Phyto-Oestrogen Excretion and Rate of Bone Loss in Postmenopausal Women

Phyto-oestrogen excretion and rate of bone loss in postmenopausal women

AFM Kardinaal1, MS Morton2, IEM BruÈggemann-Rotgans3 and ECH van Beresteijn4

1Department of Consumer Research and Epidemiology, TNO Nutrition and Food Research Institute, Zeist, The Netherlands; 2Tenovus Cancer Research Centre, University of Wales College of Medicine, Cardiff, UK; 3Department of Biopharmaceutical Analysis, TNO Nutrition and Food Research Institute, Zeist, The Netherlands; and 4Department of Nutrition and Health, Netherlands Institute for Dairy Research (NIZO), Ede, The Netherlands

Objective: The hypothesis was tested that the rate of postmenopausal bone loss is inversely associated with long- term urinary excretion of phyto-oestrogens, as a marker of habitual dietary intake. Design: Secondary analysis of a 10-year follow-up study (1979 ± 1989) among postmenopausal women in the Netherlands. Subjects: From the original population of 154 women, 32 women were selected with an annual rate of radial bone loss of 0.5% over the ®rst 5 years of the study and 35 women with a rate of 2.5% per year. Methods: The iso¯avonoids genistein, daidzein and equol, and the lignan enterolactone were determined by gas chromatography ± mass spectrometry in aggregate samples from annually collected urine samples. Cortical bone density of the radius had previously been measured annually by single-photon absorptiometry. Results: Excretion of iso¯avonoids did not differ between both groups, although in multivariate analysis equol excretion was weakly positively associated with rate of bone loss in the 5 years after the menopause. Enterolactone excretion was signi®cantly higher in the group with high rate of bone loss. This positive association remained in multivariate linear regression analysis after adjustment for age, years since menopause, body mass index and intake of calcium, vegetable protein and dietary ®bre. Conclusions: Enterolactone excretion is likely to be an indicator of consumption of grains and legumes; it is not clear whether the observed positive association with rate of bone loss is a causal one. Our results do not support a preventive effect of low, unsupplemented dietary intake of phyto-oestrogens on postmenopausal cortical bone loss. However, no conclusions can be drawn about effects of higher doses of phyto-oestrogens. Sponsorship: This study was supported by the Dutch Prevention Fund, a charity organisation. Descriptors: urinary iso¯avones; urinary lignans; menopause; bone density; osteoporosis; oestrogens

Introduction endometrial cancer. However, there is evidence that the use of oestrogen, with or without progestin, increases the Osteoporosis is a major public health problem in elderly risk of breast cancer (Collaborative Group on Hormonal populations around the world, in particular in women. Factors in Breast Cancer, 1997). This potential drawback, Recent estimates suggest that 30% of postmenopausal in combination with the low compliance to HRT observed white women in the United States and 23% of women in some populations (Ravnikar, 1987), perhaps due to an aged 50 years and over in Europe have osteoporosis aversion to the prolonged use of some kinds of medication, (Cooper and Barrett-Connor, 1996). More than half of stimulates the investigation of the role of dietary com- these women are estimated to have also sustained a low pounds in the prevention of postmenopausal bone loss. trauma fracture. Worldwide there were about 1.66 million In Italy, a number of trials support the preventive effect hip fractures in 1990 and this number is expected to of ipri¯avone on postmenopausal bone loss (Gambacciani continue to rise in the future (Melton, 1996). et al, 1997; Agnusdei et al, 1997; Valente et al, 1994). The risk of developing osteoporosis after the menopause Ipri¯avone is a synthetic iso¯avone; iso¯avones are natural is largely determined by the peak bone mass obtained at plant hormones with an oestrogen-like structure, with soy young adult age on the one hand, and the rate of bone loss products as their predominant source. The main represen- after the menopause on the other. Postmenopausal bone tatives of this class of compounds are genistein and loss is primarily