AJCN Tufts Nutrition Symposium, Boston Sept 24-26
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Atkinson, C.
Right arrow Articles by Bingham, S. A
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Atkinson, C.
Right arrow Articles by Bingham, S. A
Agricola
Right arrow Articles by Atkinson, C.
Right arrow Articles by Bingham, S. A
American Journal of Clinical Nutrition, Vol. 79, No. 2, 326-333, February 2004
© 2004 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATIONS

The effects of phytoestrogen isoflavones on bone density in women: a double-blind, randomized, placebo-controlled trial1,2,31-3

Charlotte Atkinson, Juliet E Compston, Nicholas E Day, Mitch Dowsett and Sheila A Bingham

1 From the MRC Biostatistics Unit, Institute of Public Health, Robinson Way, Cambridge, United Kingdom (CA and NED); the Metabolic Bone Unit, Addenbrooke's Hospital, Cambridge, United Kingdom (JEC); the Department of Biochemistry, Royal Marsden Hospital, London (MD); and the MRC Dunn Human Nutrition Unit, Cambridge, United Kingdom (SAB)

2 Supported by grants from the Food Standards Agency and the Medical Research Council; Novogen Ltd (Australia) provided the Promensil tablets and provided research support (to SAB).

3 Address reprint requests to SA Bingham, Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom. E-mail: sab{at}mrc-dunn.cam.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Isoflavone phytoestrogen therapy has been proposed as a natural alternative to hormone replacement therapy (HRT). HRT has a beneficial effect on bone, but few trials in humans have investigated the effects of isoflavones on bone.

Objective: The objective of the study was to determine the effect on bone density of a red clover-derived isoflavone supplement that provided a daily dose of 26 mg biochanin A, 16 mg formononetin, 1 mg genistein, and 0.5 mg daidzein for 1 y. Effects on biochemical markers of bone turnover and body composition were also studied.

Design: Women aged 49-65 y (n = 205) were enrolled in a double-blind, randomized, placebo-controlled trial; 177 completed the trial. Bone density, body composition, bone turnover markers, and diet were measured at baseline and after 12 mo.

Results: Loss of lumbar spine bone mineral content and bone mineral density was significantly (P = 0.04 and P = 0.03, respectively) lower in the women taking the isoflavone supplement than in those taking the placebo. There were no significant treatment effects on hip bone mineral content or bone mineral density, markers of bone resorption, or body composition, but bone formation markers were significantly increased (P = 0.04 and P = 0.01 for bone-specific alkaline phosphatase and N-propeptide of collagen type I, respectively) in the intervention group compared with placebo in postmenopausal women. Interactions between treatment group and menopausal status with respect to changes in other outcomes were not significant.

Conclusion: These data suggest that, through attenuation of bone loss, isoflavones have a potentially protective effect on the lumbar spine in women.

Key Words: Isoflavones • phytoestrogens • bone density • randomized controlled trial • body composition • estrogen and vitamin D receptor polymorphisms


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogens play an important role in skeletal homeostasis, and ovarian hormone deficiency is one of the most important risk factors for osteoporosis. There are clear bone-related benefits of hormone replacement therapy (HRT; 1), but compliance with such regimens is generally very poor (2, 3), partly as a result of fears about the risks of cancer (3).

Isoflavones are compounds in plant foods, particularly soybeans (4, 5), that are structurally similar to the mammalian estrogens (6, 7) and that have received considerable attention for their potential bone-sparing properties. Rates of hip fracture in Asian populations, whose traditional diets are rich in soy, are substantially lower than those in whites residing in the United States (8). However, bone mineral density (BMD) in Asian populations is comparable to that in white populations after adjustment for height and weight (9). Nevertheless, data from animal experiments provided evidence that soy protein can attenuate menopausal bone loss (10, 11), and it was suggested that isoflavones in soy might be responsible for protective effects on bone (12). In humans, some (13-17) but not all (18) cross-sectional studies in Asian populations reported significant positive associations between soy protein or isoflavone intakes and BMD. Furthermore, intervention trials in humans using either soy protein or isoflavone extracts generally reported protective effects on bone, although sample sizes were small and trials often were relatively short. In postmenopausal women, consumption of soy protein providing 90 mg isoflavones/d for 24 wk resulted in a significant increase in lumbar spine bone mineral content (BMC) and BMD (19), and a red clover isoflavone supplement providing 57 or 85.5 mg isoflavones/d for 6 mo resulted in an increase in BMD of the proximal radius and ulna (20). In perimenopausal women, soy protein that provided 80.4 mg isoflavones/d for 24 wk did not increase lumbar spine BMC or BMD, but it did lower the extent of bone loss compared with that in the control group (21). Several intervention studies reported the effects of soy protein or isoflavone extracts on markers of bone turnover. In postmenopausal women, diets rich in soyfoods resulted in significant increases in serum osteocalcin concentrations (22, 23) and decreases in urinary N-telopeptide excretion (22). In perimenopausal women, a 4-wk intervention with an isoflavone extract resulted in a significant reduction in the bone resorption marker pyridinoline (24), but other studies reported little or no effect of soy protein or isoflavone supplements on markers of bone turnover in humans (25-27).

