Urinary isoflavone phytoestrogens in German children and adolescents--a longitudinal examination in the DONALD cohort.

SCOPE: In light of concerns about hormonally active agents, it is important to assess human exposure to such compounds, especially in children as a susceptible subgroup. Estrogenic plant constituents are present in the human diet in varying levels, in particular the isoflavones daidzein (DAI) and genistein (GEN). We aimed to examine age-dependent and secular trends in phytoestrogen exposures and to investigate equol (EQ) excretion of German children using biomarker analysis in 24-h urine samples from a longitudinally designed study. METHODS AND RESULTS: The concentrations of DAI, its metabolite EQ and GEN were determined by GC-MS analysis in 24-h urines (510 samples) collected between 1985 and 2000 in 90 (47 boys) German children (6-18 years old), who are participants in the Dortmund Nutritional and Anthropometric Longitudinally Designed study. The results from the urinary biomarker analysis indicate isoflavone exposures at quite variable levels in German children: Analyte concentrations in over 500 urine samples cover the range reported previously in adults on typical German diet and with soy intake. EQ, the DAI metabolite produced by the gastrointestinal microflora, was detected in a high fraction of all samples, with 28/90 children (31%) excreting EQ in all their urines, and 62/90 children (68%) in at least one sample. Interestingly, when multiple urines obtained from individuals at different ages (6-18 years) were analyzed, EQ formation did not appear to be a constant trait over time. When stratified by sex, DAI, EQ and GEN concentrations (ng/mL) in urines and excretion rates (µg/day) were similar in boys and girls. Total isoflavone excretion rates (µg/day) increased during childhood (6-12 years) (p=0.02) and were constant during adolescence (13-18 years) (p=0.6). No clear trend for changes in dietary isoflavone exposure over the total study period was seen (p=0.7). CONCLUSIONS: In conclusion, biomarkers in urine of German children and adolescents indicate a frequent, but widely variable dietary isoflavone intake and suggest no secular increase (1985-2000) in the exposure to isoflavone phytoestrogens among German children and adolescents.

Relation of isoflavones and fiber intake in childhood to the timing of puberty.

BACKGROUND: It has been suggested that phytoestrogens and dietary fiber can affect puberty timing. OBJECTIVE: We examined whether intake of isoflavone and fiber in healthy white children before their pubertal growth spurt [age at take-off (ATO)] was associated with puberty timing. DESIGN: Multivariate regression analyses were performed in 227 DONALD (DOrtmund Nutritional and Anthropometric Longitudinally Designed) Study participants with 3-d weighed dietary records and information on potential confounders at baseline (1 and 2 y before ATO). In a subsample (n = 111), urinary isoflavones were determined in 24-h urine samples by gas chromatography-mass spectrometry analysis. Puberty timing was examined by using ATO and chronologic ages at pubertal stage 2 for breast development (B2) or gonadal development, peak height velocity (PHV), and menarche or voice break. RESULTS: Girls whose diet was in the highest dietary isoflavone tertile experienced Tanner stage 2 for breast development ap 0.7 y later and reached PHV ap 0.6 y later than did girls whose diet was in the lowest isoflavone tertile [age (95% CI) at B2: 10.7 y (10.4, 10.9 y) compared with 10.0 y ( 9.7, 10.3 y), respectively; P for trend = 0.04; age at PHV: 11.9 y (11.6, 12.2 y) compared with 11.3 y (11.0, 11.6 y), respectively; P for trend = 0.04; adjusted for body mass index z score and fiber intake]. In boys, dietary isoflavones were not associated with pubertal markers. Urinary isoflavone and dietary fiber intakes were not associated with pubertal markers. CONCLUSIONS: Girls, but not boys, with higher prepubertal isoflavone intakes appear to enter puberty at a later age. Fiber intake in this sample of healthy white girls and boys was not relevant for puberty timing.

Simultaneous determination of daidzein, equol, genistein and bisphenol A in human urine by a fast and simple method using SPE and GC-MS.

Human diet contains weakly estrogenic compounds such as daidzein (DAI) and genistein (GEN), phytoestrogens present in soy and many vegetables as well as bisphenol A (BPA), a contaminant from packing materials and plastic containers for foods and beverages. In light of concerns about hormonally active agents, biomonitoring methods are needed to assess human exposure to such compounds. A method for simultaneous determination of DAI, its metabolite equol (EQ), GEN, and BPA by GC-MS analysis was established, validated and applied to measure concentrations in human urine. Sample preparation involves enzymatic conjugate cleavage, SPE and derivatization by silylation. For GC/MS analysis, deuterated DAI and GEN and( 13)C-BPA are used as internal standards. LOD are 4, 4, 5 and 3 ng/mL urine for DAI, EQ, GEN and BPA, respectively. Interassay variations were 9% for DAI, 15% for EQ, 18% for GEN and 10% for BPA. Simple workup and accuracy of the method are suited for biomonitoring. An analysis of urine samples from 15 adults consuming typical German food revealed dietary exposure to phytoestrogens in all samples: GEN concentrations ranged between 13 and 238 ng/mL, those for DAI ranged from 12 to 356 ng/mL. More than half of the individuals excreted also the more estrogenic metabolite EQ, at levels of 8-128 ng/mL. Higher concentrations (GEN: 820, DAI: 960 and EQ: 1740 ng/mL) were measured in a 24 h urine sample upon ingestion of soy protein (50 g with 12.9 mg DAI and 25.2 mg GEN). Only urine collected after some days on strict phytoestrogen-free diet had undetectable isoflavone levels. BPA was detected in 9 of 15 urine samples ranging from 3 to 11 ng/mL, and at 55 ng/mL in one sample. In conclusion, a reliable method to determine BPA and isoflavones in urine was established and applied in a pilot study: Biomonitoring results show much higher dietary exposure to phytoestrogens than to BPA in German adults.