caused by the sharp decrease in oestrogen daidzein. Iso¯avones have been postulated to play a role levels that is inherent to menopause. Hormone replacement in the prevention of a number of chronic diet-related therapy (HRT) is effective in the prevention of bone loss in diseases (Adlercreutz & Mazur, 1997). It is also hypothe- early menopause (Recker, 1993). Oestrogens are combined sised that dietary intake of iso¯avones has a bone-sparing with low-dose progestins to prevent oestrogen-induced effect on postmenopausal bone loss. Limited evidence from animal experiments (Anderson et al, 1995, 1996); Blair, Correspondence: AFM Kardinaal, Department of Research and 1996; Fanti et al, 1996) seems to support this hypothesis. Epidemiology, TNO Nutrition and Food Research Institute, Zeist, The Netherlands. Intervention studies with iso¯avones are under way, but so Received 23 January 1998; revised 1 July 1998; accepted 9 July 1998 far results from human studies have not been reported. Bone loss in post menopausal women AFM Kardinaal et al 851 We investigated the relation between long-term excre- Body mass index was calculated from the annual mea- tion of iso¯avones, as a marker of dietary intake, and the surements of body weight and height (body weight (kg)/ rate of cortical bone loss at the radius in a 10-year follow- height2 (m2)). up study among perimenopausal women in the Netherlands. Biochemical analyses For each individual in the groups selected on low or high Methods rate of bone loss (n 67), an aggregate sample was made from the annually collected urine samples, proportional to Population the 24-h volume. Phyto-oestrogens in urine were analysed From 1979 to 1989, 154 healthy early postmenopausal by gas chromatography ± mass spectrometry (GC-MS) women participated in a prospective study of cortical (Morton et al, 1994). Urine (50 ml) was aliquoted by bone loss in relation to habitual calcium intake. None of weight into a silanised B19 test tube and 0.1 M sodium them had a history of diabetes mellitus, renal or bone acetate buffer, pH5.0 (2.0 ml) was added. A cocktail of disease, or had undergone major gynaecological surgery deuterated internal standards, containing deuterated analo- or taken any medication known to affect calcium or bone gues of enterodiol, enterolactone, equol, daidzein and metabolism (such as oestrogens, thiazide diuretics, corti- genistein, was added in methanol (50 ml). After equilibra- costeroids, vitamin D in pharmacological dosages or cal- tion at room temperature for 30 min, b-glucuronidase/aryl cium supplements of more than 500 mg/day). Participants sulphatase (Helix pomatia) was added in 0.1 M acetate were recruited in 1978, by inviting all women born between buffer. The mixture was incubated overnight at 37 C and 1922 and 1931 and living in the community of Ede, The then extracted with fractionated diethyl ether (5 ml). The Netherlands. After the ®rst examination, 264 eligible organic layer was blown to dryness under nitrogen, dis- healthy women consented to participate and were enrolled solved in a mixture of chloroform ± heptane ± methanol in the study. At the end of the follow-up period the data of (10:10:1; 200 ml) and applied to the top of a short column 43 participants could not be evaluated because they had (361 cm) of Sephadex LH20 constructed in a silanised dropped out for various reasons (such as moving to another glass Pasteur pipette. The column was washed with chloro- city). Of the remaining 221 women, 67 were put on form ± heptane ± methanol (10:10:1; 5 ml) and this wash medication known to in¯uence calcium and bone meta- was discarded. The iso¯avones and lignans present in the bolism, leaving the data of 154 women to be evaluated sample were eluted with methanol (2 ml), which was blown (Beresteijn et al, 1990). All participants used their habitual to dryness under nitrogen at 60 C. The extract was deriva- diet during the study period and care was taken to avoid tised for GC-MS by reaction with N-(t-butyldimethylsilyl)- in¯uencing them in this respect. N-methyltri¯uoroacetamide containing 1% t-butyldi- Of the population of 154 women, 32 women had a rate methylsilyl chloride (100 ml) in acetonitrile (100 ml) at of cortical bone loss at the radius of 0.5% per year or less 65 C for 2 h, to