This study was part of a multi-center intervention located in Frederiksberg (Denmark), Reading (UK), Rome (Italy), and Potsdam (Germany). Potential effects of isoflavones on gut satiety hormones and body weight were investigated in the German population. Thirty-six healthy postmenopausal women (age 59 ± 6 y, BMI 24.7 ± 2.8 kg/m2), defined as at least 12 months since the last menstrual cycle, were recruited by advertisement in the local media. Thirty-four of the subjects completed the intervention. One of the subjects was excluded because of a prolonged respiratory infection, the other one because of start of a treatment with an angiotensin-converting-enzyme inhibitor. None of the volunteers had used hormone replacement therapy for six months, antibiotics for three months, or isoflavone, vitamin, or mineral containing supplements for two months. All volunteers were non-smokers. Parameters of renal and liver function were within normal range. Subjects were classified as equol producers, when equol in a 24 h urine sample exceeded 936 nmol/liter during isoflavone treatment, which corresponds to an urinary equol excretion of > 0.45 mg/day . The study protocol was approved by the Ethics Committee of the University of Potsdam, Germany. All volunteers gave written informed consent prior to the study.
This was a randomized, double-blind, placebo-controlled, 2 × 8-wk crossover study, separated by an 8-wk washout period. Subjects were invited to the metabolic unit on 6 occasions (t0, t4, and t8 on each intervention arm), after 12-h overnight fasts. To exclude potential second-meal effects, a set low-fat evening meal (< 10 g fat) was consumed the evening before each of the study days. Recipes for the preparation of the meals were provided to the participants. Energy contents of the meals were comparable. Subjects were asked to consume two fruit cereal bars/d (Health & Diet Food, Manchester, UK), one in the morning and one in the afternoon, in addition to their normal diet. During the treatment period, cereal bars were enriched with 2 × 25 mg isoflavones/d, with a genistein to daidzein ratio of 2:1 ("Solgen 40", Solbar Plant Extracts, Ashdod, Israel). Thus, isoflavone intake in the treatment group of the present study was in the upper range of the daily isoflavone intake in traditional Asian diets (15 – 50 mg/d) . The product was tested before packaging and during the study by HPLC, to ensure stability of the isoflavones . Placebo did not contain any isoflavones. Each cereal bar (40 g) had an average nutrient content of energy (652 kJ); protein 2.6 g; carbohydrate 17.3 g; fat 8.5 g; fiber 1.8 g; sodium 0.012 g. Subjects perceived the isoflavone-enriched and placebo cereal bars as identical in taste and visual appearance. Habitual diet was assessed by estimated 3-d food records three times during the study. Diet diaries were completed at baseline (t0) and after 4 weeks (t4) of each intervention arm. All food records included two week days and one weekend day. Nutrient intake was calculated based on the German Food and Nutrient Data Base Bundeslebensmittelschlüssel BLS II.3 . To avoid weight gain, subjects were advised to replace snacks with the cereal bars. Subjects kept daily records of cereal bar consumption and well-being in a study diary. Dietary compliance was further assessed by measurement of phytoestrogen concentrations in 24-hour urine , which was collected at start and end of each intervention period. Body weight was measured at each visit.
Blood was collected in ice-chilled EDTA tubes for the analysis of glucose, ghrelin, and PYY. Following centrifugation at 1600 g for 10 minutes at 4°C, aliquots were immediately frozen at -20°C until assayed. All samples from individual subjects were measured in the same assay. Immunoreactive total ghrelin was measured by a commercially available radioimmunoassay (Phoenix Pharmaceuticals, Mountain View, CA, USA), as previously described . Immunoreactive total human PYY was measured by a commercially available radioimmunoassay (LINCO Research, Missouri, USA), using125I-labeled bioactive PYY as tracer and a PYY antiserum to determine the level of active PYY by the double antibody/PEG technique. The PYY antibody is raised in guinea pigs and recognizes both the PYY 1–36 and PYY 3–36 forms of human PYY. Intra- and inter-assay coefficient of variation was 5.3% and 7.0%, respectively. Insulin, and glucose, and urinary phytoestrogens (genistein, daidzein, equol) were analyzed as previously described .
Data are given as mean ± SEM, anthropometric data are given as mean ± SD. Changes from baseline, e.g. week-8 compared to week-0 (t8-t0), were used as the dependent variables. Data were calculated as changes from baseline on the original scale, when normally distributed. Skewed data where log transformed, and changes from baseline on the log scale were calculated, and these changes now correspond to a multiplicative change from baseline on the original scale. Subjects were included as a random factor within a linear mixed model. Fixed effects included in the final model were: baseline parameters, treatment, treatment order, and changes in BMI. Further exploratory investigation of equol group was included in the model. Pearson's correlation coefficient was calculated between baseline PYY and changes in PYY. Statistical analysis was performed using SAS 8.4 (SAS Institute Inc., Cary, NC).