The effects of phytoestrogen isoflavones on bone density in women:a double-blind, randomized, placebo-controlled trial1–3 Charlotte Atkinson, Juliet E Compston, Nicholas E Day, Mitch Dowsett, and Sheila A Bingham ABSTRACT
for their potential bone-sparing properties. Rates of hip fracture Background: Isoflavone phytoestrogen therapy has been pro-
in Asian populations, whose traditional diets are rich in soy, are posed as a natural alternative to hormone replacement therapy substantially lower than those in whites residing in the United (HRT). HRT has a beneficial effect on bone, but few trials in States (8). However, bone mineral density (BMD) in Asian humans have investigated the effects of isoflavones on bone.
populations is comparable to that in white populations after Objective: The objective of the study was to determine the effect
adjustment for height and weight (9). Nevertheless, data from on bone density of a red clover–derived isoflavone supplement that animal experiments provided evidence that soy protein can provided a daily dose of 26 mg biochanin A, 16 mg formononetin, attenuate menopausal bone loss (10, 11), and it was suggested 1 mg genistein, and 0.5 mg daidzein for 1 y. Effects on biochem- that isoflavones in soy might be responsible for protective ical markers of bone turnover and body composition were also effects on bone (12). In humans, some (13–17) but not all (18) cross-sectional studies in Asian populations reported signifi- Design: Women aged 49–65 y (n ϭ 205) were enrolled in a
cant positive associations between soy protein or isoflavone double-blind, randomized, placebo-controlled trial; 177 completed intakes and BMD. Furthermore, intervention trials in humans the trial. Bone density, body composition, bone turnover markers, using either soy protein or isoflavone extracts generally re- and diet were measured at baseline and after 12 mo.
ported protective effects on bone, although sample sizes were Results: Loss of lumbar spine bone mineral content and bone
small and trials often were relatively short. In postmenopausal mineral density was significantly (P ϭ 0.04 and P ϭ 0.03, respec- women, consumption of soy protein providing 90 mg isofla- tively) lower in the women taking the isoflavone supplement than vones/d for 24 wk resulted in a significant increase in lumbar in those taking the placebo. There were no significant treatment spine bone mineral content (BMC) and BMD (19), and a red effects on hip bone mineral content or bone mineral density, clover isoflavone supplement providing 57 or 85.5 mg isofla- markers of bone resorption, or body composition, but bone forma- vones/d for 6 mo resulted in an increase in BMD of the tion markers were significantly increased (P ϭ 0.04 and P ϭ 0.01 proximal radius and ulna (20). In perimenopausal women, soy for bone-specific alkaline phosphatase and N-propeptide of colla-gen type I, respectively) in the intervention group compared with protein that provided 80.4 mg isoflavones/d for 24 wk did not placebo in postmenopausal women. Interactions between treatment increase lumbar spine BMC or BMD, but it did lower the group and menopausal status with respect to changes in other extent of bone loss compared with that in the control group (21). Several intervention studies reported the effects of soy Conclusion: These data suggest that, through attenuation of bone
protein or isoflavone extracts on markers of bone turnover. In loss, isoflavones have a potentially protective effect on the lumbar postmenopausal women, diets rich in soyfoods resulted in Am J Clin Nutr 2004;79:326–33.
significant increases in serum osteocalcin concentrations (22,23) and decreases in urinary N-telopeptide excretion (22). In KEY WORDS
Isoflavones, phytoestrogens, bone density, perimenopausal women, a 4-wk intervention with an isofla- randomized controlled trial, body composition, estrogen and vita- vone extract resulted in a significant reduction in the bone 1 From the MRC Biostatistics Unit, Institute of Public Health, Robinson Way, Cambridge, United Kingdom (CA and NED); the Metabolic BoneUnit, Addenbrooke’s Hospital, Cambridge, United Kingdom (JEC); the INTRODUCTION
Department of Biochemistry, Royal Marsden Hospital, London (MD); and Estrogens play an important role in skeletal homeostasis, and the MRC Dunn Human Nutrition Unit, Cambridge, United Kingdom ovarian hormone deficiency is one of the most important risk factors for osteoporosis. There are clear bone-related benefits 2 Supported by grants from the Food Standards Agency and the Medical of hormone replacement therapy (HRT; 1), but compliance Research Council; Novogen Ltd (Australia) provided the Promensil tablets with such regimens is generally very poor (2, 3), partly as a and provided research support (to SAB).