form the t-butyldimethylsilyl ethers of over the ®rst 5 years of the study; 35 women had an annual the analytes and internal standards. The reaction mixture rate of cortical bone loss of 2.5% or more. Seven years after was blown to dryness under nitrogen gas and dissolved in the end of the 10-year follow-up, average urinary excretion cyclohexane (50 ml) for analysis. Calibration standards of dietary phyto-oestrogens during the study period was were derivatised in the same manner. determined in these two groups. Gas chromatography ± mass spectrometry was carried out using a Fison's Instruments (Manchester, UK) MD800 benchtop quadrupole mass spectrometer coupled Data collection to a Fison's Instruments GC8000 chromatograph. The gas During the 10-year follow-up, annual measurements were chromatograph housed a 15 m60.25 mm DB5MS (0.25 mm performed of bone mass of the radius, body weight and phase thickness) capillary column (J & W Scienti®c, CA, height, dietary intake of calcium and other nutrients, and USA) and was programmed from 250C to 280C at blood and 24-h urine samples were collected. To exclude 49.9C/min under a helium pressure of 50 kPa. The inter- seasonal variations, all measurements were performed face was maintained at 275C and the source at 250C. annually for each individual at the same time of the year Peak height ratios of analyte to internal standard were (between January and June). measured for each sample and calibration standard using Cortical bone mineral content (BMC) of the radius was MassLab software (Fison's Instruments, Manchester, UK) measured by single-photon absorptiometry (125I, Norland- and concentrations were determined by reference to stan- Cameron Bone Mineral Analyzer, type 178) and calculated dard curve. Concentrations were expressed relative to as the mean of ®ve scans of the distal third of the urinary creatinine. Biochemical markers of bone turnover nondominant radius. Results were expressed as g/cm and were determined in serum (alkaline phosphatase, osteocal- adjusted for bone width (BMD, g/cm2). The long-term cin, 1,25-dihydroxyvitamin D) and urine (hydroxyproline), coef®cient of variation of the bone mass measurements as previously described (Beresteijn et al, 1986). was 1.1%. Rates of BMD loss were calculated from individual regression equations of BMD versus time and Statistical analyses expressed as percentage of the initial value. The distributions of the phyto-oestrogen concentrations Food consumption was estimated by a trained dietitian were very much skewed and could not be normalised by using the cross-check dietary history method. Calcium and transformation. Therefore, median values and 25th and vegetable protein intake were calculated by means of a 75th centiles of the distribution are given and differences computer program based on the Dutch Food Composition between the groups with low and high rate of bone loss Table. Details of the interview procedure and validation of were tested with the Kruskall ± Wallis test. For height, the nutritional data have been described elsewhere (Bere- weight and BMI, calcium and vegetable protein intake, steijn et al, 1987). and biochemical parameters measured in serum or urine, Bone loss in post menopausal women AFM Kardinaal et al 852 the average values over the 10-year follow-up period were Table 2 Concentration of phyto-oestrogens in urine (mg/g creatinine) calculated; differences between the groups were tested with Student's t-test. The associations between the following Bone loss 0.5%/year Bone loss 2.5%/year variables and phyto-oestrogen excretion (genistein, daid- Phyto-oestrogen 25th ± 75th 25th ± 75th zein, equol and enterolactone, respectively) were evaluated compound Median centiles Median centiles by Pearson correlation analysis: age, years since meno- Genistein 124 82 ± 167 88 51 ± 167 pause, height, weight, body mass index, dietary intake of Daidzein 363 261 ± 497 282 189 ± 383 calcium, vegetable protein and dietary ®bre, serum con- Equol 15 5 ± 51 12 9 ± 73 centrations of alkaline phosphatase, osteocalcin and 1,25- Total iso¯avones 410 259 ± 567 350 194 ± 460 dihydroxyvitamin D, and urinary hydroxyproline/creatinine Enterolactone 838 572 ± 1228 1108* 737 ± 1869 ratio. The association between phyto-oestrogen excretion *P < 0.05. and