We aimed to determine the effects of consuming a red clover-derived isoflavone supplement (in a daily dose providing 26 mg biochanin A, 16 mg formononetin, 1 mg genistein, and 0.5 mg daidzein) for 1 y on the BMC and BMD of the lumbar spine and hip, on biochemical markers of bone turnover, and on body composition.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Between November 1997 and May 1999, women aged 49-65 y were recruited from the Breast Screening Unit of Addenbrooke's Hospital (Cambridge, United Kingdom). The primary outcome measure was breast density, and women were selected for the study according to the extent of dense tissue seen on their most recent mammogram; mammograms from 1908 women were classified according to their Wolfe pattern (28), and women with P2 or DY Wolfe breast patterns (ie, dense breast patterns; n = 1149, 60% of all mammograms classified) were sent a recruitment letter, which contained a short description of the study and a reply slip. The rate of response to the letter was 68% (n = 781); of the responders, 205 (18% of the women who received letters) were eligible and were randomly assigned to receive an isoflavone or placebo tablet (Figure 1Go). Women were not eligible to participate if they had a personal history of breast cancer or major breast surgery or if they were currently taking HRT. Women who expressed an interest in taking part were visited at home. During this initial home visit, the study was explained in detail, and all women who wished to participate were asked for written informed consent. A total of 205 women were randomly assigned to receive isoflavone or placebo tablets (Figure 1Go). Additional home visits were made after {approx}5.5 and 11 mo on the study. All study procedures were approved by the Dunn Human Nutrition Unit Ethics Committee and the Cambridge Local Research Ethics Committee.



View larger version (38K):
[in this window]
[in a new window]
  		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 who completed the trial but was excluded from all analyses because she had taken oral contraceptives.

 
Participants were randomly assigned to receive daily either a red clover-derived isoflavone tablet that provided 26 mg biochanin A, 16 mg formononetin, 1 mg genistein, and 0.5 mg daidzein (Promensil; Novogen Ltd, Sydney, Australia) or a placebo of identical appearance. We did not measure the isoflavone content of the Promensil tablets in our laboratory, but an independent study showed that the manufacturer's statements as to the quantity and type of isoflavones in the tablets were correct (29). Randomization was performed by using random number generation in MICROSOFT EXCEL (version 2.2; Microsoft Corp, Redmond, WA), and researchers and study participants remained blinded to the tablet allocation throughout the study. Participants were asked to take 1 tablet/d. The code identifying those who had been taking the isoflavone tablets and those who had been taking the placebo was broken when all participants had completed all stages of the study.

Urine samples
Women were asked to make 24-h urine collections at baseline and 12 mo. To check compliance, women also were asked to make a 24-h urine collection at 6 mo. The completeness of all urine collections was assessed by using the p-aminobenzoic acid (PABA)-check test (30). Samples containing 85-110% of the ingested PABA were designated satisfactory. For samples with PABA recoveries of between 70% and 85%, which indicated that all tablets had been taken but that the urine collection was incomplete, the urinary excretion of isoflavones was adjusted to 93% PABA recovery (31). Samples with <70% recovery were designated incomplete. Samples with >110% PABA recovery were considered unsatisfactory, because additional sources of PABA (eg, a multivitamin) may have been consumed, and an accurate determination of sample completeness could not be made.

Urinary excretion of genistein, daidzein, formononetin, and biochanin A was measured by using HPLC and a modification of the method of Setchell et al (32) and Franke et al (33). Briefly, samples were incubated with ß-glucuronidase for 20-72 h at 37 °C. Phenolic components were extracted into an ethyl:acetate (6:4 by vol) solvent mixture. After vortex mixing for 30 s and subsequent 10-min centrifugation at 2320 x g and 20 °C, the organic phase was transferred to a 2-mL vial and evaporated to dryness under vacuum at 43 °C. Extraction residues were reconstituted in 100 µL 50% isopropanol solution and centrifuged for 10 min at 2320 x g and 20 °C. A 5-µL aliquot was then injected directly onto the HPLC column, which consisted of an Alltima 250 x 2.1-mm, 5 µmol/L, C-18 stationary phase (Alltech Associates, New South Wales, Australia), and a mobile phase (acetonitrile and water) containing 0.05% trifluoroacetic acid with a gradient of acetonitrile from 25-100%. Detection and quantification were performed via photo diode array detector. Flavone was used as the internal standard; the limit of detection (LOD) was 0.05 µg/mL, and the limit of quantitation (LOQ) was 0.1 µg/mL.

Total and free pyridinoline and deoxypyridinoline, markers of bone resorption, were measured in urine samples collected at baseline and when nearing completion at 12 mo with the use of a rapid automated assay technique according to the method of Pratt et al (34). Briefly, for total pyridinoline and deoxypyridinoline, 0.5 mL urine was hydrolyzed with an equal volume of 12 mmol/L HCl for 18 h at 107 °C. Hydrolysates were centrifuged for 2 min at 13 500 x g and room temperature, and hydroxypyridinium cross-links were extracted from 0.5 mL of the supernatant by solid-phase extraction (employing cellulose). Pyridinoline and deoxypyridinoline were separated and quantified by using HPLC. Free pyridinoline and deoxypyridinoline were measured without the hydrolysis step. The within-batch CV was <3% for pyridinoline and <5% for deoxypyridinoline measurements; baseline and 12-mo urine samples from individual participants were analyzed together in the same batch. All urine samples were analyzed for their creatinine content (Kone autoanalyser; Jaffe method), and excretion of pyridinoline and deoxypyridinoline was corrected for creatinine excretion.

