (n = 21 (spine) and n = 22 (hip)), and corticosteroid use (n = 48 (spine) and n = 50 (hip)). Dietary data quality control exclusions (refer to the following Measurements discussion) numbered 123 for the spine sample and 128 for the hip sample. Finally, for 143 women in the spine sample and 146 in the hip sample, at least one final model covariate was missing, leaving for 19961997 multivariable analyses 1,927 women in the spine sample and 1,967 in the hip sample, representing 79.9 and 81.5 percent, respectively, of all participants at SWAN BMD sites. By ethnic group for each bone density site (lumbar spine or hip), this analysis contained data from, respectively, 83.9 and 85.8 percent of Caucasian, 72.4 and 74.3 percent of African-American, 80.0 and 81.2 percent of Chinese, and 80.8 and 81.1 percent of Japanese participants at SWAN BMD sites. (Exclusions do not sum to 100 percent because 32 women in the spine sample and 33 in the hip sample had more than one reason for being omitted from final analyses.)
Measurements
The study outcome measures were lumbar spine, femoral neck, and total hip BMD measured in grams/centimeter squared. Measurements were made by using Hologic 2000 (at the Pittsburgh and Oakland sites) and 4500A (at the Boston, Detroit, and Los Angeles sites) instruments (Hologic, Inc., Bedford, Massachusetts). A standard quality control program included daily phantom measurement, cross-calibration with a 6-monthly circulating anthropomorphic spine measurement, and site-level review of all participant scans for specified flagging criteria. All flagged scans were reviewed by using the Hologic-MDM (now Synarc) data quality assurance service (Hologic, Inc.) and were accepted as is, were reanalyzed, or were rejected.
The primary exposure variables were dietary genistein (measured in micrograms/day) and dietary daidzein, assessed by using a modified Block FFQ (18). This modified FFQ was adapted to measure macro- and micronutrients in our ethnically diverse sample (e.g., to be able to accommodate Asian-style mixed dishes). Determination of the genistein and daidzein content of foods was based on the method of Rienli and Block (12
); because the Pearson correlation coefficient between calculated intakes of these two phytoestrogens in our sample was 0.98, only genistein was used as the exposure variable in our relational analyses. Diet quality control exclusions were as follows: recorded intakes of less than four or more than 17 solid foods per day, omission of more than 10 foods when responding to the FFQ, and estimated kilocalorie intakes of less than 500 or more than 5,000 daily.
Standardized, self-report questionnaires were used to assess the covariates of age (years, continuous), current smoking (yes/no) (19), place of birth (US or non-US location), duration of residence in the United States (years), physical activity (summary score of active living, home, and recreational physical activity) (20
), history of hyperthyroidism (yes/no), ever use (yes/no) of any estrogen or estrogen/progestin pill or patch (including oral contraceptives or postmenopausal hormone therapy), and menstrually defined menopause status (premenopausal (regular menses), early perimenopausal (menses within the prior 3 months but less predictable)). Intakes of calcium (milligrams/day), protein (kilocalories/day), and alcohol (kilocalories/day) were estimated by using the modified FFQ (18
).
Data analysis
First, dietary genistein and daidzein intakes were calculated for each of the four ethnic groups. Among African-American and Caucasian women, the amount of these isoflavones consumed was very low (table 1). Thus, we did not continue to evaluate the relation between genistein and BMD for these two ethnic groups. For Chinese and Japanese women, crude and adjusted mean BMD values were compared by ethnic-specific tertile of genistein intake. Because consumption of most nutrients is positively related to total energy intake, it is necessary to adjust for total energy. Therefore, our relational analyses used energy-adjusted genistein as the primary exposure variable (21). Energy-adjusted genistein was calculated separately for Chinese and Japanese women by using a linear regression model with total energy intake as the independent variable and genistein intake as the dependent variable. Residuals from these regressions were then used as the genistein exposure variable in models relating genistein intake to BMD. This residual technique is considered the optimal method of energy adjustment (21
). Analysis of variance (unadjusted) and analysis of covariance (adjusted) models were used to assess the relation between genistein and BMD. Adjusted mean BMD values were controlled for age, current smoking, physical activity, dietary calcium, dietary alcohol, dietary protein, height, weight, and menopause status. The following were also considered as final model covariates and were so used if statistically significant: duration of residence in the United States, self-reported ever use of estrogen or estrogen/progestin (largely the oral contraceptive pill), self-report of over- or underactive thyroid, and self-reported use of thyroid pills. Interactions between menopause status and genistein intake were tested in each model and were reported if statistically significant.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Japanese and Chinese women were similar in age, calcium intake, and hormone use but differed according to several other relevant characteristics (table 2). Table 2 presents data for Japanese pre- and perimenopausal women separately, because the relational analyses of genistein to BMD, described subsequently, found different results according to menstrual status of these women.
|
|
|
The relation between BMD and genistein intake differed for Chinese and Japanese participants: we found no association for Chinese women but a substantial, positive association for premenopausal Japanese women. Although the Japanese women, on average, reported higher genistein consumption than did the Chinese women, approximately 85 percent of participants in each of these ethnic groups reported overlapping genistein intakes in the range of 80030,000 µg daily. Thus, in this subgroup with overlapping genistein intakes, a dose effect could be distinguished from an ethnicity effect. We modeled the relation between BMD and energy-adjusted dietary genistein (continuously), adjusted for all covariates, and included a genistein-by-ethnicity interaction term. In this combined Japanese and Chinese model, at the femoral neck, higher genistein intake was associated with higher adjusted BMD only in Japanese women; no relation was found among the Chinese women (p = 0.0204 for genistein-by-ethnicity interaction). At the spine, the genistein-ethnicity interaction was also statistically significant (p = 0.0284).