rate of cortical bone loss was estimated by linear regression analysis, with adjustment for age, years since menopause and body mass index. Rate of cortical bone loss level in 10 samples (5 from the LR and 5 from the HR over the whole study period (1980 ± 1989) was measured group). Enterolactone excretion was signi®cantly higher in for 63 subjects: two subjects in both groups had incomplete the HR group (P < 0.05). data due to drop-out, hysterectomy or death. All analyses Excretion of the individual iso¯avonoids was not asso- were performed with the SAS statistical package. ciated with any of the covariates measured; total iso¯avone excretion was positively associated with body weight (r 0.26) and body mass index (r 0.25). Enterolactone Results excretion was inversely correlated with body mass index (r 7 0.24, P 0.05) and positively correlated with Women with a low rate of bone loss (LR) had higher body intake of vegetable protein (r 0.23, P 0.07) and dietary weight and BMI than women with a high rate of bone loss ®bre (r 0.28, P < 0.05). Iso¯avonoids and enterolactone (HR) (Table 1). The HR group was on average slightly excretion were not associated with radial bone density at longer after menopause (P 0.07). Concentrations of the start of the study in 1980, nor with biochemical markers serum osteocalcin and 1,25-hydroxyvitamin D and of urin- of bone turnover. ary hydroxyproline were on average signi®cantly higher in The relation between phyto-oestrogen excretion and the the HR group. The initial cortical bone density at the radius rate of cortical bone loss was examined in a multiple linear was similar in both groups; there were no differences in regression model, with adjustment for age, years since usual intake of calcium and vegetable protein. menopause and body mass index (Table 3). The equol Excretion of the iso¯avonoids did not differ between the excretion was a signi®cant determinant of the variation in LR and HR group (Table 2), although median excretion bone loss over the period 1980/1985, but not for the whole was consistently higher in the LR group (for daidzein, study period 1980/1989. Higher enterolactone excretion P 0.09). Equol concentrations were below the detection was associated with higher rates of bone loss in the period 1980/1989 (P < 0.05). Additional adjustment for intake of calcium, vegetable protein or dietary ®bre did Table 1 Characteristics of women with different rates of bone loss not substantially change the results, although the contribu- (meanÆ s.e.m.) in 1980 ± 1985 tion of enterolactone was no longer statistically signi®cant when ®bre intake was included in the regression model. Bone loss Bone loss 0.5%/year 2.5%/year (n 32) (n 35)

Age in 1989 (y) 63.4Æ 0.4 62.8Æ 0.4 Discussion Time since menopause (in 9.3Æ 0.4 10.2Æ 0.2 This observational longitudinal study among healthy, early 1989) (y) Body weight (kg)a 75.1Æ 2.2 64.5Æ 6.5**** postmenopausal women in the Netherlands evaluated Height (cm)a 163.1Æ 1.0 163.6Æ 0.9 whether the intake of phyto-oestrogens in a habitual, low- BMI (kg/m2)a 28.3Æ 0.8 24.1Æ 0.4**** phyto-oestrogen diet, assessed by urinary phyto-oestrogen Rate of bone loss 1980-1989 0.27Æ 0.08 1.91Æ 0.08**** excretion, is related to the rate of cortical bone loss around (%/year)b BMD radius 1980 0.692Æ 0.008 0.702Æ 0.008 Calcium intake (mg/d)a 1140Æ 59 1210Æ 53 a Dietary ®bre intake (g/d) 24.8Æ 1.2 27.0Æ 5.6 Table 3 Multivariate linear regression modelsa for the relation between Vegetable protein intake 20.7Æ 0.9 22.0Æ 0.7 urinary phyto-oestrogen excretion and rate of bone loss (bÆ s.e.) (g/d)a Serum alkaline phosphatase 34.7Æ 1.6 34.7Æ 1.0 Association with Association with a (U/l) rate of bone rate of bone Serum osteocalcin 6.7Æ 0.3 8.6Æ 0.4*** Phyto-oestrogen loss (in %/year) loss (in %/year) a,c (ng/ml) (mg/g creatinine over 1980 ± 1985 over 1980 ± 1989 Serum 1,25-dihydroxy- 58.5Æ 1.8 65.2Æ 2.4* 6 100) (bÆ s.e.) (bÆ s.e.) vitamin D (pmol/l)a Urinary 20.2Æ 0.6 22.3Æ 0.5** Genistein 0.132Æ 0.074 0.044Æ 0.043 hydroxyproline/creatinine Daidzein 0.061Æ 0.043 0.014Æ 0.025 ratio (mmol/mmol)a Equol 0.490Æ 0.180** 0.142Æ 0.108 Total iso¯avones 0.098Æ 0.061 0.042Æ 0.036 aMean over follow-up period. Enterolactone 0.014Æ 0.008 0.009Æ 0.004* bn 30 and 33, respectively cn 22 and 