Blood samples
Fasting blood samples were taken at baseline and 12 mo. Participants were asked to refrain from eating or drinking (except water) from midnight until after the sample had been taken the following morning. A total of 35.5 mL blood was drawn at each visit, including 9.0 mL blood that was drawn into a lithium heparin tube and 9.0 mL that was drawn into a serum tube. The serum tube was left at room temperature for >=1 h before centrifugation to allow clotting. After centrifugation for 10 min at 2000 rpm and 5 °C, aliquots of plasma and serum were stored at -20 °C. Before centrifugation, {approx}1 mL whole blood was removed from the lithium heparin tube, and DNA was extracted by using a Qiagen kit (Qiagen Ltd, Crawley, United Kingdom) and was used for genotyping of estrogen receptor and vitamin D receptor gene polymorphisms (PvuII and BsmI, respectively).

Plasma bone-specific alkaline phosphatase (bone ALP), a marker of bone formation, was measured by using the Metra kit (Quidel Ltd, Oxford, United Kingdom; 35). The intraassay and interassay CVs were 3.9% and 10.9%, respectively. Plasma concentrations of the N-propeptide of collagen type I (PINP), also a marker of bone formation, were measured by using the Orion Diagnostica radioimmunoassay kit (Espoo, Finland; 36), for which the intraassay and interassay CVs were 9.3% and 12.5%, respectively. For both assays, baseline and 12-mo samples from each participant were run together on the same plate to minimize variability.

Serum estradiol was measured by using a previously described method (37), and serum follicle-stimulating hormone (FSH) was measured by using an enzyme immunoassay on an Abbott AxSYM automated analyzer (Abbott Diagnostics, Maidenhead, United Kingdom). Menopausal status was determined by using baseline concentrations of estradiol and FSH as follows; women were classified as premenopausal if they had <30 IU FSH/L and >100 pmol estradiol/L, as postmenopausal if they had >30 IU FSH/L and <100 pmol estradiol/L, and as perimenopausal if they had >30 IU FSH/L and >100 pmol estradiol/L or <30 IU FSH/L and <100 pmol estradiol/L. However, if a woman had noted on the questionnaire completed at the initial home visit that she was currently menstruating, but her baseline hormone profile was that of a postmenopausal woman (ie, >30 IU FSH/L and <100 pmol estradiol/L), she was classified as perimenopausal.

Bone density, body composition, and calcium and vitamin D intakes
Bone density and body composition were assessed by dual-energy X-ray absorptiometry (DXA) at baseline and after {approx}12 mo with the use of an Hologic QDR-4500A scanner (Hologic Inc, Waltham, MA). The precision of the in vivo measurement of BMC by DXA in the Metabolic Bone Unit at Addenbrooke's Hospital is 1% in the spine and 2-3% in the proximal femur.

Individual scans of the lumbar spine (L1-L4), hip (femoral neck, trochanter, and intertrochanteric region), and whole body were taken. If a scan revealed signs of osteoporosis (ie, a BMD T-score <-2.5), the participant was notified in writing and advised to visit her general practitioner. Follow-up spine data were not available for one woman in the isoflavone group, and follow-up bone density and body-composition data were not available for one woman in the placebo group. Six women from the isoflavone group and 8 women from the placebo group were excluded from all analyses regarding bone, because they had taken medications during the study that can affect bone density (eg, bisphosphonates, statins, or calcium and vitamin D supplements). Participants' height and weight were measured at the time of the DXA scans, and body mass index (BMI) was calculated as [weight (kg)/height (m2)]; baseline height was unavailable for one woman in the isoflavone group.

Participants were asked to complete a food-frequency questionnaire (FFQ) at baseline and after 12 mo on the study. Participants were given verbal and written instructions on how to complete the FFQ, and calculation of calcium and vitamin D intakes was based on published food-composition tables (38).

Sample size and data analysis
A sample of 100 in each treatment group gave 80% power to detect a >=1.3% difference between groups with respect to the change in BMC over 1 y, assuming an SD of 3.2% and setting {alpha} at 0.05. Interactions between treatment group and polymorphisms in the estrogen receptor and vitamin D receptor genes are not reported because of a lack of power to detect significant associations. Results are available from the authors on request.

Statistical analyses were performed by using SAS statistical software (version 6.12; SAS Institute, Cary, NC) under the Windows operating system. A P value of <0.05 was considered significant. Changes in BMC, BMD, markers of bone turnover, body composition, and intakes of calcium and vitamin D were calculated as 12-mo data minus baseline data. We used Student's t tests to test for differences between treatment groups for changes in BMC, BMD, markers of bone turnover, and body composition. Data on changes in calcium and vitamin D intakes were skewed, and therefore the nonparametric Wilcoxon's rank-sum test was used to test for differences between treatment groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Baseline characteristics are shown in Table 1Go. Differences between treatment groups were nonsignificant (P > 0.05). Sixteen women withdrew from the isoflavone group and 12 withdrew from the placebo group (Figure 1Go). The principal reasons for withdrawal were commencement of HRT and work commitments or family problems that prevented completion of study activities. Other reasons included feeling no beneficial effects of the intervention or having no interest in continuing on the trial, heavy menstrual bleeding, or illnesses preventing completion of study activities (eg, severe hip pain, skin irritation and sores, vomiting, and diarrhea). One woman in the isoflavone group was diagnosed with an interval cancer of the breast (ie, a cancer detected in the interval after a negative mammographic result) 2 mo after the start of the intervention and was withdrawn from the study. The difference between treatment groups in the number of withdrawals was not significant ({chi}2 = 1.123, P = 0.29).


