The prevention of osteoporosis by nutritional phytoestrogens

Simplified methods for the assessment of O-desmethylangolensin (O-DMA) and equol in human biological fluids have been developed. Currently, expensive and cumbersome GC-MS methodologies have to be used to ensure adequate sensitivity. These methods are based on time-resolved fluoroimmunoassay (TR FIA) using a europium chelate as a label. Immunoassays have the advantage of allowing a high thruput at relatively low costs with equipment that most laboratories will be able to afford. The use of these methods has a validity that goes beyond the present project. Indeed, the presence of equol rather than O-DMA in urine is considered a marker of decreased cancer risk. In the first method, after the synthesis of 4-O-carboxymethyl-O-DMA, this compound is coupled to bovine serum albumin, and then used as antigen in immunization of rabbits. The tracers with the europium chelate are synthesized using the same 4-O-derivative of the α-methyldeoxybenzoin. After enzymatic hydrolysis and ether extraction the immunoassay is carried out by time resolved fluoroimmunoassay (TR FIA). Cross-reactivity was tested with angolensin, dihydrogenistein, dihydrodaidzein, equol, 6-OH-angolensin, trans-4-OH-equol, 6-OH-O-DMA, cis-4-OH-equol and 5-OH-equol. The antiserum cross-reacted only with angolensin. This cross-reactivity seems not to influence the results, which were highly specific. The correlation coefficient between the plasma TR FIA results and the GC-MS results was high; r value was 0.985. The intra-assay coefficients of variation (CVs) for plasma O-DMA concentrations and for urine O-DMA concentrations at three different concentrations varied 2.8-7.7 and 3.0-6.0%, respectively and the inter-assay CVs varied 3.8 - 8.9 and 4.4-6.6%, respectively. The working range of the plasma and urine O-DMA assays was 0.5-512nmol/l. In the second method, after synthesis of 4-O-carboxymethylequol the compound is coupled to BSA, and then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4-O-derivative of the isoflavan. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a GC-MS method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula is used to correct the urine values making them comparable to the GC-MS results. The correlation coefficients between the TR-FIA methods and GC-MS methods were high; r values, respectively for the plasma and urine method, were 0.98 and 0.91. The intra-assay coefficient of variation (CV %) for the TR-FIA plasma and urine method at three different concentrations vary between 5.5-6.5 and 3.4-6.9, respectively. The inter-assay CV % vary between 5.4-9.7 and 7.4-7.7, respectively. The working ranges of the plasma and urine assay are 1.27-512 nmol/l and 1.9-512 nmol/l, respectively. The development of these methods has great potential for commercial exploitation and allows simplifying the implementation of studies of phytoestrogen effects in humans.

The success of IF-enriched foods is dependent on the level of acceptance of the product(s) in the target group. This study describes the acceptance of IF-enriched food products that are not on the market yet among women in five European countries: Italy, France, Finland, UK and the Netherlands. Based on focus group interviews, a questionnaire was developed. In total 2500 women 45-70 years of age from the five countries completed a self- administered questionnaire on attitudes, social influence, barriers, and claim acceptance towards IF-enriched products. Mean age of the women was 56.1 y (SD 7.2). A self-administered questionnaire was used, including 34 questions on the following themes: - Reduction of risk of osteoporosis through dietary adjustments; - Knowledge of phyto-oestrogens / isoflavones; - Attitude towards products enriched with isoflavones (IF) in general; - Preference for type of IF-enriched products; - Preference for properties of IF-enriched products; - Perceived consequences of IF-enriched products; - Personal values in reference to IF-enriched products; - Attitude towards various claims; - Personal data. Stages of change towards reduction of osteoporosis through dietary adjustment showed that most women were in precontemplation (44%) and maintenance (39%). Breakfast was the preferred time of use for IF-enriched foods (70%). According to the women the most suitable for fortification were dairy products, bread and fruit juices. Most important product properties were detailed information, good taste and available in the supermarket. 50% of the variance in intention to use isoflavone enriched food products could be explained by concerns, social influence, barriers and self-efficacy. There were several differences between countries in acceptance of IF-enriched foods. This study showed many differences in perceptions between the countries. Therefore, for product development and marketing it is important that strategies and products will be designed that take account of the cultural diversity. Whether this should be focused on international differences or cross-cultural segments needs further research.

A randomised, double blind, placebo controlled parallel multi-centre trial was conducted in The Netherlands (TNO Quality of Life, Zeist), Italy (INRAN, Rome) and France (INRA, Clermont-Ferrant) to investigate the effects of 1-year IF-enriched food/s consumption on bone mineral metabolism, bone density and on hormonal status in early postmenopausal women. 91 subjects completed the study in the Netherlands, 78 in Italy and 68 in France. Subjects were randomly allocated to a treatment group (IF+) and a control group (IF-). The IF+ group were administered a mean daily isoflavone intake of 110mg. Urine, serum and plasma samples are still available. More refined techniques could be used to evaluate biological effects of isoflavones, for example using proteomic and metabonomic techniques.

A randomised, double blind, placebo controlled parallel multi-centre trial was conducted in The Netherlands (TNO Quality of Life, Zeist), Italy (INRAN, Rome) and France (INRA, Clermont-Ferrant) to investigate the effects of 1-year IF-enriched food/s consumption on bone mineral metabolism, bone density and on hormonal status in early postmenopausal women. 91 subjects completed the study in the Netherlands, 78 in Italy and 68 in France. Subjects were randomly allocated to a treatment group (IF+) and a control group (IF-). The IF+ group were administered a mean daily isoflavone intake of 110 mg. The following SAS datasets are available: - Data1 Screening Parameters - Data2 Safety Parameters - Data3 Body weight - Data4 Adverse events - Data5 Compliance - Data6 3-Day dietary record - Data7 Short Food Questionnaire continue variables - Data7 Short Food Questionnaire consumption yes/no - Data8 Physical Activity Questionnaire - Data9 Climacteric Symptoms Questionnaire - Data10 Bone mineral density (2 separate datasets combined) - Data11 Bone markers in plasma - Data12 Hormones in serum I - Data12 Hormones in serum II - Data13 Isoflavones in plasma - Data14 Genotyping - Data15 Bone markers in urine - Data16 Isoflavones in urine nmol/24h - Data16 Urine volume and equol nmol/L - Data18 Vaginal smears The data have been analysed and reported, but can be further be exploited to analyse the effects of isoflavone supplements and of background genetic and lifestyle factors on different biological parameters in post-menopausal women.

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