21, respectively aAdjusted for age, years since menopause and body mass index; n 63. *P 0.05; **P 0.01; ***P 0.001; ****P 0.0001. *P < 0.05, ** P < 0.01. Bone loss in post menopausal women AFM Kardinaal et al 853 menopause. In contrast with the hypothesis, women with a symptoms. The lower incidences of breast, prostate and relatively high rate of bone loss had signi®cantly higher colon cancer, and the low prevalence of menopausal urinary excretion of enterolactone than women with a low symptoms in Asian populations coincide with a much rate of bone loss, while equol excretion was also weakly higher intake of phyto-oestrogens, mainly iso¯avones positively associated with rate of cortical bone loss. The from soy. Although such associations do not form evidence difference between the 25th and 75th centiles of equol for a causal relationship, it is not unlikely that potential excretion is associated with 0.26% bone loss per year, in bene®cial effects can be expected only at levels of intake the years immediately after the menopause. For enterolac- that are higher than habitual intakes from Western diets. tone this difference is associated with an increase in rate of Recent estimates indicate intakes of 20 ± 50 mg iso¯avones bone loss of 0.07% per year. Excretion of the main per day in Japan and China (Coward et al, 1993; Fukutake iso¯avones genistein and daidzein was not associated et al, 1996), while the intake in Western countries is with the rate of cortical bone loss. assumed to be less than 1 mg/day. However, a recent The women in this study consumed a normal Dutch diet; case-control study of phyto-oestrogens and breast cancer there were no vegetarians among them. The main food (Ingram et al, 1997) detected a signi®cant inverse associa- source of iso¯avonoids in the present study is unknown. tion in a population of Australian women, where phyto- Iso¯avonoids are present in soy products, but these are oestrogen intake is likely to be relatively low in comparison consumed by only 6% of Dutch women aged 50 ± 65 years with Asian women, and where the difference in phyto- (Anonymous, 1993). Soy protein is added to many manu- oestrogen excretion in the lowest and highest quartiles of factured products and could thus add to the dietary intake in excretion was only about a factor 3. this population. Enterolactone is produced by bacteria in Phyto-oestrogens, in particular the iso¯avones present in the colon from precursors mainly in grains, legumes, soybeans, have also been suggested to have a bene®cial oilseeds and vegetables (Thompson et al, 1991), which effect on bone preservation, although the mechanism has form a more substantial part of the diet. not yet been cleared up. The evidence from experimental or The low intake of iso¯avonoid sources is re¯ected in the observational studies is very limited so far. More evidence low level of urinary excretion of iso¯avonoids in our is available for ipri¯avone, a synthetic iso¯avone, which population. Urinary excretion of iso¯avonoids is dose has been shown to inhibit postmenopausal bone loss in a dependent at low to moderate levels of soy-protein con- number of clinical trials. When metabolized, ipri¯avone sumption (Karr et al, 1997). Equol is a metabolite of partly breaks down to daidzein, which can be detected in daidzein, but is excreted by some individuals and not by the urine after supplementation with ipri¯avone. The usual others (Setchell et al, 1984); this ®nding is reproduced in dose in these studies was 600 mg per day, often given in our study, where equol excretion was below the detection three daily doses of 200 mg. In placebo-controlled studies level in 10 subjects. Since phyto-oestrogen excretion with a duration of 1 or 2 years, this dose has been shown to depends on recent dietary intake and there may be other inhibit the loss of bone mineral density of the spine sources of within-subject variation, in this study a pooled (Valente et al, 1994; Agnusdei et al, 1997; de Aloysio et sample was prepared from yearly samples collected over a al, 1997; Gambacciani et al, 1997; Gennari et al, 1997) and 10-year period, as a better indicator of usual intake than a radius (Adami et al, 1997; Gennari et al, 1997) that was single measure. Phyto-oestrogen excretion was expressed observed in the