In both studies, repeated overnight urine samples were collected during the luteal phase, aliquoted into 2 mL containers, and stored at -80°C. Ascorbic and boric acid were added to the urine collection containers to control bacterial growth. For the BEAN1 study, the baseline and the final samples were analyzed for 188 women after 8-11 years of storage. If no sample was available at month 24, the month 12 sample was used instead. For the 79 BEAN2 participants, 3 samples (baseline and at the end of low-soy [month 6] and high-soy [month 13] diets) were analyzed after 1-4 years of storage. For one woman, only months 0 and 13 samples were available for this analysis.
In both studies, the most predominant steroidal estrogens in premenopausal women,14 (link) namely E1, E2, 2-OHE1, 2-OHE2, 2-MeOE1, 4-OHE1, E3, 16keto-E2, 16α-OHE1 were measured by LCMS (model Exactive, Thermo Fisher Scientific, Waltham, MA) using 5 labeled internal standards as described in detail previously.15 (link) Ascorbic acid was added during hydrolysis and during derivatization to prevent artificial oxidation of sensitive analytes.15 (link) Analysis of an external urine pool from premenopausal women repeated on 9 different days revealed coefficients of variation of 4-21% depending on the analyte concentrations. Urinary creatinine concentrations were measured using a Roche-Cobas MiraPlus clinical chemistry autoanalyzer (Roche Diagnostics, Switzerland). All estrogen and isoflavonoid measurements were expressed per mg creatinine to adjust for urine volume.
Urinary isoflavonoids as a biomarker for soy intake were measured previously by high-pressure liquid chromatography in BEAN116 (link) and by LCMS in BEAN2.17 (link) The isoflavonoid equol was assessed by LCMS in both studies, but in BEAN1, it was measured at the same time as the estrogen metabolites in 2 urine samples only, whereas in BEAN2 equol was measured together with genistein and dadzein in 8 urine samples per woman. Since equol producers are thought to experience more protective effects of isoflavones than non-producers,20 (link) equol producer status was determined based on 2 criteria: urinary daidzein excretion ≥2 nmol/mg creatinine and a urinary equol to daidzein ratio ≥0.018.21 ;22 In BEAN1, 23 women met the criteria at least once and were considered equol producers: 7 were of Asian ethnicity and 16 were non-Asian. In BEAN2, 41 women were equol producers, of which 10 were of Asian ethnicity.
In both studies, the most predominant steroidal estrogens in premenopausal women,14 (link) namely E1, E2, 2-OHE1, 2-OHE2, 2-MeOE1, 4-OHE1, E3, 16keto-E2, 16α-OHE1 were measured by LCMS (model Exactive, Thermo Fisher Scientific, Waltham, MA) using 5 labeled internal standards as described in detail previously.15 (link) Ascorbic acid was added during hydrolysis and during derivatization to prevent artificial oxidation of sensitive analytes.15 (link) Analysis of an external urine pool from premenopausal women repeated on 9 different days revealed coefficients of variation of 4-21% depending on the analyte concentrations. Urinary creatinine concentrations were measured using a Roche-Cobas MiraPlus clinical chemistry autoanalyzer (Roche Diagnostics, Switzerland). All estrogen and isoflavonoid measurements were expressed per mg creatinine to adjust for urine volume.
Urinary isoflavonoids as a biomarker for soy intake were measured previously by high-pressure liquid chromatography in BEAN116 (link) and by LCMS in BEAN2.17 (link) The isoflavonoid equol was assessed by LCMS in both studies, but in BEAN1, it was measured at the same time as the estrogen metabolites in 2 urine samples only, whereas in BEAN2 equol was measured together with genistein and dadzein in 8 urine samples per woman. Since equol producers are thought to experience more protective effects of isoflavones than non-producers,20 (link) equol producer status was determined based on 2 criteria: urinary daidzein excretion ≥2 nmol/mg creatinine and a urinary equol to daidzein ratio ≥0.018.21 ;22 In BEAN1, 23 women met the criteria at least once and were considered equol producers: 7 were of Asian ethnicity and 16 were non-Asian. In BEAN2, 41 women were equol producers, of which 10 were of Asian ethnicity.