View this table:
[in this window]
[in a new window]
  		TABLE 1 . Baseline characteristics and time between measurements in subjects receiving isoflavone or placebo tablets1

 
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% had PABA 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, formononetin, and biochanin A) were nonsignificant at baseline, but differences were highly significant at 6 and 12 mo (Figure 2Go). The data shown do not include women with <70% or >110% PABA recovery. Inclusion of these women did not alter the results. Among women in the placebo group, isoflavone excretion did not change significantly from baseline to 6 mo (P = 0.80) or from baseline to 12 mo (P = 0.15; Figure 2Go).



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 2. Mean (±SEM) urinary isoflavone excretion (sum of daidzein, genistein, formononetin, and biochanin A) by treatment group at baseline, 6 mo, and 12 mo, excluding samples with <70% or >110% p-aminobenzoic acid recovery. At baseline: isoflavone group, n = 66; placebo group, n = 72. At 6 mo: isoflavone group, n = 70; placebo group, n = 73. At 12 mo: isoflavone group, n = 76; placebo group, n = 79. The difference in isoflavone excretion between treatment groups was nonsignificant at baseline (P = 0.23), but differences between treatment groups at 6 and 12 mo were highly significant (both: P < 0.001).

 
There were no significant differences between treatment groups with respect to changes in dietary intakes of calcium and vitamin D from baseline to 12 mo; mean (±SD) changes in calcium intakes for women in the isoflavone and placebo groups, respectively, were - 43 ± 224 and - 54 ±165 mg/d (P = 0.32), and changes in vitamin D intakes for women in the isoflavone and placebo groups, respectively, were - 0.24 ± 1.6 and - 0.19 ± 2.3 µg/d (P = 0.52).

Changes in spine and hip BMC and BMD at 1 y by treatment group are shown in Figure 3Go. Interactions between treatment group and menopausal status for the changes in spine or hip BMC and BMD were not significant (P > 0.05). Differences between treatment groups for losses of spine BMC and BMD were significant. Percentage change in spine BMC and BMD among women in the isoflavone and placebo groups, respectively, showed similar trends: mean (±SEM) BMC: -1.42 ± 0.36% and -2.35 ± 0.37% (P = 0.07); BMD: -1.08 ± 0.27% and -1.86 ± 0.29% (P = 0.05). Decreases in BMC and BMD of the hip were generally greater among women in the placebo group than among women in the isoflavone group, but differences between treatment groups were not significant (Figure 3Go). Similar results were found when considering percentage changes (data not shown). In an analysis of covariance on changes from baseline to 12 mo, with baseline data (BMC or BMD) as a covariate, the effect of treatment group on the changes in spine BMC and BMD remained significant (P = 0.05, and P = 0.03, respectively). Similarly, for changes in hip BMC and BMD, the effect of treatment group remained nonsignificant (P = 0.38 and P = 0.85, respectively).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 3. Mean (±SEM) changes in spine and hip bone mineral content (BMC) and bone mineral density (BMD) at 1 y by treatment group. Isoflavone group, n = 77 (n = 78 for hip); placebo group, n = 81. Interactions between menopausal status and treatment group for changes in BMC or BMD were nonsignificant. Differences between treatment groups with respect to changes in spine BMC and BMD were significant (P = 0.04 and P = 0.03, respectively); differences between treatment groups with respect to changes in hip BMC and BMD were nonsignificant (P = 0.48 and P = 0.30, respectively).

 
Markers of bone turnover at baseline and 12 mo by treatment group are shown in Table 2Go. The difference between treatment groups with respect to the change in PINP was of borderline significance. The interaction between treatment group and menopausal status was significant for the changes in bone ALP and PINP (P = 0.05 and P = 0.03, respectively). When grouped by menopausal status, differences between treatment groups were significant among postmenopausal women; mean (±SD) changes in bone ALP among postmenopausal women in the isoflavone and placebo groups, respectively, were 5.65 ± 5.92 and 3.71 ± 2.92 U/L (P = 0.04), and changes in PINP among postmenopausal women in the isoflavone and placebo groups, respectively, were 9.72 ± 25.19 and -1.40 ± 19.12 mg/L (P = 0.01). No other changes differed significantly between treatment groups. Results similar to those for total pyridinoline and deoxypyridinoline were obtained for free pyridinoline and deoxypyridinoline (data not shown). There were no significant differences between treatment groups with respect to the changes in whole-body BMC or BMD or body-composition measures (Table 3Go), and interactions between treatment group and menopausal status were not significant (P > 0.05).


