This randomized, double-blind, placebo-controlled with two parallel arms was approved by the Institutional Review Board of the University of Texas Medical Branch (UTMB). Written informed consent was obtained from all subjects. The primary purpose was to study the biological effects of soy isoflavones in women, and those trial results will be reported separately. A secondary purpose was to validate riboflavin as a biomarker of adherence to daily ingestion of study pills in both placebo and active treatment arms of the study, as reported here. Trial registration: www.clinicaltrials.gov and the identifier is NCT00204490.
The study subjects were 197 premenopausal women between 30 and 42 years of age with regular monthly menstrual cycles. Four baseline visits consisted of two visits during each of two separate luteal phases not more than 6 months apart, after which 197 qualified subjects were randomized to treatment with isoflavone or placebo pills. Each placebo pill contained a 246 mg carbohydrate filler (90% maltodextrin and 10% Sethness caramel color). Each isoflavone pill contained 246 mg Nova Soy, which consisted of soy glycones (daidzin, genistin, and glycitin) and aglycones (daidzein, genistein, and glycitein) at a 9:1 molar ratio; the total aglycone equivalent amounts were 30 mg daidzein, 30 mg genistein, and 8.3 mg glycitein. Both the placebo pill and isoflavone pill also contained 15 mg riboflavin, 60 mg sorbitol, 3 mg magnesium stearate, and 676 mg dicalcium phosphate to give a final tablet weight of 1000 mg. Both pills were identical in appearance, designed by Dr. Brent Flickinger, and generously provided for this study at no cost by Archer Daniel Midland Co. (Decatur, IL).
Subjects were randomized in blocks of six using the PLAN procedure in SASã (SAS Version 9.3, SAS Institute, Inc. Cary, NC), which assured equal sizes of the study groups. All subjects, research staff, and investigators were blinded to the treatment assignments, which were known only to research pharmacists who dispensed supplies of the study pills but were not involved in other aspects of the trial. At each study visit subjects were given a three month supply of study pills in blister packs. Each blister for daily dosing contained two study pills and one prenatal vitamin pill (Rugby Prenavite Prenatal Formula, Swanson Health Products, Duluth, GA) that met the required daily intake of vitamins and minerals. Subjects were instructed to avoid vitamin supplements not provided by the study, and to take the pills contained in one blister pack daily for 5 days per week for up to 2 years.
Administration of study pills began on the second day of the menstrual bleeding following the fourth baseline visit. Study visits then occurred at 3-month intervals and between 20 to 24 days after onset of menstrual bleeding. Before each visit the subjects collected urine for 12 hours overnight in a 3 liter, light-protected container containing 1 gm sodium azide and 10 mL glycerol as preservatives. Aliquots of urine were stored at −20°C until analyzed for riboflavin by a high performance liquid chromatography (HPLC) with fluorescence detection (Chen et al., 2005 , Ramanujam et al., 2011 ), and for daidzein and genistein by a previously described gas chromatography-flame ionization detection (GC-FID) method (Lu et al., 1995 ).
All baseline and treatment phase urine samples were analyzed for riboflavin, daidzein, and genistein by one of the investigators who was blinded to treatment assignment. Riboflavin results were used in discussions with the study participants regarding their adherence to ingestion of study pills, whereas the isoflavone results remained blinded to the subjects and members of the study team until the trial ended and data were ready for analysis.
The study subjects were 197 premenopausal women between 30 and 42 years of age with regular monthly menstrual cycles. Four baseline visits consisted of two visits during each of two separate luteal phases not more than 6 months apart, after which 197 qualified subjects were randomized to treatment with isoflavone or placebo pills. Each placebo pill contained a 246 mg carbohydrate filler (90% maltodextrin and 10% Sethness caramel color). Each isoflavone pill contained 246 mg Nova Soy, which consisted of soy glycones (daidzin, genistin, and glycitin) and aglycones (daidzein, genistein, and glycitein) at a 9:1 molar ratio; the total aglycone equivalent amounts were 30 mg daidzein, 30 mg genistein, and 8.3 mg glycitein. Both the placebo pill and isoflavone pill also contained 15 mg riboflavin, 60 mg sorbitol, 3 mg magnesium stearate, and 676 mg dicalcium phosphate to give a final tablet weight of 1000 mg. Both pills were identical in appearance, designed by Dr. Brent Flickinger, and generously provided for this study at no cost by Archer Daniel Midland Co. (Decatur, IL).
Subjects were randomized in blocks of six using the PLAN procedure in SASã (SAS Version 9.3, SAS Institute, Inc. Cary, NC), which assured equal sizes of the study groups. All subjects, research staff, and investigators were blinded to the treatment assignments, which were known only to research pharmacists who dispensed supplies of the study pills but were not involved in other aspects of the trial. At each study visit subjects were given a three month supply of study pills in blister packs. Each blister for daily dosing contained two study pills and one prenatal vitamin pill (Rugby Prenavite Prenatal Formula, Swanson Health Products, Duluth, GA) that met the required daily intake of vitamins and minerals. Subjects were instructed to avoid vitamin supplements not provided by the study, and to take the pills contained in one blister pack daily for 5 days per week for up to 2 years.
Administration of study pills began on the second day of the menstrual bleeding following the fourth baseline visit. Study visits then occurred at 3-month intervals and between 20 to 24 days after onset of menstrual bleeding. Before each visit the subjects collected urine for 12 hours overnight in a 3 liter, light-protected container containing 1 gm sodium azide and 10 mL glycerol as preservatives. Aliquots of urine were stored at −20°C until analyzed for riboflavin by a high performance liquid chromatography (HPLC) with fluorescence detection (Chen et al., 2005 , Ramanujam et al., 2011 ), and for daidzein and genistein by a previously described gas chromatography-flame ionization detection (GC-FID) method (Lu et al., 1995 ).
All baseline and treatment phase urine samples were analyzed for riboflavin, daidzein, and genistein by one of the investigators who was blinded to treatment assignment. Riboflavin results were used in discussions with the study participants regarding their adherence to ingestion of study pills, whereas the isoflavone results remained blinded to the subjects and members of the study team until the trial ended and data were ready for analysis.