Although this study focused on a nutrient-level analysis, genistein and daidzein food sources among the Japanese and Chinese SWAN participants are summarized in appendix table 1. Fermented soy foods (listed in italics) were more highly represented in the Japanese compared with the Chinese women's diets.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To compare our results with those found in the literature, the capability of the FFQ to estimate genistein intake must be addressed. It is acknowledged that absolute quantities of nutrients consumed cannot be calculated from FFQs. Furthermore, errors in existing databases of the isoflavone content of foods, missing isoflavone food sources, and known small-area variation in the isoflavone content of soy crops combined to limit our confidence in the accuracy of any dietary estimation method of isoflavone intake (11, 12
). In our study, the relative ranking of genistein intake should not have been impacted by these constraints; however, because of these known problems with estimation, direct genistein exposure should not be compared with that found in other studies. Genistein dose-response contrasts should be interpreted very cautiously and primarily in relative terms. Finally, because genistein and daidzein are highly correlated, only one of them could be used in mathematical analyses. Because genistein and daidzein are both metabolically active, and most interventional research has administered genistein and daidzein in combination, our data on dietary exposure to isoflavones can be roughly compared with prior work by using our aggregate median values of dietary genistein and daidzeinabout 12 mg/day in Japanese women and 5 mg/day in Chinese women.
When what is generally considered the moderate-to-high dose range of genistein and daidzein is used (16), animal and human intervention studies corroborate a positive relation between isoflavones and BMD (23
, 24
). Fanti et al. tested the ability of three genistein doses (1 µg, 5 µg, and 25 µg per gram of body weight) to prevent ovariectomy-induced bone loss in rats; the lowest dose was ineffective, while the higher two were equally effective (23
). Similarly, a 24-week, randomized controlled trial compared the bone density effects of two doses of isoflavones (predominantly genistein and daidzein) in postmenopausal women (24
). No BMD effect was found in the women who received 56 mg/day of genistein/daidzein, but those randomized to 90 mg/day of genistein/daidzein evidenced an approximate 2 percent increase in spinal BMD compared with baseline measurements. Consonant results emerge from two other studies that tested single doses of predominantly genistein and daidzein isoflavone compounds (25
, 26
). In a 2-year study of ovariectomized monkeys, 28 mg of isoflavones daily did not prevent postoophorectomy bone loss (25
). However, a 24-week intervention with an isoflavone dose of 80.4 mg/day preserved BMD in perimenopausal women (26
). In aggregate, although dose-response curves are not readily comparable across species, these intervention studies suggest that above some threshold dose, the isoflavones genistein and daidzein have a positive effect on BMD. Our results in premenopausal Japanese women further point to the possibility of a dose-response relation, with higher amounts of genistein leading to higher BMD.
Results of models that included Chinese and Japanese women and considered an interaction term between ethnicity and genistein supported the hypothesis that these two groups of women have different BMD responses to similar amounts of dietary genistein. How might ethnicity modify the bone tropism exerted by genistein? Japanese women could have a higher density of osteoblast estrogen receptors compared with Chinese women and thus have a greater bone effect for a given dose. To our knowledge, evidence for ethnic differences in estrogen receptor density in bone cells is lacking, but ethnic variations have been reported for the classic estrogen receptor in breast tissue (27, 28
). Second, the Japanese diet may contain more readily absorbed forms of isoflavones. It has been proposed that isoflavone aglycones (forms devoid of a sugar moiety) are absorbed faster and in greater amounts than are the glycoside (with sugar attached) forms (29
). Compared with the Chinese diet, the traditional Japanese diet is higher in fermented soy products, such as tempeh, miso, and soy paste; fermented soy sources are rich in aglycones (30
). Correspondingly, in our sample, fermented soy food sources represented a vastly greater proportion of isoflavone intake for Japanese compared with Chinese women. It is also plausible that, compared with Chinese women, Japanese women may have higher amounts of the ß glucosidase enzyme. If Japanese women consume more readily absorbed forms of phytoestrogens or metabolize them more efficiently, then estimates of physiologic exposure based purely on dietary content will underestimate effects of isoflavones in Japanese women.
Bone-trophic effects of phytoestrogens are biologically plausible; mechanisms of action include binding to estrogen receptors, stimulating formation of sex hormone-binding globulin, and inhibiting tyrosine kinase (31, 32
). However, our analysis explored more than the simple hypothesis that genistein and daidzein would benefit bone; we postulated the more complex theory that the bone effect of phytoestrogens might vary according to a woman's menopause status, a proxy for her endogenous hormonal milieu. This idea for effect modification by endogenous hormone status derives from the concept that phytoestrogens are selective estrogen receptor modulators. They demonstrate differential binding and activity to the alpha and beta estrogen receptors (32
) based on interactions with the receptor binding pocket and the resultant conformational change of the receptor (33
). Given that phytoestrogen-estrogen receptor interactions will necessarily compete with those of the cognate ligand, the result will likely depend in part on concentrations of endogenous sex steroids (9
). The present study used self-reported premenopausal (defined as regular menses) status and early perimenopausal (defined as less-predictable menses) status as proxies of endogenous sex steroids and found a positive effect of genistein on BMD only in regularly menstruating women. Additional work in SWAN, using actual measured hormone levels, may be able to explore this observation further.
Given the lack of a criterion standard to measure diet, the validity of nutrient estimates based on an FFQ should be considered in the context of this analysis. Measurement of genistein exposure in SWAN relied on a modification of the Health Habits and History Questionnaire ("Block FFQ") (18). The FFQ was adapted to accommodate Asian-style mixed dishes and was programmed to calculate genistein and daidzein intakes based on a comprehensive compilation of published food sources (12
). Concordant with other diet measurement techniques (34
), this study found that consumption of genistein and daidzein was almost perfectly correlated. We assessed the validity of the daidzein and genistein estimates computed from the modified Block FFQ in an independent sample of 58 Japanese and 18 Caucasian volunteers (35
). Four 24-hour urine collections were made, and high-pressure liquid chromatography (HPLC) determinations of daidzein and genistein were made in each. The Spearman correlation between the four urinary assays of daidzein and the FFQ-estimated daidzein intake was 0.49, and the urinary-FFQ correlation for genistein was 0.30. These correlations are higher than many others that have been reported between dietary measures and biomarkers (36
38
) and are certainly in the range considered adequate for ranking of nutritional intakes (39
).
The foremost limitation of this study is the incompleteness of the database of isoflavone food sources (11, 12
), which may have led to underestimation of true intake; however, this shortcoming should not have diminished our ability to rank participants. We may have captured the phyto-estrogen food sources of Japanese women more completely than those of Chinese women, producing biased estimates. Nonetheless, negatively biased estimates for the Chinese women would not explain the lack of genistein's effect on BMD in Chinese women in the combined Japanese and Chinese model. It must be acknowledged that the modified Block FFQ (18
) was not validated against urine or serum estimates of phytoestrogens in Chinese women. It is also possible that unmeasured dietary constituents or confounders could have been responsible for our results. This analysis was cross-sectional, which constrained causal inference. Finally, because our finding of a positive relation between dietary genistein and BMD was confined to only the premenopausal subgroup of Japanese women, these results may be chance findings produced by multiple statistical testing.
In summary, this study found a strong, positive, dose-response relation between dietary soy isoflavones (assessed by genistein) and BMD in premenopausal Japanese women. It also suggests that the absent effect of soy isoflavones on BMD in Chinese women was not due to their lower average intake compared with Japanese women and that dietary differences in fermented soy food sources may be one reason for this ethnic difference. Further work is needed to confirm these provocative findings and to understand the mechanisms underlying these complex relations.
![]() |
ACKNOWLEDGMENTS |
---|
Clinical centers: University of Michigan, Ann Arbor, MichiganDr. MaryFran Sowers, Principal Investigator (PI) (grant U01 NR04061); Massachusetts General Hospital, Boston, MassachusettsDr. Robert Neer, PI, 19951999; Dr. Joel Finkelstein, PI, 1999present (grant U01 AG12531); Rush University, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IllinoisDr. Lynda Powell, PI (grant U01 AG12505); University of California, Davis/Kaiser Permanente, Oakland, CaliforniaDr. Ellen Gold, PI (grant U01 AG12554); University of California, Los AngelesDr. Gail A. Greendale, PI (grant U01 A12539); University of Medicine and DentistryNew Jersey Medical School, Newark, New JerseyDr. Gerson Weiss, PI (grant U01 AG12535); and the University of Pittsburgh, Pittsburgh, PennsylvaniaDr. Karen Matthews, PI (grant U01 AG12546). Central laboratory: University of Michigan, Ann Arbor, MichiganDr. Rees Midgley, PI, 19952000; Dr. Dan McConnell, PI, 2000present (grant U01 AG12495, Central Ligand Assay Satellite Services). Coordinating center: New England Research Institutes, Watertown, MassachusettsDr. Sonja McKinlay, PI, 19951999; Dr. Kay Johannes, PI, 1999present (grant U01 AG12553). Steering committee: Dr. Chris Gallagher, Chair, 19951996; Dr. Jenny Kelsey, Chair, 1996present. NIH project offices: National Institute on Aging, Bethesda, MarylandDrs. Sherry Sherman and Marcia Ory; National Institute of Nursing Research, Bethesda, MarylandDr. Carole Hudgings.
The manuscript was reviewed by the Publications and Presentations Committee of SWAN and has its endorsement.
The authors thank the study staff at each SWAN site.
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|