3 Address reprint requests to SA Bingham, Dunn Human Nutrition Unit, result of fears about the risks of cancer (3).
Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Isoflavones are compounds in plant foods, particularly soy- Kingdom. E-mail:
beans (4, 5), that are structurally similar to the mammalian estrogens (6, 7) and that have received considerable attention Accepted for publication August 8, 2003.
Am J Clin Nutr 2004;79:326 –33. Printed in USA. 2004 American Society for Clinical Nutrition FIGURE 1. Flow chart describing the progress of the volunteers during the trial. HRT, hormone replacement therapy. *Includes 2 women who completed
the trial but were excluded from all analyses because they had taken oral contraceptives or had been treated for alcoholism. **Includes 1 woman whocompleted the trial but was excluded from all analyses because she had taken oral contraceptives.
resorption marker pyridinoline (24), but other studies reported response to the letter was 68% (n ϭ 781); of the responders, little or no effect of soy protein or isoflavone supplements on 205 (18% of the women who received letters) were eligible and markers of bone turnover in humans (25–27).
were randomly assigned to receive an isoflavone or placebo We aimed to determine the effects of consuming a red tablet (Figure 1). Women were not eligible to participate if
clover–derived isoflavone supplement (in a daily dose provid- they had a personal history of breast cancer or major breast ing 26 mg biochanin A, 16 mg formononetin, 1 mg genistein, surgery or if they were currently taking HRT. Women who and 0.5 mg daidzein) for 1 y on the BMC and BMD of the expressed an interest in taking part were visited at home.
lumbar spine and hip, on biochemical markers of bone turn- During this initial home visit, the study was explained in detail, and all women who wished to participate were asked forwritten informed consent. A total of 205 women were ran-domly assigned to receive isoflavone or placebo tablets (Figure SUBJECTS AND METHODS
1). Additional home visits were made after Ϸ5.5 and 11 mo on Subjects
the study. All study procedures were approved by the Dunn Between November 1997 and May 1999, women aged Human Nutrition Unit Ethics Committee and the Cambridge 49–65 y were recruited from the Breast Screening Unit of Addenbrooke’s Hospital (Cambridge, United Kingdom). The Participants were randomly assigned to receive daily either a primary outcome measure was breast density, and women were red clover–derived isoflavone tablet that provided 26 mg bio- selected for the study according to the extent of dense tissue chanin A, 16 mg formononetin, 1 mg genistein, and 0.5 mg seen on their most recent mammogram; mammograms from daidzein (Promensil; Novogen Ltd, Sydney, Australia) or a 1908 women were classified according to their Wolfe pattern placebo of identical appearance. We did not measure the isofla- (28), and women with P2 or DY Wolfe breast patterns (ie, vone content of the Promensil tablets in our laboratory, but an dense breast patterns; n ϭ 1149, 60% of all mammograms independent study showed that the manufacturer’s statements classified) were sent a recruitment letter, which contained a as to the quantity and type of isoflavones in the tablets were short description of the study and a reply slip. The rate of correct (29). Randomization was performed by using random number generation in MICROSOFT EXCEL (version 2.2; Mi- content (Kone autoanalyser; Jaffe method), and excretion of crosoft Corp, Redmond, WA), and researchers and study par- pyridinoline and deoxypyridinoline was corrected for creati- ticipants remained blinded to the tablet allocation throughout the study. Participants were asked to take 1 tablet/d. The codeidentifying those who had been taking the isoflavone tablets Blood samples
and those who had been taking the placebo was broken when Fasting blood samples were taken at baseline and 12 mo.
all participants had completed all stages of the study.