View this table:
[in this window]
[in a new window]
  		TABLE 2 . Markers of bone turnover at baseline and at 12 mo according to treatment group1

 

View this table:
[in this window]
[in a new window]
  		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

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is one of the longest and largest studies to date to examine the effects of an isoflavone supplement derived from red clover on spine and hip BMC and BMD, biochemical markers of bone turnover, and body composition. We observed an attenuation of lumbar spine bone loss among women taking the isoflavone supplement compared with that among women taking the placebo. Trends for losses of hip BMC and BMD were similar to those seen in the lumbar spine, but differences between treatments were not significant. However, this was not entirely surprising, given that the hip contains a relatively large proportion of cortical bone, which is metabolically less active than the trabecular bone of which the spine is primarily composed (39, 40). Furthermore, precision of the measurement of hip BMC is lower than that of the measurement of spine BMC ({approx}2-3% for the proximal femur compared with 1% for the spine; JE Compston, personal communications, 1997 and 2002), and therefore the power to detect significant differences between treatment groups was lower as a result of slow bone turnover and lower measurement precision.

Potter et al (19) previously reported an increase in lumbar spine BMC and BMD among postmenopausal women whose diets were supplemented daily for 24 wk with soy protein isolate providing 90 mg, but not 56 mg, isoflavones/d. In our study, we did not see an increase in BMC or BMD with the isoflavone supplement, but the daily dose of isoflavones was lower than that used by Potter et al (19). However, in a study of perimenopausal women, Alekel et al (20) did not see an increase in lumbar spine BMC or BMD with a daily dose of 80.4 mg isoflavones (as soy protein) for 24 wk, but, similar to our study, they saw a reduction in the extent of bone loss. In a recent study of 28 premenopausal women (mean age: 24 y) there were no significant effects of a soy protein supplement that provided either 0 or 90 mg isoflavones/d for 12 mo (41). This suggests that isoflavones may be beneficial in attenuating age-associated bone loss rather than in enhancing peak bone mass in younger premenopausal women.

Promensil tablets contain isoflavones derived from red clover and, unlike soy protein, relatively large proportions of biochanin A and formononetin, in addition to daidzein and genistein (29). It has been observed that the use of isoflavone extracts does not always result in lipid-lowering effects (42, 43) that are the same as those seen with intact soy protein (44), and it has been suggested that the isoflavone extraction process may remove some component of the food that has synergistic effects in combination with isoflavones or that isoflavones may become inactivated during the isolation and purification process (45, 46). However, the combination of isoflavones in a food or dietary supplement also may be important in terms of their potential beneficial health effects. It was suggested that daidzein may be more important than other isoflavones in terms of relieving menopausal symptoms (47), and the tablets used in the present study were previously shown to produce beneficial effects on bone (20), but conflicting data have been presented with respect to blood lipid concentrations (42, 43).

Because of both the structural similarity of isoflavones to mammalian estrogens and their ability to bind to estrogen receptors (7), it is widely hypothesized that the actions of isoflavones are mediated via the estrogen receptors. However, the exact mechanism of action of isoflavones on bone remains to be fully elucidated. In our study, the 2 bone formation markers increased in postmenopausal women taking the isoflavone supplement. This is somewhat in agreement with studies in ovariectomized rats suggesting that soy or isoflavones stimulate bone formation (10, 48). However, we did not see overall increases in BMC or BMD, but such discord between markers of bone turnover and BMD adds to the ongoing controversy surrounding the use of biochemical markers as predictors of BMD (49).

Prospective and case-control studies showed positive associations between weight and the risk of breast cancer among postmenopausal women and negative associations among premenopausal women (reviewed in 50). Furthermore, increased abdominal fat has consistently been associated with increased mortality and with the incidence of cardiovascular disease, hypertension, and diabetes (51, 52). The menopause is associated with a potentially detrimental shift in the distribution of fat toward a more central location (53-55), but in users of HRT, this shift is minimized, and HRT users are more likely to have less abdominal fat than do nonusers (56-58). In the present study, we did not see a significant effect of the isoflavone supplement on the distributions of fat and lean tissue within the body after 1 y.

Despite being one of the longest intervention studies with an isoflavone supplement to date, a potential limitation of this study is that it was relatively short-term with respect to the outcome measures; longer-term studies are needed to determine the effects of isoflavones (both as intact soy protein and as dietary supplements) on hip BMC and BMD and also on longer-term outcome measures such as fracture rates. Nevertheless, our findings suggest that, through attenuation of bone loss, the isoflavone supplement has a potentially protective effect on the lumbar spine.


   ACKNOWLEDGMENTS
 
We thank Sue Gardner, Nicola Duffy, Nasima Siddiqui, Jane Bettany, and Jayne Girvan for their assistance with the day-to-day running of the trial; Shirley Love for the measurement of bone density and body composition; Simon P Robins, Phyllis Nicol, and Alexander Duncan for the bone marker assays; Alison Dunning, Catherine Healey, and Alex Loktionov for the genotyping; and Addenbrooke's Hospital Pharmacy for administering the tablets. Novogen Ltd performed the urinary isoflavone analyses.