control groups. Several studies (Agnusdei per gram creatinine and not per 24 h, to take into account et al, 1995; de Aloysio et al, 1997; Gambacciani et al, possible incomplete urine collection by some subjects and 1997) have also evaluated the effect of ipri¯avone in evaporation of part of the samples during storage. The urine combination with low-dose conjugated oestrogens. Low samples in which the phyto-oestrogens were determined doses of oral conjugated oestrogens (0.3 mg/day) can had been stored at 7 20C for 6 ± 16 years. There is little counteract neurovegetative menopausal symptoms, but information about the stability of these compounds over higher doses (0.625 mg/day) are required to prevent bone such a period. However, it is unlikely that any deterioration loss in postmenopausal women. The combination of of phyto-oestrogens would differ between the groups with 600 mg ipri¯avone and 0.3 mg oral conjugated oestrogens either low or high rates of bone loss. Loss during storage (Agnusdei et al, 1995) or 25 mg 17-b-oestradiol transder- could therefore not explain our ®ndings of a difference in mally per day (de Aloysio et al, 1997) has been shown equol and enterolactone levels. effective in the prevention of bone loss, observed at low Phyto-oestrogens are an example of bioactive compo- doses of oestrogen. Such a combined therapy is suggested nents in food that have been suggested to play a role in the as an approach to prevent or treat all symptoms related to prevention of a range of chronic, diet-related diseases menopause. A recent report (Gambacciani et al, 1997) (Adlercreutz & Mazur, 1997; Murkies et al, 1998). Most showed that the bone-sparing effect of 600 mg ipri¯avone of the epidemiological and experimental research so far has could also be obtained with the combined administration of focused on the potential role of iso¯avonoids and lignans in 400 mg/day of ipri¯avone and 0.3 mg/day of conjugated cancer protection via different mechanisms (Adlercreutz, oestrogens. 1995). More recently, evidence from animal studies sug- There are two reports of a relation between soy protein gests that positive effects of phytoestrogens might be and bone density. Bone loss in ovariectomized rats was expected on cardiovascular risk factors (Anthony et al, prevented by a soy-protein diet, in comparison with a casein 1996) and short-term interventions in postmenopausal diet (Arjmandi et al, 1996). Erdman et al, (1996) have women indicate possible bene®cial effects on menopausal reported a signi®cant increase in bone mineral content and symptoms such as hot ¯ushes (Brzezinski et al, 1997; bone mineral density of the lumbar spine in postmenopausal Albertazzi et al, 1998). The potential preventive effects women receiving a daily dose of 40 g soy protein containing of phyto-oestrogens are supported by cross-cultural com- 2.25 mg iso¯avones/g protein for 6 months, in comparison parisons between Asian and Western populations, with with a control group receiving protein from casein/nonfat respect to risk of speci®c types of cancer and menopausal dry milk. Preliminary reports (Anderson et al, 1996; Blair, Bone loss in post menopausal women AFM Kardinaal et al 854 1996; Fanti et al, 1996) indicate that genistein is able to Anderson JJB, Garner SC, Ambrose WW & Ohue T (1996): Genistein and prevent bone loss in ovariectomized rats. Another prelimin- bone: studies in rat models and bone cell lines. Second International Symposium on the Role of Soy in Preventing and Treating Chronic ary report (Anderson et al, 1995) compares the effect of Disease, 1996, Brussels (abstract). genistein on the retention of cancellous bone tissue in rats, Anonymous (1993): Zo eet Nederland, 1992; resultaten van de Voedselcon- in contrast to Premarin, an oestrogen with established bone- sumptiepeiling 1992. Den Haag: Voorlichtingsbureau voor de Voeding. retaining properties, and control diets. Genistein was given Anthony MS, Clarkson TB, Hughes CL, Morgan TM & Burke GL (1996): in three doses (1.0, 3.2 and 10.0 mg/d). Genistein at the Soybean iso¯avones improve cardiovascular risk factors without affect- ing the reproductive system of peripubertal rhesus monkeys. J. Nutr. lowest dose had a similar effect to Premarin in maintaining 126, 43 ± 50. trabecular bone tissue, but at higher doses it had essentially Arjmandi BH, Alekel L, Hollis BW, Amin D, Stacewicz-Sapuntzakis M, no retentive effect on bone tissue. Guo P & Kukreja SC (1996): Dietary soybean protein prevents bone Although a preventive effect of phyto-oestrogens on loss in an ovariectomized rat model of osteoporosis. J. Nutr. 126, 161 ± 167. bone loss is biologically plausible, given their structural Beresteijn ECH van, Hof MA van 't Waard H de, Dekker PR, Neeter R, similarity to oestradiol, there are as yet no results from Winkeldermaat HJ, Visser RM, Schaafsma G, Schaik M van & human studies to support this. Our study ®nds no relation Duursma SA (1986): Design and data quality of a mixed longitudinal between genistein or daidzein excretion and the rate of study to elucidate the role of dietary calcium and phosphorus on bone cortical bone loss. This lack of association may be mineralization in pre-, peri-, and postmenopausal women. Am. J. Clin. Nutr. 43, 538 ± 548. explained by the low levels of intake, probably too low Beresteijn ECH van, van 't Hof MA, van der Heiden-Winkeldermaat JH, to compensate in any way the drastic decrease in natural ten Have Witjes A & Neeter R (1987): Evaluation of the usefulness of oestrogens after the menopause. For equol and enterolac- the cross-check dietary history method in longitudinal studies. J. Chron. tone we unexpectedly found that higher excretion is asso- Dis. 40, 1051 ± 1058. ciated with higher rate of bone loss. This is an indication Beresteijn ECH van, van 't Hof MA, Schaafsma G, de Waard H & Duursma SA (1990): Habitual dietary calcium intake and cortical bone that the group with high bone loss consumes larger quan- loss in perimenopausal women: a longitudinal study. Calcif. Tissue Int. tities of grains and legumes (including soy protein), which 47, 338 ± 344. is supported by the positive correlation between enterolac- Blair HC (1996): Action of genistein and other tyrosine kinase inhibitors in tone excretion and intake of dietary ®bre. However, the preventing osteoporosis. Second International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, 1996, Brussels positive association between equol and enterolactone and (abstract). rate of bone loss remained after adjustment for intake of Brzezinski A, Adlercreutz H, Shaoul R, RoÈsler A, Shmueli A, Tanos V & dietary ®bre. Whether this could be a causal relation can Schenker JG (1997): Short-term effect of phyto-estrogen-rich diet on only be speculated upon. However, it is more likely that postmenopausal women. Menopause 4, 89 ± 94. equol and enterolactone in this population are markers of Collaborative Group on Hormonal Factors in Breast Cancer (1997): Breast cancer and hormone replacement therapy: collaborative reanalysis of unmeasured differences in diet and lifestyle that are asso- data from 51 epidemiological studies of 52,705 women with breast ciated with differences in rate of bone loss, but could not be cancer and 108,411 women without breast cancer. Lancet 350, 1047 ± adjusted for. 1059. In conclusion, our results suggest that unsupplemented Cooper C & Barrett-Connor E (1996): Epidemiology of osteoporosis. In dietary intake of phyto-oestrogens has no preventive effect Osteoporosis 1996, Proceedings of the 1996 World Congress on Osteoporosis, Amsterdam, 1996, ed. SE Papapoulos, et al, pp 75 ± 77. on early postmenopausal cortical bone loss. No conclusions Amsterdam: Elsevier. can be drawn about effects of higher doses of phyto- Coward L, Barnes NC, Setchell KDR & Barnes S (1993): Genistein, oestrogens, either through dietary intervention or use of daidzein, and their beta-glycoside conjugatesÐ antitumor iso¯avones in supplements, or about effects on trabecular bone. soybean foods from American and Asian diets. J. Agr. Food Chem. 41, 1961 ± 1967. de Aloysio D, Gambacciani M, Altieri P, Ciaponi M, Ventura V, Mura M, Acknowledgements ÐThe authors thank RJGM Meijer for preparing the Genazzani AR & Bottiglioni F (1997): Bone density changes in pooled urine samples; Mrs M Kuilman, and Mrs JH den Breeijen, for postmenopausal women with the administration of ipri¯avone alone performing the data analysis; and Dr C Arts for discussing the manuscript. or in association with low-dose ERT. Gynecol. Endocrinol. 11, The study was supported by research grant 28-1528-1 of the Dutch 289 ± 293. Prevention Fund. Erdman JW, Stillman RJ, Lee KF & Potter SM (1996): Short-term effects of soybean iso¯avones on bone in postmenopausal women. Second International Symposium on the Role of Soy in Preventing and Treating References Chronic Disease, 1996, Brussels (abstract). Adami S, Bufalino L, Cervetti R, Di Marco C, Di Munno O, Fantasia GC, Fanti O, Faugere MC, Gang Z, Schmidt J, Cohen D & Malluche HH Serni U, Vecchiet L & Passeri M (1997): Ipri¯avone prevents radial (1996): Systematic administration of genistein partially prevents bone bone loss in postmenopausal women with low bone mass over 2 years. loss in ovariectomized rats in a non-oestrogen-like mechanism. Second Osteoporos: Int. 7, 119 ± 125. International Symposium on the Role of Soy in Preventing and Treating Adlercreutz H (1995): Phytoestrogens: epidemiology and a possible role in Chronic Disease, 1996, Brussels (abstract). cancer protection. Environ. Health Perspect. 103, Suppl.7, 103 ± 112. Fukutake M, Takahashi M, Ishida K, Kawamura H, Sugimura T & Adlercreutz H & Mazur W (1997): Phyto-oestrogens and Western dis- Wakabayashi K (1996): Quanti®cation of genistein and genistin in eases. Ann. Med. 29, 95 ± 120. soybeans and soybean products. Food Chem. Toxicol. 34, 457 ± 461. Agnusdei D, Gennari C & Bufalino L (1995): Prevention of early Gambacciani M, Ciaponi M, Cappagli B, Piaggesi L & Genazzani AR postmenopausal bone loss using low doses of conjugated estrogens (1997): Effects of combined low dose of iso¯avone derivative ipri- and the non-hormonal, bone-active drug ipri¯avone. Osteoporos. Int. 5, ¯avone and estrogen replacement on bone mineral density and meta- 462 ± 466. bolism in postmenopausal women. Maturitas 28, 75 ± 81. Agnusdei D, Crepaldi G, Isaia G, Mazzuoli G, Ortolani S, Passeri M, Gennari C, Adami S, Agnusdei D, Bufalino L, Cervetti R, Crepaldi G, Di Bufalino L & Gennari C (1997): A double-blind, placebo-controlled Marco C, Di Munno O, Fantasia L, Isaia GC, Mazzuoli GF, Ortolani S, trial of ipri¯avone for prevention of postmenopausal spinal bone loss. Passeri M, Serni U & Vecchiet L (1997): Effect of chronic treatment Calcif. Tissue Int. 61, 142 ± 147. with ipri¯avone in postmenopausal women with low bone mass. Calcif. Albertazzi P, Pansini F, Bonaccorsi G, Zanotti L, Forini E & De Aloysio D Tissue Int. 61, Suppl.1, S19 ± 22. (1998): The effect of dietary soy supplementation on hot ¯ushes. Obstet. Ingram D, Sanders K, Kolybaba M & Lopez D (1997): Case-control study Gynecol. 91, 6 ± 11. of phyto-oestrogens and breast cancer. Lancet 350, 990 ± 994. Anderson JJ, Ambrose WW & Garner SC (1995): Orally dosed genistein Karr SC, Lampe JW, Hutchins AM & Slavin JL (1997): Urinary iso- from soy and prevention of cancellous bone loss in two ovariectomized ¯avonoid excretion in humans is dose dependent at low to moderate rat models. J. Nutr. 125, Suppl., 799S (abstract). levels of soy-protein consumption. Am. J. Clin. Nutr. 66, 46 ± 51. Bone loss in post menopausal women AFM Kardinaal et al 855 Melton III LJ (1996): Global aspects of osteoporosis epidemiology. In Recker RR (1993): Current therapy for osteoporosis. J. Clin. Endocrinol. Osteoporosis 1996. Proceedings of the 1996 World Congress on Metab. 76, 14 ± 16. Osteoporosis, Amsterdam, 1996, ed. SE Papapoulos, et al, pp 79 ± 86. Setchell KDR, Borriello SP, Hulme P, Kirk DN & Axelson M (1984): Amsterdam: Elsevier. Nonsteroidal estrogens of dietary origin: possible role in hormone- Morton MS, Wilcox G, Wahlqvist ML & Grif®ths K (1994): Determina- dependent disease. Am. J. Clin. Nutr. 40, 569 ± 578. tion of lignans and iso¯avonoids in human female plasma following Thompson LU, Robb P, Serraino M & Cheung F (1991): Mammalian dietary supplementation. J. Endocrinol. 142, 251 ± 259. lignan production from various foods. Nutr. Cancer 16, 43 ± 52. Murkies AL, Wilcox G & Davis SR (1998): Clinical review 92: phyto- Valente M, Bufalino L, Castiglione GN, D'Angelo R, Mancuso A, Galoppi estrogens. J. Clin. Endocrinol. Metab. 83, 297 ± 303. P & Zichella L (1994): Effects of 1-year treatment with ipri¯avone on Ravnikar V (1987): Compliance with hormone therapy. Am. J. Obstet. bone in postmenopausal women with low bone mass. Calcif. Tissue Int. Gynecol. 156, 1332 ± 1334. 5, 377 ± 380.