Participants were asked to refrain from eating or drinking Urine samples
(except water) from midnight until after the sample had beentaken the following morning. A total of 35.5 mL blood was Women were asked to make 24-h urine collections at base- drawn at each visit, including 9.0 mL blood that was drawn into line and 12 mo. To check compliance, women also were asked a lithium heparin tube and 9.0 mL that was drawn into a serum to make a 24-h urine collection at 6 mo. The completeness of tube. The serum tube was left at room temperature for Ն1 h all urine collections was assessed by using the p-aminobenzoic before centrifugation to allow clotting. After centrifugation for acid (PABA)–check test (30). Samples containing 85–110% of 10 min at 2000 rpm and 5 °C, aliquots of plasma and serum the ingested PABA were designated satisfactory. For samples were stored at Ϫ20 °C. Before centrifugation, Ϸ1 mL whole with PABA recoveries of between 70% and 85%, which indi- blood was removed from the lithium heparin tube, and DNA cated that all tablets had been taken but that the urine collection was extracted by using a Qiagen kit (Qiagen Ltd, Crawley, was incomplete, the urinary excretion of isoflavones was ad- United Kingdom) and was used for genotyping of estrogen justed to 93% PABA recovery (31). Samples with Ͻ70% receptor and vitamin D receptor gene polymorphisms (PvuII recovery were designated incomplete. Samples with Ͼ110% PABA recovery were considered unsatisfactory, because addi- Plasma bone-specific alkaline phosphatase (bone ALP), a tional sources of PABA (eg, a multivitamin) may have been marker of bone formation, was measured by using the Metra kit consumed, and an accurate determination of sample complete- (Quidel Ltd, Oxford, United Kingdom; 35). The intraassay and interassay CVs were 3.9% and 10.9%, respectively. Plasma Urinary excretion of genistein, daidzein, formononetin, and concentrations of the N-propeptide of collagen type I (PINP), biochanin A was measured by using HPLC and a modification also a marker of bone formation, were measured by using the of the method of Setchell et al (32) and Franke et al (33).
Orion Diagnostica radioimmunoassay kit (Espoo, Finland; 36), Briefly, samples were incubated with ␤-glucuronidase for for which the intraassay and interassay CVs were 9.3% and 20–72 h at 37 °C. Phenolic components were extracted into an 12.5%, respectively. For both assays, baseline and 12-mo sam- ethyl:acetate (6:4 by vol) solvent mixture. After vortex mixing ples from each participant were run together on the same plate for 30 s and subsequent 10-min centrifugation at 2320 ϫ g and 20 °C, the organic phase was transferred to a 2-mL vial and Serum estradiol was measured by using a previously de- evaporated to dryness under vacuum at 43 °C. Extraction res- scribed method (37), and serum follicle-stimulating hormone idues were reconstituted in 100 ␮L 50% isopropanol solution (FSH) was measured by using an enzyme immunoassay on an and centrifuged for 10 min at 2320 ϫ g and 20 °C. A 5-␮L Abbott AxSYM automated analyzer (Abbott Diagnostics, aliquot was then injected directly onto the HPLC column, Maidenhead, United Kingdom). Menopausal status was deter- which consisted of an Alltima 250 ϫ 2.1-mm, 5 ␮mol/L, C-18 mined by using baseline concentrations of estradiol and FSH as stationary phase (Alltech Associates, New South Wales, Aus- follows; women were classified as premenopausal if they had tralia), and a mobile phase (acetonitrile and water) containing Ͻ30 IU FSH/L and Ͼ100 pmol estradiol/L, as postmenopausal 0.05% trifluoroacetic acid with a gradient of acetonitrile from if they had Ͼ30 IU FSH/L and Ͻ100 pmol estradiol/L, and as 25–100%. Detection and quantification were performed via perimenopausal if they had Ͼ30 IU FSH/L and Ͼ100 pmol photo diode array detector. Flavone was used as the internal estradiol/L or Ͻ30 IU FSH/L and Ͻ100 pmol estradiol/L.
standard; the limit of detection (LOD) was 0.05 ␮g/mL, and the However, if a woman had noted on the questionnaire com- limit of quantitation (LOQ) was 0.1 ␮g/mL.
pleted at the initial home visit that she was currently menstru- Total and free pyridinoline and deoxypyridinoline, markers ating, but her baseline hormone profile was that of a postmeno- of bone resorption, were measured in urine samples collected at pausal woman (ie, Ͼ30 IU FSH/L and Ͻ100 pmol estradiol/L), baseline and when nearing completion at 12 mo with the use of she was classified as perimenopausal.
a rapid automated assay technique according to the method ofPratt et al (34). Briefly, for total pyridinoline and deoxypyr-idinoline, 0.5 mL urine was hydrolyzed with an equal volume Bone density, body composition, and calcium and vitamin
of 12 mmol/L HCl for 18 h at 107 °C. Hydrolysates were D intakes
centrifuged for 2 min at 13 500 ϫ g and room temperature, and Bone density and body composition were assessed by dual- hydroxypyridinium cross-links were extracted from 0.5 mL of energy X-ray absorptiometry (DXA) at baseline and after Ϸ12 the supernatant by solid-phase extraction (employing cellu- mo with the use of an Hologic QDR-4500A scanner (Hologic lose). Pyridinoline and deoxypyridinoline were separated and Inc, Waltham, MA). The precision of the in vivo measurement quantified by using HPLC. Free pyridinoline and deoxypyr- of BMC by DXA in the Metabolic Bone Unit at Addenbrooke’s idinoline were measured without the hydrolysis step. The with- Hospital is 1% in the spine and 2–3% in the proximal femur.
in-batch CV was Ͻ3% for pyridinoline and Ͻ5% for deoxy- Individual scans of the lumbar spine (L1–L4), hip (femoral pyridinoline measurements; baseline and 12-mo urine samples neck, trochanter, and intertrochanteric region), and whole body from individual participants were analyzed together in the same were taken. If a scan revealed signs of osteoporosis (ie, a BMD batch. All urine samples were analyzed for their creatinine T-score ϽϪ2.5), the participant was notified in writing and TABLE 1
Baseline characteristics and time between measurements in subjects receiving isoflavone or placebo tablets1
Total pyridinoline (nmol/mmol creatinine) Total deoxypyridinoline (nmol/mmol creatinine) 1 DXA, dual-energy X-ray absorptiometry; BMC, bone mineral content; BMD, bone mineral density; ALP, alkaline phosphatase; PINP, N-propeptide of collagen type I. Differences between treatment groups werenonsignificant (P Ͼ 0.05).
3 Menopausal status unknown for 3 women in the isoflavone group; percentages may not add to 100% 4 Isoflavone group, n ϭ 78; Placebo group, n ϭ 81.
5 Isoflavone group, n ϭ 81; Placebo group, n ϭ 88.
6 Isoflavone group, n ϭ 77; Placebo group, n ϭ 81.
advised to visit her general practitioner. Follow-up spine data Windows operating system. A P value of Ͻ0.05 was consid- were not available for one woman in the isoflavone group, and ered significant. Changes in BMC, BMD, markers of bone follow-up bone density and body-composition data were not turnover, body composition, and intakes of calcium and vita- available for one woman in the placebo group. Six women min D were calculated as 12-mo data minus baseline data. We from the isoflavone group and 8 women from the placebo used Student’s t tests to test for differences between treatment group were excluded from all analyses regarding bone, because groups for changes in BMC, BMD, markers of bone turnover, they had taken medications during the study that can affect and body composition. Data on changes in calcium and vitamin bone density (eg, bisphosphonates, statins, or calcium and D intakes were skewed, and therefore the nonparametric Wil- vitamin D supplements). Participants’ height and weight were coxon’s rank-sum test was used to test for differences between measured at the time of the DXA scans, and body mass index (BMI) was calculated as [weight (kg)/height (m2)]; baselineheight was unavailable for one woman in the isoflavone group.
Participants were asked to complete a food-frequency ques- tionnaire (FFQ) at baseline and after 12 mo on the study.
Baseline characteristics are shown in Table 1. Differences
Participants were given verbal and written instructions on how between treatment groups were nonsignificant (P Ͼ 0.05).
to complete the FFQ, and calculation of calcium and vitamin D Sixteen women withdrew from the isoflavone group and 12 intakes was based on published food-composition tables (38).
withdrew from the placebo group (Figure 1). The principalreasons for withdrawal were commencement of HRT and work Sample size and data analysis
commitments or family problems that prevented completion of A sample of 100 in each treatment group gave 80% power to study activities. Other reasons included feeling no beneficial detect a Ն1.3% difference between groups with respect to the effects of the intervention or having no interest in continuing change in BMC over 1 y, assuming an SD of 3.2% and setting on the trial, heavy menstrual bleeding, or illnesses preventing ␣ at 0.05. Interactions between treatment group and polymor- completion of study activities (eg, severe hip pain, skin irrita- phisms in the estrogen receptor and vitamin D receptor genes tion and sores, vomiting, and diarrhea). One woman in the are not reported because of a lack of power to detect significant isoflavone group was diagnosed with an interval cancer of the associations. Results are available from the authors on request.
breast (ie, a cancer detected in the interval after a negative Statistical analyses were performed by using SAS statistical mammographic result) 2 mo after the start of the intervention software (version 6.12; SAS Institute, Cary, NC) under the and was withdrawn from the study. The difference between FIGURE 2. Mean (ϮSEM) urinary isoflavone excretion (sum of daid-
zein, genistein, formononetin, and biochanin A) by treatment group atbaseline, 6 mo, and 12 mo, excluding samples with Ͻ70% or Ͼ110%p-aminobenzoic acid recovery. At baseline: isoflavone group, n ϭ 66;placebo group, n ϭ 72. At 6 mo: isoflavone group, n ϭ 70; placebo group, n ϭ 73. At 12 mo: isoflavone group, n ϭ 76; placebo group, n ϭ 79. Thedifference in isoflavone excretion between treatment groups was nonsig-nificant at baseline (P ϭ 0.23), but differences between treatment groupsat 6 and 12 mo were highly significant (both: P Ͻ 0.001).
treatment groups in the number of withdrawals was not signif- icant (␹2 ϭ 1.123, P ϭ 0.29).
According to the PABA-check method, 58%, 72%, and 77% of the women had complete urine collections at baseline, 6 mo,and 12 mo, respectively. A further 22%, 11%, and 13% hadPABA recoveries between 70% and 85% at baseline, 6 mo, and 12 mo, respectively. Differences between treatment groups in
sum isoflavone excretion (sum of daidzein, genistein, for-
mononetin, and biochanin A) were nonsignificant at baseline,
but differences were highly significant at 6 and 12 mo (Figure
). The data shown do not include women with Ͻ70% or
FIGURE 3. Mean (ϮSEM) changes in spine and hip bone mineral
Ͼ110% PABA recovery. Inclusion of these women did not content (BMC) and bone mineral density (BMD) at 1 y by treatment group.
Isoflavone group, n ϭ 77 (n ϭ 78 for hip); placebo group, n ϭ 81.
alter the results. Among women in the placebo group, isofla- Interactions between menopausal status and treatment group for changes in vone excretion did not change significantly from baseline to 6 BMC or BMD were nonsignificant. Differences between treatment groups mo (P ϭ 0.80) or from baseline to 12 mo (P ϭ 0.15; Figure 2).
with respect to changes in spine BMC and BMD were significant (P ϭ 0.04 There were no significant differences between treatment and P ϭ 0.03, respectively); differences between treatment groups with groups with respect to changes in dietary intakes of calcium respect to changes in hip BMC and BMD were nonsignificant (P ϭ 0.48and P ϭ 0.30, respectively).
and vitamin D from baseline to 12 mo; mean (ϮSD) changesin calcium intakes for women in the isoflavone and placebogroups, respectively, were – 43 Ϯ 224 and – 54 Ϯ165 mg/d ences between treatment groups were not significant (Figure (P ϭ 0.32), and changes in vitamin D intakes for women in the 3). Similar results were found when considering percentage isoflavone and placebo groups, respectively, were – 0.24 Ϯ 1.6 changes (data not shown). In an analysis of covariance on and – 0.19 Ϯ 2.3 ␮g/d (P ϭ 0.52).
changes from baseline to 12 mo, with baseline data (BMC or Changes in spine and hip BMC and BMD at 1 y by treatment BMD) as a covariate, the effect of treatment group on the group are shown in Figure 3. Interactions between treatment
changes in spine BMC and BMD remained significant (P ϭ group and menopausal status for the changes in spine or hip 0.05, and P ϭ 0.03, respectively). Similarly, for changes in hip BMC and BMD were not significant (P Ͼ 0.05). Differences BMC and BMD, the effect of treatment group remained non- between treatment groups for losses of spine BMC and BMD significant (P ϭ 0.38 and P ϭ 0.85, respectively).
were significant. Percentage change in spine BMC and BMD Markers of bone turnover at baseline and 12 mo by treatment among women in the isoflavone and placebo groups, respec- group are shown in Table 2. The difference between treatment
tively, showed similar trends: mean (ϮSEM) BMC: Ϫ1.42 Ϯ groups with respect to the change in PINP was of borderline 0.36% and Ϫ2.35 Ϯ 0.37% (P ϭ 0.07); BMD: Ϫ1.08 Ϯ 0.27% significance. The interaction between treatment group and and Ϫ1.86 Ϯ 0.29% (P ϭ 0.05). Decreases in BMC and BMD menopausal status was significant for the changes in bone ALP of the hip were generally greater among women in the placebo and PINP (P ϭ 0.05 and P ϭ 0.03, respectively). When group than among women in the isoflavone group, but differ- grouped by menopausal status, differences between treatment TABLE 2
Markers of bone turnover at baseline and at 12 mo according to treatment group1
1 x៮ Ϯ SD. ALP, alkaline phosphatase; PINP, N-propeptide of collagen type I.
2 n ϭ 80 for pyridinoline and deoxypyridinoline.
groups were significant among postmenopausal women; mean than the trabecular bone of which the spine is primarily com- (ϮSD) changes in bone ALP among postmenopausal women in posed (39, 40). Furthermore, precision of the measurement of the isoflavone and placebo groups, respectively, were 5.65 Ϯ hip BMC is lower than that of the measurement of spine BMC 5.92 and 3.71 Ϯ 2.92 U/L (P ϭ 0.04), and changes in PINP (Ϸ2–3% for the proximal femur compared with 1% for the among postmenopausal women in the isoflavone and placebo spine; JE Compston, personal communications, 1997 and groups, respectively, were 9.72 Ϯ 25.19 and Ϫ1.40 Ϯ 19.12 2002), and therefore the power to detect significant differences mg/L (P ϭ 0.01). No other changes differed significantly between treatment groups was lower as a result of slow bone between treatment groups. Results similar to those for total turnover and lower measurement precision.
pyridinoline and deoxypyridinoline were obtained for free pyr- Potter et al (19) previously reported an increase in lumbar idinoline and deoxypyridinoline (data not shown). There were spine BMC and BMD among postmenopausal women whose no significant differences between treatment groups with re- diets were supplemented daily for 24 wk with soy protein spect to the changes in whole-body BMC or BMD or body- isolate providing 90 mg, but not 56 mg, isoflavones/d. In our composition measures (Table 3), and interactions between
study, we did not see an increase in BMC or BMD with the treatment group and menopausal status were not significant isoflavone supplement, but the daily dose of isoflavones was lower than that used by Potter et al (19). However, in a studyof perimenopausal women, Alekel et al (20) did not see an DISCUSSION
increase in lumbar spine BMC or BMD with a daily dose of This is one of the longest and largest studies to date to 80.4 mg isoflavones (as soy protein) for 24 wk, but, similar to examine the effects of an isoflavone supplement derived from our study, they saw a reduction in the extent of bone loss. In a red clover on spine and hip BMC and BMD, biochemical recent study of 28 premenopausal women (mean age: 24 y) markers of bone turnover, and body composition. We observed there were no significant effects of a soy protein supplement an attenuation of lumbar spine bone loss among women taking that provided either 0 or 90 mg isoflavones/d for 12 mo (41).
the isoflavone supplement compared with that among women This suggests that isoflavones may be beneficial in attenuating taking the placebo. Trends for losses of hip BMC and BMD age-associated bone loss rather than in enhancing peak bone were similar to those seen in the lumbar spine, but differences mass in younger premenopausal women.
between treatments were not significant. However, this was not Promensil tablets contain isoflavones derived from red clo- entirely surprising, given that the hip contains a relatively large ver and, unlike soy protein, relatively large proportions of proportion of cortical bone, which is metabolically less active biochanin A and formononetin, in addition to daidzein and TABLE 3
BMI, body composition, and total-body bone mineral content (BMC) and bone mineral density (BMD) at
baseline and at 12 mo by treatment group1
1 x៮ Ϯ SD. Differences between treatment groups with respect to changes from baseline to 12 mo were 2 Placebo group, n ϭ 90.
3 Isoflavone group, n ϭ 78; placebo group, n ϭ 80.
genistein (29). It has been observed that the use of isoflavone All authors participated fully in the preparation of the manuscript. CA extracts does not always result in lipid-lowering effects (42, was responsible for subject recruitment and day-to-day running of the trial, 43) that are the same as those seen with intact soy protein (44), sample analyses, and statistical analyses; JEC was responsible for bone and it has been suggested that the isoflavone extraction process densitometry; NED was responsible for the data analysis; MD was respon-sible for the hormone assays; and SAB was responsible for the design of may remove some component of the food that has synergistic the experiment, sample analysis, and significant advice and consultation effects in combination with isoflavones or that isoflavones may regarding all aspects of the trial. None of the authors had conflicts of become inactivated during the isolation and purification pro- cess (45, 46). However, the combination of isoflavones in afood or dietary supplement also may be important in terms oftheir potential beneficial health effects. It was suggested that REFERENCES
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