All authors participated fully in the preparation of the manuscript. CA was responsible for subject recruitment and day-to-day running of the trial, sample analyses, and statistical analyses; JEC was responsible for bone densitometry; NED was responsible for the data analysis; MD was responsible for the hormone assays; and SAB was responsible for the design of the experiment, sample analysis, and significant advice and consultation regarding all aspects of the trial. None of the authors had conflicts of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Castelo-Branco C, Figueras F, Sanjuan A, Pons F, Vicente JJ, Vanrell JA. Long-term postmenopausal hormone replacement therapy effects on bone mass: differences between surgical and spontaneous patients. Eur J Obstet Gynecol Reprod Biol 1999;83:207-11.[Medline]
  2. Pilon D, Castilloux AM, LeLorier J. Estrogen replacement therapy: determinants of persistence with treatment. Obstet Gynecol 2001;97:97-100.[Abstract/Free Full Text]
  3. Castelo-Branco C, Figueras F, Sanjuan A, et al. Long-term compliance with estrogen replacement therapy in surgical postmenopausal women: benefits to bone and analysis of factors associated with discontinuation. Menopause 1999;6:307-11.[Medline]
  4. Reinli K, Block G. Phytoestrogen content of foods—a compendium of literature values. Nutr Cancer 1996;26:123-48.[Medline]
  5. Liggins J, Bluck LJ, Runswick S, Atkinson C, Coward WA, Bingham SA. Daidzein and genistein contents of vegetables. Br J Nutr 2000;84:717-25.[Medline]
  6. Setchell KDR, Adlercreutz H. Mammalian lignans and phyto-oestrogens: recent studies on their formation, metabolism and biological role in health and disease. In: Rowland I, ed. Role of the gut flora in toxicity and cancer. London: Academic Press, 1988:315-45.
  7. Setchell KD, Cassidy A. Dietary isoflavones: biological effects and relevance to human health. J Nutr 1999;129:758S-67S.
  8. Ling X, Lu A, Zhao X, Chen X, Cummings SR. Very low rates of hip fracture in Beijing, People's Republic of China: the Beijing Osteoporosis Project. Am J Epidemiol 1996;144:901-7.[Abstract/Free Full Text]
  9. Lau EM, Cooper C. The epidemiology of osteoporosis. The oriental perspective in a world context. Clin Orthop 1996;323:65-74.
  10. Arjmandi BH, Alekel L, Hollis BW, et al. Dietary soybean protein prevents bone loss in an ovariectomized rat model of osteoporosis. J Nutr 1996;126:161-7.
  11. Harrison E, Adjei A, Ameho C, Yamamoto S, Kono S. The effect of soybean protein on bone loss in a rat model of postmenopausal osteoporosis. J Nutr Sci Vitaminol (Tokyo) 1998;44:257-68.[Medline]
  12. Arjmandi BH, Getlinger MJ, Goyal NV, et al. Role of soy protein with normal or reduced isoflavone content in reversing bone loss induced by ovarian hormone deficiency in rats. Am J Clin Nutr 1998;68(suppl):1358S-63S.[Abstract]
  13. Greendale GA, FitzGerald G, Huang MH, et al. Dietary soy isoflavones and bone mineral density: results from the study of women's health across the nation. Am J Epidemiol 2002;155:746-54.[Abstract/Free Full Text]
  14. Mei J, Yeung SS, Kung AW. High dietary phytoestrogen intake is associated with higher bone mineral density in postmenopausal but not premenopausal women. J Clin Endocrinol Metab 2001;86:5217-21.[Abstract/Free Full Text]
  15. Horiuchi T, Onouchi T, Takahashi M, Ito H, Orimo H. Effect of soy protein on bone metabolism in postmenopausal Japanese women. Osteoporos Int 2000;11:721-4.[Medline]
  16. Ho SC, Chan SG, Yi Q, Wong E, Leung PC. Soy intake and the maintenance of peak bone mass in Hong Kong Chinese women. J Bone Miner Res 2001;16:1363-9.[Medline]
  17. Somekawa Y, Chiguchi M, Ishibashi T, Aso T. Soy intake related to menopausal symptoms, serum lipids, and bone mineral density in postmenopausal Japanese women. Obstet Gynecol 2001;97:109-15.[Abstract/Free Full Text]
  18. Nagata C, Shimizu H, Takami R, Hayashi M, Takeda N, Yasuda K. Soy product intake and serum isoflavonoid and estradiol concentrations in relation to bone mineral density in postmenopausal Japanese women. Osteoporos Int 2002;13:200-4.[Medline]
  19. Potter SM, Baum JA, Teng H, Stillman RJ, Shay NF, Erdman JW Jr. Soy protein and isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am J Clin Nutr 1998;68(suppl):1375S-9S.[Abstract]
  20. Clifton-Bligh PB, Baber RJ, Fulcher GR, Nery ML, Moreton T. The effect of isoflavones extracted from red clover (Rimostil) on lipid and bone metabolism. Menopause 2001;8:259-65.[Medline]
  21. Alekel DL, Germain AS, Peterson CT, Hanson KB, Stewart JW, Toda T. Isoflavone-rich soy protein isolate attenuates bone loss in the lumbar spine of perimenopausal women. Am J Clin Nutr 2000;72:844-52.[Abstract/Free Full Text]
  22. Scheiber MD, Liu JH, Subbiah MT, Rebar RW, Setchell KD. Dietary inclusion of whole soy foods results in significant reductions in clinical risk factors for osteoporosis and cardiovascular disease in normal postmenopausal women. Menopause 2001;8:384-92.[Medline]
  23. Chiechi LM, Secreto G, D'Amore M, et al. Efficacy of a soy rich diet in preventing postmenopausal osteoporosis: the Menfis randomized trial. Maturitas 2002;42:295-300.[Medline]
  24. Uesugi T, Fukui Y, Yamori Y. Beneficial effects of soybean isoflavone supplementation on bone metabolism and serum lipids in postmenopausal Japanese women: a four-week study. J Am Coll Nutr 2002;21:97-102.[Abstract/Free Full Text]
  25. Upmalis DH, Lobo R, Bradley L, Warren M, Cone FL, Lamia CA. Vasomotor symptom relief by soy isoflavone extract tablets in postmenopausal women: a multicenter, double-blind, randomized, placebo-controlled study. Menopause 2000;7:236-42.[Medline]
  26. Knight DC, Howes JB, Eden JA, Howes LG. Effects on menopausal symptoms and acceptability of isoflavone-containing soy powder dietary supplementation. Climacteric 2001;4:13-8.[Medline]
  27. Hale GE, Hughes CL, Robboy SJ, Agarwal SK, Bievre M. A double-blind randomized study on the effects of red clover isoflavones on the endometrium. Menopause 2001;8:338-46.[Medline]
  28. Wolfe JN. Breast patterns as an index of risk for developing breast cancer. AJR Am J Roentgenol 1976;126:1130-7.[Abstract]
  29. Setchell KD, Brown NM, Desai P, et al. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr 2001;131:1362S-75S.[Abstract/Free Full Text]
  30. Bingham S, Cummings JH. The use of 4-aminobenzoic acid as a marker to validate the completeness of 24 h urine collections in man. Clin Sci 1983;64:629-35.[Medline]
  31. Johansson G, Bingham S, Vahter M. A method to compensate for incomplete 24-hour urine collections in nutritional epidemiology studies. Public Health Nutr 1999;2:587-91.[Medline]
  32. Setchell KD, Welsh MB, Lim CK. High-performance liquid chromatographic analysis of phytoestrogens in soy protein preparations with ultraviolet, electrochemical and thermospray mass spectrometric detection. J Chromatogr 1987;386:315-23.[Medline]
  33. Franke AA, Custer LJ, Cerna CM, Narala K. Rapid HPLC analysis of dietary phytoestrogens from legumes and from human urine. Proc Soc Exp Biol Med 1995;208:18-26.[Abstract]
  34. Pratt DA, Daniloff Y, Duncan A, Robins SP. Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography. Anal Biochem 1992;207:168-75.[Medline]
  35. Gomez B Jr, Ardakani S, Ju J, et al. Monoclonal antibody assay for measuring bone-specific alkaline phosphatase activity in serum. Clin Chem 1995;41:1560-6.[Abstract/Free Full Text]
  36. Melkko J, Kauppila S, Niemi S, et al. Immunoassay for intact amino-terminal propeptide of human type I procollagen. Clin Chem 1996;42:947-54.[Abstract/Free Full Text]
  37. Dowsett M, Goss PE, Powles TJ, et al. Use of the aromatase inhibitor 4-hydroxyandrostenedione in postmenopausal breast cancer: optimization of therapeutic dose and route. Cancer Res 1987;47:1957-61.[Abstract/Free Full Text]
  38. Bingham SA, Welch AA, McTaggart A, et al. Nutritional methods in the European Prospective Investigation of Cancer in Norfolk. Public Health Nutr 2001;4:847-58.[Medline]
  39. Cooper C. Bone mass throughout life: bone growth and involution. In: Francis RM, Dick WC, eds. Osteoporosis: pathogenesis and management. Dordrecht, Netherlands: Kluwer Academic Publishers, 1990:1-26.
  40. Reeve J. Clinical assessment of the physical properties of bone. In: Compston JE, ed. Osteoporosis: new perspectives on causes, prevention and treatment. London: Royal College of Physicians of London, 1996:41-53.
  41. Anderson JJ, Chen X, Boass A, et al. Soy isoflavones: no effects on bone mineral content and bone mineral density in healthy, menstruating young adult women after one year. J Am Coll Nutr 2002;21:388-93.[Abstract/Free Full Text]
  42. Nestel PJ, Yamashita T, Sasahara T, et al. Soy isoflavones improve systemic arterial compliance but not plasma lipids in menopausal and perimenopausal women. Arterioscler Thromb Vasc Biol 1997;17:3392-8.[Abstract/Free Full Text]
  43. Hodgson JM, Puddey IB, Beilin LJ, Mori TA, Croft KD. Supplementation with isoflavonoid phytoestrogens does not alter serum lipid concentrations: a randomized controlled trial in humans. J Nutr 1998;128:728-32.[Abstract/Free Full Text]
  44. Anderson JW, Johnstone BM, Cook-Newell ME. Meta-analysis of the effects of soy protein intake on serum lipids. N Engl J Med 1995;333:276-82.[Abstract/Free Full Text]
  45. Barnes S. Phytoestrogens and breast cancer. Baillieres Clin Endocrinol Metab 1998;12:559-79.[Medline]
  46. Clarkson TB, Anthony MS. Phytoestrogens and coronary heart disease. Baillieres Clin Endocrinol Metab 1998;12:589-604.[Medline]
  47. Eden J. Phytoestrogens and the menopause. Baillieres Clin Endocrinol Metab 1998;12:581-7.[Medline]
  48. Fanti O, Faugere MC, Gang Z, Schmidt J, Cohen D, Malluche HH. Systematic administration of genistein partially prevents bone loss in ovariectomized rats in a nonestrogen-like mechanism. Am J Clin Nutr 1998;68(suppl):1517S(abstr).
  49. Looker AC, Bauer DC, Chesnut CH III, et al. Clinical use of biochemical markers of bone remodeling: current status and future directions. Osteoporos Int 2000;11:467-80.[Medline]
  50. Cleary MP, Maihle NJ. The role of body mass index in the relative risk of developing premenopausal versus postmenopausal breast cancer. Proc Soc Exp Biol Med 1997;216:28-43.[Abstract]
  51. Bjorntorp P. Abdominal fat distribution and disease: an overview of epidemiological data. Ann Med 1992;24:15-8.[Medline]
  52. Pi-Sunyer FX. Health implications of obesity. Am J Clin Nutr 1991;53(suppl):1595S-603S.[Abstract/Free Full Text]
  53. Poehlman ET, Toth MJ, Gardner AW. Changes in energy balance and body composition at menopause: a controlled longitudinal study. Ann Intern Med 1995;123:673-5.[Abstract/Free Full Text]
  54. Svendsen OL, Hassager C, Christiansen C. Age- and menopause-associated variations in body composition and fat distribution in healthy women as measured by dual-energy X-ray absorptiometry. Metabolism 1995;44:369-73.[Medline]
  55. Tremollieres FA, Pouilles JM, Ribot CA. Relative influence of age and menopause on total and regional body composition changes in postmenopausal women. Am J Obstet Gynecol 1996;175:1594-600.[Medline]
  56. Perrone G, Liu Y, Capri O, et al. Evaluation of the body composition and fat distribution in long-term users of hormone replacement therapy. Gynecol Obstet Invest 1999;48:52-5.[Medline]
  57. Reubinoff BE, Wurtman J, Rojansky N, et al. Effects of hormone replacement therapy on weight, body composition, fat distribution, and food intake in early postmenopausal women: a prospective study. Fertil Steril 1995;64:963-8.[Medline]
  58. Sites CK. Hormone replacement therapy: cardiovascular benefits for aging women. Coron Artery Dis 1998;9:789-93.[Medline]
Received for publication December 3, 2002. Accepted for publication August 8, 2003.




This article has been cited by other articles:


Home page
 Menopause IntHome page
T. J Powles, A. Howell, D G. Evans, E. V McCloskey, S. Ashley, R. Greenhalgh, J. Affen, L. A. Flook, and A. Tidy
Red clover isoflavones are safe and well tolerated in women with a family history of breast cancer
Menopause Int, March 1, 2008; 14(1): 6 - 12.
[Abstract] [Full Text] [PDF]

Home page
 Agron. J.Home page
K. A. Nelson, G. E. Rottinghaus, and T. E. Nelson
Effect of Lactofen Application Timing on Yield and Isoflavone Concentration in Soybean Seed
Agron. J., April 4, 2007; 99(3): 645 - 649.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Coll. Nutr.Home page
D. Bunout, G. Barrera, L. Leiva, V. Gattas, M. P. de la Maza, F. Haschke, P. Steenhout, P. Klassen, C. Hager, E. Offord, et al.
Effect of a Nutritional Supplementation on Bone Health in Chilean Elderly Subjects with Femoral Osteoporosis
J. Am. Coll. Nutr., June 1, 2006; 25(3): 170 - 177.
[Abstract] [Full Text] [PDF]

Home page
 The Annals of PharmacotherapyHome page
A. M. Whelan, T. M Jurgens, and S. K Bowles
Natural Health Products in the Prevention and Treatment of Osteoporosis: Systematic Review of Randomized Controlled Trials
Ann. Pharmacother., May 1, 2006; 40(5): 836 - 849.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
T. Clavel, M. Fallani, P. Lepage, F. Levenez, J. Mathey, V. Rochet, M. Serezat, M. Sutren, G. Henderson, C. Bennetau-Pelissero, et al.
Isoflavones and Functional Foods Alter the Dominant Intestinal Microbiota in Postmenopausal Women
J. Nutr., December 1, 2005; 135(12): 2786 - 2792.
[Abstract] [Full Text] [PDF]

Home page
 Arch Intern MedHome page
X. Zhang, X.-O. Shu, H. Li, G. Yang, Q. Li, Y.-T. Gao, and W. Zheng
Prospective Cohort Study of Soy Food Consumption and Risk of Bone Fracture Among Postmenopausal Women
Arch Intern Med, September 12, 2005; 165(16): 1890 - 1895.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
C. M. Weaver and J. M. K. Cheong
Soy Isoflavones and Bone Health: The Relationship Is Still Unclear
J. Nutr., May 1, 2005; 135(5): 1243 - 1247.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
M. Messina
Western soy intake is too low to produce health effects
Am. J. Clinical Nutrition, August 1, 2004; 80(2): 528 - 529.
[Full Text] [PDF]

Home page
 J. Nutr.Home page
C. Atkinson, W. Oosthuizen, S. Scollen, A. Loktionov, N. E. Day, and S. A. Bingham
Modest Protective Effects of Isoflavones from a Red Clover-Derived Dietary Supplement on Cardiovascular Disease Risk Factors in Perimenopausal Women, and Evidence of an Interaction with ApoE Genotype in 49-65 Year-Old Women
J. Nutr., July 1, 2004; 134(7): 1759 - 1764.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Atkinson, C.
Right arrow Articles by Bingham, S. A
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Atkinson, C.
Right arrow Articles by Bingham, S. A
Agricola
Right arrow Articles by Atkinson, C.
Right arrow Articles by Bingham, S. A

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS