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The mass ratio of urinary 2-hydroxyestrone to 16-α-hydroxyestrone (2:16) is hypothesized as a biomarker of breast cancer risk in premenopausal women, with higher ratios being theoretically protective. Cruciferous vegetable intake has been associated with higher urinary 2:16 in some studies. We investigated whether a whole-food supplement made from dried Brussels sprouts and kale would increase urinary 2:16 in comparison with placebo or cruciferous vegetables in women. This randomized, parallel arm, placebo-controlled, partly blinded study included 78 healthy premenopausal women (38-50 y) with screening urinary 2:16 ≤3.0. Subjects received either six capsules containing 550 mg dried Brussels sprouts and kale per capsule, 40 g daily alternating broccoli or Brussels sprouts, or placebo for eight weeks. Urinary 2:16 and creatinine were measured at baseline, four, and eight weeks. Intent-to-treat repeated measures-ANOVA with multiple imputation ( n =100) for missing values identified no treatment effect ( P =0.9) or treatment-by-time interaction ( P =0.6); however, a significant time effect was noted ( P =0.02). Per-protocol analyses including complete cases found no treatment effect ( P =1) or treatment-by-time interaction ( P =0.6); however, the significant time effect remained ( P =0.03). Restricting analysis to subjects with >80% compliance maintained the time effect ( P =0.02). Using Pearson correlations, android-pattern and android:gynoid fat were predictive of change ( P ≤0.05). In conclusion, neither cruciferous supplements nor an added vegetable serving altered urinary 2:16 in premenopausal women with eight weeks treatment. This ratio did vary with time, which is important for designing future trials.

Background: Prostate cancer (PCa) is the most common cancer in North American men. Beyond the established contribution of androgens to disease progression, growing evidence suggest that oestrogen-related pathways might also be of clinical importance. The aim of this study was to explore the association of urinary oestrogen levels with clinical outcomes., Methods: Urine samples from the prospective multi-institutional PROCURE cohort were collected before RP for discovery (n = 259) and validation (n = 253). Urinary total oestrogens (unconjugated + conjugated), including oestrone and oestradiol, their bioactive and inactive catechol and methyl derivatives (n = 15), were measured using mass spectrometry (MS)., Results: The median follow-up time for the discovery and replication cohorts was 7.6 and 6.5 years, respectively. Highly significant correlations between urinary oestrogens were observed; however, correlations with circulating oestrogens were modest. Our findings indicate that higher levels of urinary oestriol and 16-ketoestradiol were associated with lower risk of BCR. In contrast, higher levels of 2-methoxyestrone were associated with an increased risk of development of metastasis/deaths., Conclusions: Our data suggest that urinary levels of oestriol and 16-ketoestradiol metabolites are associated with a more favourable outcome, whereas those of 2-methoxyestrone are associated with an elevated risk of metastasis after RP. Further studies are required to better understand the impact of oestrogens on disease biology and as easily accessible urine-based risk-stratification markers.

Obesity is a common comorbidity for pulmonary arterial hypertension (PAH). Additionally, oestrogen and its metabolites are risk factors for the development of PAH. Visceral adipose tissue (VAT) is a major site of oestrogen production; however, the influence of obesity-induced changes in oestrogen synthesis and metabolism on the development of PAH is unclear. To address this we investigated the effects of inhibiting oestrogen synthesis and metabolism on the development of pulmonary hypertension in male and female obese mice.We depleted endogenous oestrogen in leptin-deficient ( ob / ob ) mice with the oestrogen inhibitor anastrozole (ANA) and determined the effects on the development of pulmonary hypertension, plasma oestradiol and urinary 16α-hydroxyestrone (16αOHE1). Oestrogen metabolism through cytochrome P450 1B1 (CYP1B1) was inhibited with 2,2',4,6'-tetramethoxystilbene (TMS). ob / ob mice spontaneously develop pulmonary hypertension, pulmonary vascular remodelling and increased reactive oxygen species production in the lung; these effects were attenuated by ANA. Oestradiol levels were decreased in obese male mice; however, VAT CYP1B1 and 16αOHE1 levels were increased. TMS also attenuated pulmonary hypertension in male ob / ob mice. Intra-thoracic fat from ob / ob mice and VAT conditioned media produce 16αOHE1 and can contribute to oxidative stress, effects that are attenuated by both ANA and TMS.Obesity can induce pulmonary hypertension and changes in oestrogen metabolism, resulting in increased production of 16αOHE1 from VAT that contributes to oxidative stress. Oestrogen inhibitors are now in clinical trials for PAH. This study has translational consequences as it suggests that oestrogen inhibitors may be especially beneficial in treating obese individuals with PAH., Competing Interests: Conflict of interest: K.M. Mair has nothing to disclose. Conflict of interest: K.Y. Harvey has nothing to disclose. Conflict of interest: A.D. Henry has nothing to disclose. Conflict of interest: D.Z. Hillyard has nothing to disclose. Conflict of interest: M. Nilsen has nothing to disclose. Conflict of interest: M.R. MacLean has nothing to disclose., (Copyright ©ERS 2019.)

Purpose: Diindolylmethane (DIM), a bioactive metabolite of indole-3-carbinol found in cruciferous vegetables, has proposed cancer chemoprevention activity in the breast. There is limited evidence of clinically relevant activity of DIM or long-term safety data of its regular use. A randomized, double-blind, placebo-controlled trial was conducted to determine the activity and safety of combined use of BioResponse DIM® (BR-DIM) with tamoxifen., Methods: Women prescribed tamoxifen (n = 130) were randomly assigned oral BR-DIM at 150 mg twice daily or placebo, for 12 months. The primary study endpoint was change in urinary 2/16α-hydroxyestrone (2/16α-OHE1) ratio. Changes in 4-hydroxyestrone (4-OHE1), serum estrogens, sex hormone-binding globulin (SHBG), breast density, and tamoxifen metabolites were assessed., Results: Ninety-eight women (51 placebo, 47 DIM) completed intervention; compliance with treatment was >91%. BR-DIM increased the 2/16α-OHE1 ratio (+3.2 [0.8, 8.4]) compared to placebo (-0.7 [-1.7, 0.8], P < 0.001). Serum SHBG increased with BR-DIM compared to placebo (+25 ± 22 and +1.1 ± 19 nmol/L, respectively). No change in breast density measured by mammography or by MRI was observed. Plasma tamoxifen metabolites (endoxifen, 4-OH tamoxifen, and N-desmethyl-tamoxifen) were reduced in women receiving BR-DIM versus placebo (P < 0.001). Minimal adverse events were reported and did not differ by treatment arm., Conclusion: In patients taking tamoxifen for breast cancer, daily BR-DIM promoted favorable changes in estrogen metabolism and circulating levels of SHBG. Further research is warranted to determine whether BR-DIM associated decreases in tamoxifen metabolites, including effects on endoxifen levels, attenuates the clinical benefit of tamoxifen., Trial Registration: ClinicalTrials.gov NCT01391689.

Hormonal exposures are known to influence breast cancer risk among women with a BRCA1 mutation. Thus, dietary factors that increase the 2-hydroxyestrone (OHE):16α-OHE ratio, a biomarker inversely related to breast cancer development, may also influence cancer risk. We conducted a dietary intervention study to evaluate the ability of 300 mg/day of 3,3'-diindolylmethane (DIM) to increase the urinary 2:16α-OHE ratio in 20 women with a BRCA1 mutation. BRCA1 mutation carriers (n = 15) were assigned to receive 300 mg/day of Rx Balance BioREsponse DIM for 4-6 weeks (intervention group) and five BRCA1 mutation carriers did not take DIM (control group). The urinary 2:16α-OHE ratio was assessed at baseline and after 4-6 weeks by immunoassay. There was no significant effect of DIM on the 2:16α-OHE ratio (2.4 at baseline vs. 3.0 after the intervention, P = 0.35). A short dietary intervention with DIM did not significantly increase the 2:16α-OHE ratio in female BRCA1 mutation carriers. Larger studies investigating the effect of dietary or lifestyle interventions on circulating hormone levels in these high-risk women are warranted.

Purpose: Two main estradiol metabolites have different biological behavior with tumorigenic features of 16-OHE1 and antiproliferative characteristics of 2-OHE1. We investigated the ratio of these estradiol metabolites in pre- and postmenopausal patients with breast cancer (BC) within two case-control studies., Methods: From 41 premenopausal patients with (cases) and without (controls N = 211) BC and 207 postmenopausal patients with and without BC (N = 206), urine samples were collected. Urine samples were collected prior to surgery and stored at -20 °C until measurement by ELISA. The multiple linear regression test with two interactions was performed to evaluate the influence of different factors on the metabolic ratio., Results: In premenopausal patients, log ratio of 2-OHE1/16-OHE1 was 0.25 (CI 0.20;0.29) and 0.21 (CI 0.11;0.31) for controls and cases without significant difference. In postmenopausal patients, log ratio was 0.22 (CI 0.17;0.26) and 0.11 (CI 0.07;0.15) in controls and cases, respectively, and was statistically significantly lower (p = 0.0002). Log ratio was significantly influenced by BMI, but only in postmenopausal patients, an increased BMI resulted in a significantly (p < 0.042) decreased ratio., Conclusions: Our case control studies suggest that in postmenopausal women a different metabolism of estrogens may play a role in the tumorigenesis of breast cancer. This genetically determined metabolism could be influenced by the exogenic factor BMI. In premenopausal women different hormone levels at different time points of the menstrual cycle may be an explanation that why we could not find an influence of estrogen metabolism.

Introduction: Variations in inflammatory markers have been reported in adult women during the luteal phase, but whether these findings are observed during adolescence is unknown. We postulate that higher ultrasensitive C-reactive protein (usCRP) and lower 2-hydroxyestrone (2OHE) levels, an estrogen metabolite with cardioprotective actions, are present during the luteal phase in young women., Aim: To evaluate usCRP levels during the menstrual cycle and to determine its association with 2OHE and 16α-hydroxyestrone (16OHE) in adolescents., Methods: Healthy postmenarcheal adolescents (N = 37) were studied during one menstrual cycle in follicular phase (FP) and luteal phase-like period (LP-L)., Results: Elevations in usCRP levels in the LP-L were observed in the entire group and in anovulatory cycles (1.9 ± 1.1 mg/L in FP to 2.5 ± 1.8 mg/L in LP-L; p < 0.0001). Increases in estrone, estradiol, free and bioavailable estradiol, testosterone, usCRP and 2OHE levels were observed in LP-L compared with FP (p < 0.01), with a borderline elevation in IFG-I levels (p = 0.06)., Conclusions: We report an elevation of usCRP and 2OHE levels during the luteal phase in healthy adolescents. Elevations of this inflammatory marker in anovulatory adolescents without an increase in 2OHE may play a role in metabolic risks associated with chronic anovulation.

Unlabelled: The urinary ratio 2-hydroxyoestrone/16-hydroxyoestrone (URME), has been proposed in various populations on the world as a risk indicator for breast cancer (BC), however in the Mexican population has never been determined., Objective: To determine URME Mexican women and establish its relationship with risk factors for BC., Material and Methods: Cross-sectional study of 142 premenopausal and 42 posmenopausal women. The URME was determined with the kit ESTRAMETTM and was related to risk factors for BC. Correlations and linear regressions were performed., Results: The median URME was 0.90 (RIQ 0.64-1.18). The body mass index (BMI) and early menarche contribute 5.4% of their variability (F=5.17; p., (Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.)

Background: Mammographic density is a strong predictor of breast cancer risk. The total amount and the metabolism of endogenous estrogens, e.g., the ratio of 2-hydroxyestrone (2-OHE(1)) and 16α-OHE(1) may influence breast cancer risk. This study examined the association of urinary estrogen metabolites with breast density in premenopausal women., Methods: Urine samples were collected at baseline and after 2 years, analyzed for 11 estrogen metabolites plus progesterone and testosterone by liquid chromatography mass spectrometry, and adjusted for creatinine levels. Mixed-effects regression was applied to examine the association of estrogens with breast density., Results: Total estrogen metabolites (181 ± 113 vs. 247 ± 165 pmol/mg creatinine, p=0.01) and the 2/16α-OH ratio (8.4 ± 10.4 vs. 13.0 ± 17.1, p=0.02) were lower in the 74 Asian than in the 114 non-Asian women. In adjusted models, positive associations of total estrogen metabolites (p=0.002) and the 2/16α-OHE(1) ratio (p=0.08) with percent density were detected in Asians only. In all women, mammographic density was positively associated with the 2-OH pathway (p=0.01), inversely related to the 16α-OH pathway (p=0.01), and not associated with the 4-OH pathway, testosterone, and progesterone. Results for the size of the dense area weakly reflected the findings for percent density, while associations with the non-dense area were in the opposite direction., Conclusions: The findings that the 2-OH pathway is associated with higher and the 16α-OH pathway with lower breast density contradicts the hypothesized risk profile of these metabolites, but, if a relation between estrogen metabolites and breast cancer risk exists, it may be mediated through pathways other than mammographic density., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: The objective of the study was to evaluate in premenopausal women the relationships of physically active and sedentary behaviors reported for adulthood and adolescence with a comprehensive profile of estrogen metabolism., Methodology: Fifteen estrogens and estrogen metabolites (jointly termed EM) were measured using liquid chromatography-tandem mass spectrometry in luteal phase urines from 603 premenopausal women in the Nurses' Health Study II. Geometric means of individual EM, metabolic pathway groups, and pathway ratios were examined by level of exposure after adjustment for age, body mass index, alcohol intake, menstrual cycle length, and sample collection timing., Results: High overall physical activity in adulthood (42+ metabolic equivalent h/wk vs. <3 metabolic equivalent h/wk) was associated with a 15% lower level of urinary estradiol (Ptrend=0.03) and 15% lower level of 16-hydroxylation pathway EM (Ptrend=0.03). Levels of 2- and 4-hydroxylation pathway EM did not differ significantly by physical activity. High overall activity was also positively associated with four ratios: 2-pathway EM to parent estrogens (Ptrend=0.05), 2-pathway catechols to parent estrogens (Ptrend=0.03), 2-pathway catechols to methylated 2-pathway catechols (Ptrend<0.01), and 2-hydroxyestrone to 16α-hydroxyestrone (Ptrend=0.01). Similar patterns of association were noted for walking and vigorous physical activity, but there was little evidence of associations with sedentary behaviors or activity during adolescence., Conclusions: High levels of physical activity were associated with lower levels of parent estrogens and 16-hydroxylation pathway EM and preferential metabolism to 2-pathway catechols. The results of our analysis, the largest, most comprehensive examination of physical activity and estrogen metabolism to date, may be useful in future studies investigating the etiology of diseases linked to both physical activity and endogenous estrogen.

Background: Soy consumption may protect against breast cancer through modification of estrogen metabolism., Objectives: We examined the effect of soy foods on urinary estrogens and the 2-hydroxy (OH)/16α-OH estrone (E(1)) ratio in two dietary interventions with premenopausal women., Subjects/methods: The Breast, Estrogens, And Nutrition (BEAN1) study was a 2-year randomized trial and BEAN2 a 13-month randomized crossover study. In both interventions, study participants consumed a high-soy diet with 2 soy food servings/day and a low-soy diet with <3 servings of soy/week. Urine samples were collected at baseline and at the end of the diet periods, analyzed for nine estrogen metabolites by liquid chromatography mass spectrometry, and adjusted for creatinine levels. For BEAN1, two samples for 188 participants and for BEAN2, three samples for 79 women were analyzed. We applied mixed-effects regression models with log-transformed values of estrogen metabolites and soy intake as the exposure variable., Results: In BEAN1, no effect of the high-soy diet on individual estrogen metabolites or hydroxylation pathways was observed. The median 2-OH/16α-OHE(1) ratio decreased non-significantly in the intervention group from 6.2 to 5.2 as compared with 6.8 and 7.2 in the control group (P=0.63). In BEAN2, only 4-OHE(1) was significantly lower after the high-soy diet. Interaction terms of the high-soy diet with equol producer status, ethnicity and weight status revealed no significant effect modification., Conclusions: Contrary to our hypothesis and some previous reports, the results from two well-controlled dietary interventions do not support an effect of a high-soy diet on a panel of urinary estrogen metabolites and the 2-OH/16α-OHE(1) ratio.

Menopause is associated with changes in bone, muscle and fat mass. The importance of postmenopausal estrogen metabolism in bone health has been established. However, its relationship to body composition in postmenopausal women remains undetermined. The objective of this study is to determine the association between estrogen metabolism and body composition in postmenopausal women. This is a cross sectional study of 97 postmenopausal Caucasian women, 49-80 y.o., ≥1 year from the last normal menstrual period or those who have had oophorectomy. Inactive [2-hydroxyestrone (2OHE(1))] and active [16α-hydroxyestrone (16α-OHE(1))] urinary metabolites of estrogen were measured by ELISA. The whole and regional body composition was measured by DXA. We have found that both 2OHE(1), and 2OHE(1)/16α-OHE(1) ratio were negatively correlated with % total fat, and % truncal fat but positively correlated with % total lean mass. Comparing the fat and lean parameters of body composition according to tertiles of 2OHE(1) and 2OHE(1)/16αOHE(1) ratio showed that subjects in the lowest tertiles, had the highest % total fat, and % truncal fat and the lowest % total lean mass. Multiple regression analysis also showed 2OHE(1) and calcium intake as statistically significant predictors of all body composition parameters. In conclusion, in postmenopausal women, an increase in the metabolism of estrogen towards the inactive metabolites is associated with lower body fat and higher lean mass than those with predominance of the metabolism towards the active metabolites., (Published by Elsevier Ireland Ltd.)

Two groups of breast cancer patients (53±2 years) in clinical remission receiving no specific therapy were examined: group 1, with BRCA1 gene mutations (N=11) and group 2, without mutations of this kind (N=11). The two groups did not differ by insulinemia and glycemia, insulin resistance index, blood levels of thyrotropic hormone, sex hormone-binding globulin, insulin-like growth factor-1, triglycerides, or lipoproteins. In group 1, blood estradiol level was higher. Intensive glucose-induced generation of reactive oxygen species in these patients was associated with a decrease of cholesterolemia, of the C-peptide/insulin proportion, and a trend to higher urinary excretion of 4-hydroxyestrone, one of the most genotoxic catecholestrogens. BRCA1 gene mutations in breast cancer patients were associated with signs of estrogenization and a pro-genotoxic shift in the estrogen and glucose system, which could modulate the disease course and requires correction.

One of the hypothesized protective mechanisms of soy against breast cancer involves changes in estrogen metabolism to 2-hydroxy (OH) and 16α-OH estrogens. The current analysis examined the effect of soy foods on the 2:16α-OH E(1) ratio among premenopausal women during a randomized, crossover intervention study; women were stratified by equol producer status, a characteristic thought to enhance the protective effects of soy isoflavones. The study consisted of a high-soy diet with 2 soy food servings/day and a low-soy diet with <3 servings of soy/wk for 6 mo each; estrogen metabolites were measured in 3 overnight urines (baseline and at the end of the low- and high-soy diet) using gas chromatography mass spectrometry for the 82 women who completed the study. Urinary isoflavonoids were assessed by liquid chromatography mass spectrometry. When applying mixed models, the 2:16α-OH E(1) ratio increased (P = 0.05) because of a nonsignificant decrease in 16α-OH E(1) (P = 0.21) at the end of the high-soy diet. Similar nonsignificant increases in the 2:16α-OH E(1) ratio were observed in equol producers (P = 0.13) and nonproducers (P = 0.23). These findings suggest a beneficial influence of soy foods on estrogen metabolism regardless of equol producer status.

Background: To evaluate the metabolic changes in urinary steroids in pre- and post-menopausal women and men with papillary thyroid carcinoma (PTC)., Methods: Quantitative steroid profiling combined with gas chromatography-mass spectrometry was used to measure the urinary concentrations of 84 steroids in both pre- (n = 21, age: 36.95 ± 7.19 yr) and post-menopausal female (n = 19, age: 52.79 ± 7.66 yr), and male (n = 16, age: 41.88 ± 8.48 yr) patients with PTC. After comparing the quantitative data of the patients with their corresponding controls (pre-menopause women: n = 24, age: 33.21 ± 10.48 yr, post-menopause women: n = 16, age: 49.67 ± 8.94 yr, male: n = 20, age: 42.75 ± 4.22 yr), the levels of steroids in the patients were normalized to the mean concentration of the controls to exclude gender and menopausal variations., Results: Many urinary steroids were up-regulated in all PTC patients compared to the controls. Among them, the levels of three active androgens, androstenedione, androstenediol and 16α-hydroxy DHEA, were significantly higher in the pre-menopausal women and men with PTC. The corticoid levels were increased slightly in the PTC men, while progestins were not altered in the post-menopausal PTC women. Estrogens were up-regulated in all PTC patients but 2-hydroxyestrone and 2-hydroxy-17β-estradiol were remarkably changed in both pre-menopausal women and men with PTC. For both menopausal and gender differences, the 2-hydroxylation, 4-hydroxylation, 2-methoxylation, and 4-methoxylation of estrogens and 16α-hydroxylation of DHEA were differentiated between pre- and post-menopausal PTC women (P < 0.001). In particular, the metabolic ratio of 2-hydroxyestrone to 2-hydroxy-17β-estradiol, which could reveal the enzyme activity of 17β-hydroxysteroid dehydrogenase, showed gender differences in PTC patients (P < 1 × 10-7)., Conclusions: These results are expected be helpful for better understanding the pathogenic differences in PTC according to gender and menopausal conditions.

Flaxseed is a rich source of dietary lignans. It has been hypothesized that lignans may decrease breast cancer risk through modulation of endogenous hormone levels. The aim of this study was to determine the effect of flaxseed supplementation on urinary levels of estrogen metabolites that may be involved in the development of breast cancer. Forty-three postmenopausal women participated in this 12-wk preintervention-postintervention study. Participants consumed 7.5 g/day of ground flaxseed for 6 wk, followed by 15 g/day for an additional 6 wk. The mean urinary level of 16alpha -hydroxyestrone (16alpha -OHE1) was higher at the end of 12 wk compared to baseline (change of 1.32 ug/day, P = 0.02). There was no significant change in 2-OHE1 excretion. The mean urinary level of the 2-OHE1/16alpha -OHE1 ratio was lower at the end of 12 wk compared to baseline (change of -1.1, P = 0.02). Mean urinary excretion of 2-methoxyestradiol was also lower at 12 wk than at baseline (P = 0.03). Based on the current paradigm of the effects of estrogen metabolism on breast cancer risk, the regimen of dietary flaxseed intake used in this study did not appear to favorably alter breast cancer risk through shifts in estrogen metabolism pathways in postmenopausal women.

Objective: To investigate prostate cancer (Pca) risk in relation to estrogen metabolism, expressed as urinary 2-hydroxyestrone (2-OHE1), 16alpha-hydroxyestrone (16alpha-OHE1) and 2-OHE1 to 16alpha-OHE1 ratio., Methods: We conducted a case-control study within the Western New York Health Cohort Study (WNYHCS) from 1996 to 2001. From January 2003 through September 2004, we completed the re-call and follow-up of 1092 cohort participants. Cases (n = 26) and controls (n = 110) were matched on age, race and recruitment period according to a 1:4 ratio. We used the unconditional logistic regression to compute crude and adjusted odds ratios (OR) and 95% confident interval (CI) of Pca in relation to 2-OHE1, 16alphaOHE1 and 2-OHE1 to 16alpha-OHE1 by tertiles of urine concentrations (stored in a biorepository for an average of 4 years). We identified age, race, education and body mass index as covariates. We also conducted a systematic review of the literature which revealed no additional studies, but we pooled the results from this study with those from a previously conducted case-control study using the DerSimonian-Laird random effects method., Results: We observed a non-significant risk reduction in the highest tertile of 2-OHE1 (OR 0.72, 95% CI 0.25-2.10). Conversely, the odds in the highest tertile of 16alpha-OHE1 showed a non-significant risk increase (OR 1.76 95% CI 0.62-4.98). There was a suggestion of reduced Pca risk for men in the highest tertile of 2-OHE1 to 16alpha-OHE1 ratio (OR 0.56, 95% CI 0.19-1.68). The pooled estimates confirmed the association between an increased Pca risk and higher urinary levels of 16alpha-OHE1 (third vs. first tertile: OR 1.82, 95% CI 1.09-3.05) and the protective effect of a higher 2-OHE 1 to 16alpha-OHE1 ratio (third vs. first tertile: OR 0.53, 95% CI 0.31-0.90)., Conclusion: Our study and the pooled results provide evidence for a differential role of the estrogen hydroxylation pathway in Pca development and encourage further study.

Objective: This study explored whether average urinary estrogen metabolites in breast cancer high-risk women can be used to identify a subgroup of women at particularly high risk to develop breast cancer, to which prevention strategies should be addressed., Methods: The population consisted of 77 high-risk women, 30 breast cancer patients and 41 controls. All subjects answered a standardized questionnaire; height and weight and spot urine samples were also obtained. Urine hydroxyestrogen metabolites were measured in triplicate by enzyme immunoassay, and the estrogen metabolite ratios for each individual were calculated., Results: The 2:16 OHE ratio (2-hydroxyestrone/16-alpha-hydroxyestrone) in women at high risk for breast cancer was similar to that observed in the breast cancer group (1.76 +/- 2.33 versus 1.29 +/- 0.80) and lower than in controls (2.47 +/- 1.14; P = 0.00). At the multivariate linear regression model, the 2:16 OHE ratio was significantly associated with diagnosis (P = 0.000 for both the high risk and breast cancer group versus the controls) and body mass index (P = 0.005), but not with age (P = 0.604), or smoking history (P = 0.478)., Conclusions: This study suggests that lower urinary 2:16 OHE ratios are predictors of breast cancer risk. Profiling estrogen metabolites may identify women who are more probably to develop breast cancer within a population of women with known risk factors and may help to further elucidate the clinical relevance of urinary 2:16 OHE ratios as clinical markers and prognostic indicators in this population.

Background: Mammographic breast density is an established marker of breast cancer risk, and is hormonally sensitive. Studies suggest that production of the daidzein metabolites equol and O-Desmethylangolensin (ODMA) may be associated with hormones and hormonally mediated factors, but few studies have assessed relationships between the capacity to produce these metabolites and breast density., Objective: To evaluate the relationship between equol- and ODMA-producer phenotypes and breast density in premenopausal women in the United States., Design: Two hundred and three women attended a clinic visit and 200 provided a urine sample following a 3 day soy challenge. Samples were analyzed for isoflavones by GC-MS to determine daidzein-metabolizing phenotypes. Percent density on recent (<14 month prior to their clinic visit) mammograms was assessed by one reader using a computer-assisted method. Multiple regression analysis was used to assess relationships between the production of equol and ODMA and breast density. Results 55(27.5%) and 182(91%) women were classed as equol- and ODMA-producers (>87.5 ng/ml urine), respectively. In unadjusted and adjusted analyses, there were no differences in breast density between producers and non-producers of either equol or ODMA (P > 0.05)., Conclusion: In this population of low-soy consuming premenopausal women, there were no associations between daidzein-metabolizing phenotypes and breast density, suggesting that these phenotypes per se do not influence premenopausal breast density.

Catechol-O-methyltransferase (COMT) enzyme catalyzes the methylation of the 2- or 4-hydroxyestrogens to 2- or 4-methoxyestrogens. Both the hydroxyestrogens and methoxyestrogens have been shown to block or enhance the effects of estrogen respectively. Our objective was to investigate the potential role of COMT in parturition and cervical ripening using a rat model. Immunohistochemistry was conducted to detect and localize the COMT protein in rat uterine tissues during pregnancy. We measured the longitudinal changes in urinary 2-hydroxyestrogen before, during, and after pregnancy in rats. Animal studies were conducted to determine the effect of treatment with a selective COMT inhibitor on (1) mifepristone-induced preterm birth and (2) cervical resistance to stretch in pregnant rats. The intensity of staining for the COMT protein differed within the luminal epithelium, uterine gland epithelium, endometrium, and myometrium during pregnancy. Levels of staining for the COMT protein in rat myometrium were highest on day 1 and lowest on days 8 and 13, but high levels returned by days 16 and 19 of pregnancy. The levels of urinary 2-hydroxyestrogen gradually increased in the first 2 weeks of pregnancy, peaked from days 16 to 18 of pregnancy, and then gradually returned to pre-pregnancy levels after delivery. The percentage of pups retained in the uterus of pregnant rats treated with both mifepristone and COMT inhibitor (48 +/- 15%) was significantly higher (P < 0.05) when compared with the value of pregnant rats treated with mifepristone alone (12 +/- 4%). The resistance to stretch was significantly higher (P < 0.05) in cervical tissues from the pregnant rats treated with COMT inhibitor (0.28) when compared with cervical tissues taken from rats treated with vehicle control (0.18). Modulation of COMT activity may play a role in the regulation of myometrial contractility and cervical ripening during pregnancy.

Specific estrogen metabolites may initiate and promote hormone-related cancers. In epidemiological studies, significantly lower excretion of urinary estradiol (E2) and lower ratio of urinary 2-hydroxy estrogens to 16alpha-hydroxyestrone (2:16 OH-E1) have been reported in prostate cancer cases compared to controls. Although soy supplementation has been shown to increase the ratio 2:16 OH-E1 in women, no studies to our knowledge have investigated the effects of soy supplementation on estrogen metabolism in men. The objective of this randomized controlled trial was to determine the effects of soy protein isolate consumption on estrogen metabolism in men at high risk for developing advanced prostate cancer. Fifty-eight men supplemented their habitual diets with 1 of 3 protein isolates: 1) isoflavone-rich soy protein isolate (SPI+) (107 mg isoflavones/d); 2) alcohol-washed soy protein isolate (SPI-) (<6 mg isoflavones/d); or 3) milk protein isolate (MPI), each providing 40 g protein/d. At 0, 3, and 6 mo of supplementation, the urinary estrogen metabolite profile was measured by GC-MS. Both soy groups had higher E2 excretion than the MPI group at 3 and 6 mo. After 6 mo of supplementation, the SPI+ group had a significantly higher urinary 2:16 OH-E1 ratio than the MPI group. Increased urinary E2 excretion and 2:16 OH-E1 ratio in men consuming soy protein isolate are consistent with studies in postmenopausal women and suggest that soy consumption may be beneficial in men at high risk of progressing to advanced prostate cancer as a result of effects on endogenous estrogen metabolism.

Objective: To evaluate the effects of soygerm isoflavones extracts on blood lipoproteins, antioxidative capacity and urinary estrogen metabolites in postmenopausal women who receive hormone therapy (HT)., Method: Thirty-nine volunteers receiving HT were recruited, and 33 completed the study. All subjects received 6 g of soygerm extracts per day for 4 weeks. Blood and urine samples were collected for study at the beginning and at the end of study., Result: Plasma HDL-C levels increased markedly with significant decreases of plasma LDL-C/HDL-C ratio and LDL-TG levels. The lag time of conjugated dienes formation prolonged for 9.9% and thiobarbituric acid reactive substances production in copper-catalyzed oxidation of LDL decreased. The differences were statistically significant. Urinary ratio of 2-OHE(1) to 16alpha-OHE(1) increased without statistical significance., Conclusion: Soygerm extracts may improve serum lipid profile in postmenopausal Taiwanese women who receive HT, and probably provide a favorable effect on estrogen metabolism.

Background: Soy isoflavones may lower breast cancer risk through altered hepatic estrogen metabolism, leading to increased urinary excretion ratios of 2-hydroxyestrone (20HE1) to 16a-hydroxyestrone (16alphaOHE1)., Materials and Methods: Urinary excretion of 20HE1/16alphaOHE1 was measured in 36 healthy, pre-menstrual women before and after ingestion of a soy-protein formula containing 120 mg of isoflavone daily for one month. Since isoflavone absorption and metabolism depends on intestinal bacteria, effects of co-administration of Lactobacillus GG (2 x 10(12)) on estrogen ratios and isoflavone excretion were studied. Urinary isoflavone excretion measurements assessed compliance., Results: Soy isoflavone ingestion induced quantitative differences in urinary excretion of estrogen metabolites and isoflavones but failed to alter 20HE1/16alphaOHE1 ratios. Co-administration of Lactobacillus GG with soy reduced excretion of total and individual isoflavones by 40% (p=0.08), without altering 2OHE1/16alphaOHE1 ratios., Conclusion: Isoflavone-rich soy protein administration alone, or with probiotic supplement, did not alter urinary excretion of estrogen metabolites in premenopausal women. However, adding concentrated probiotics may alter isoflavone bioavailability.

We examined associations between polymorphisms in genes related to estrogen metabolism (CYP1B1 codon 432G --> C rs#1056836, CYP1B1 codon 453A --> G rs#1800440, COMT codon 158G --> A rs#4680) and biosynthesis (CYP17 T --> C promoter rs#743572, CYP19 exon 4 TTTA repeat) and urinary estrogen metabolites (2-hydroxyestrogens (2-OHE), 16alpha-hydroxyestrone (16alpha-OHE1), and their ratio) in a pilot study of 64 pre- and post-menopausal women with a family history of breast cancer. Women were participants in the Metropolitan New York Registry of Breast Cancer Families, one of six international sites of the National Cancer Institute's Breast Cancer Family Registry. We used linear regression to examine the effects of genetic variants on log-transformed urinary estrogen metabolites. After adjusting for menopausal status, BMI, and age, carriers of the CYP1B1 codon 453G variant allele had 31.0% lower levels of 2-OHE (P-value = 0.05) and 40.2% lower levels of 16alpha-OHE1 (P = 0.01). Results were similar after restricting the analyses to pre-menopausal women (n = 41). Consistent with other studies, among pre-menopausal women, carriers of the COMT codon 158A variant allele had increased 2-OHE levels (P = 0.03) and an increased 2-OHE/16alpha-OHE1 ratio (P = 0.04); carriers of the CYP17 C promoter variant allele had increased 2-OHE levels (P = 0.08). To our knowledge this is the first report showing associations between the CYP1B1 codon 453G variant allele and urinary 2-OHE and 16alpha-OHE1 metabolites. Further larger studies should be conducted to confirm these results. Future identification of individuals with genetic polymorphisms that affect estrogen metabolism and biosynthesis may help characterize women at higher breast cancer risk and could guide breast cancer prevention strategies for those individuals.

When the same subjects or laboratory animals are observed across a set of different conditions or over time, we are usually interested in studying change. In these study designs, each subject serves as its own control. In this chapter, we consider different ways to assess change over time, for example, analyses for evaluating changes from a baseline condition. Study designs and analyses for single group studies and studies with two groups are discussed in detail. Examples come from published data. Statistical methods used in the examples include paired t-tests and analysis of covariance. The use of difference scores is discussed relative to analysis of covariance.

Lysozymes (3.2.1.17) from goose (Anser anser) egg white, turkey (Melagris gallopavo) egg white, phage T4 and human milk were compared with hen egg white lysozyme in their ability to clear a suspension of Micrococcus lysodeikticus. All of the lysozymes, except hen egg white lysozyme, catalysed the clearing of the Micrococcus lysodeikticus suspension in a biphasic fashion. Compared to hen egg white lysozyme, the total absorbance or transmission change over 5 and 20 minutes was less in all cases, except for human lysozyme. Human lysozyme was, therefore, a potential alternative, more rapid signal generator for the measurement of urinary estrone glucuronide excretion rates because of its structural similarity to hen egg white lysozyme. The apparent K(M) values for hen egg white lysozyme increased with the enzyme concentration.

Background: Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) react with DNA to form depurinating--N7Gua and--N3Ade adducts. This damage leads to mutations that can initiate breast and prostate cancer. To determine whether this damage occurs in humans, urine samples from men with prostate cancer and benign urological conditions, and healthy controls were analyzed. The objective was determining whether any of the cancer patients had formed the depurinating 4-OHE1(E2)-1-N3Ade adducts., Methods: The adducts were extracted from samples by using affinity columns equipped with a monoclonal antibody developed for detecting 4-OHE1(E2)-1-N3Ade adducts. Eluted extracts were separated by capillary electrophoresis with field-amplified sample stacking and/or ultraperformance liquid chromatography. Absorption/luminescence spectroscopies and mass spectrometry were used to identify the adducts., Results: 4-OHE1-1-N3Ade was detected at higher levels in samples from subjects with prostate cancer (n = 7) and benign urological conditions (n = 4) compared to healthy males (n = 5)., Conclusion: This is the first demonstration that CEQ-derived DNA adducts are present in urine samples from subjects with prostate cancer., ((c) 2006 Wiley-Liss, Inc.)

Objective: To determine if levels of endogenous estrogen or estrogen metabolites are associated with an increased risk of developing knee osteoarthritis (OA) in women., Methods: Serum estradiol (E2) and 2 urinary estrogen metabolites (2-hydroxyestrone and 16alpha-hydroxyestrone) with radiographically defined prevalent and incident knee OA in 842 white and African American women from the Southeast Michigan Arthritis Cohort., Results: The mean age and body mass index (BMI) of women in the cohort were 42.3 years and 28.5 kg/m2, respectively. Women who developed radiographically defined knee OA had significantly greater odds of having baseline endogenous early follicular phase estradiol concentrations in the lowest tertile (<47 pg/ml; odds ratio [OR] 1.88, 95% confidence interval [95% CI] 1.07-3.51) compared with those with estradiol concentrations in the middle tertile [47-77 pg/ml]), after adjustment for age, BMI, and other covariates. Women who developed knee OA also had greater odds of having baseline urinary concentrations of 2-hydroxyestrone in the lowest tertile (OR 2.9, 95% CI 1.49-5.68) compared with women with 2-hydroxyestrone concentrations in the middle tertile), after adjustment for covariates. Women who developed knee OA were more likely to have a ratio of 16alpha-hydroxyestrone to 2-hydroxyestrone in the highest tertile (>0.86; OR 1.86, 95% CI 1.01-3.44 compared with women with ratios in the 0.54-0.86 range), after adjustment for other covariates., Conclusion: There were significant associations of lower baseline serum estradiol and urinary 2-hydroxyestrone with developing knee OA in middle-aged women.

Diet and lifestyle factors, body size, and smoking behavior may influence estrogen metabolism, but the nature of these relations may vary according to race/ethnic groups. We evaluated the association of lifestyle factors with estrogen metabolites 2-hydroxyestrone (2-OHE1) and 16alpha-hydroxyestrone (16alpha-OHE1) in a racially diverse population. With a cross-sectional study design, urine samples from 1881 African-American, Caucasian, Chinese, Japanese, and Hispanic women, aged 42-52 y, from the Study of Women's Health Across the Nation (SWAN) were assayed by EIA for 2-OHE1 and 16alpha-OHE1. Dietary factors and beverages were measured using a modified Block FFQ. Dietary fiber, vegetable and fruit servings, Brassica vegetables, polyphenols, coffee, caffeine, green and black tea, and total alcohol and wine were related to metabolite values using multiple variable regression analyses. In adjusted analyses, 2-OHE1 concentrations were significantly associated with race/ethnicity, weight, smoking, and consumption of hydroxybenzoic acid, anthocyanidins, wine, and caffeine (P < 0.05). Regression models incorporating these variables explained 19-20% of the variation in 2-OHE1 concentrations. Regression models for 16alpha-OHE1, which explained 16-17% of the variability, included race/ethnicity, smoking, caffeine, total dietary fiber, and fiber from fruits and vegetables as variables. These associations may reflect why increased consumption of polyphenol-containing foods and fruit as well as decreased smoking, caffeine intake, and body size would be consistent with hypothesized benefits and risks for selected health outcomes.

Objective: To illustrate a relationship between proliferative thyroid disease and estrogen metabolism through the analysis of urinary estrogen metabolites., Study Design and Setting: Case-control study of 49 subjects with proliferative thyroid disorders and matching them to 49 controls. Urinary estrogen metabolite ratios were obtained, measuring 2-hydroxyestrone, an anti-proliferative metabolite, to 16alpha-hydroxyestrone, a proliferative metabolite. The patients were stratified into low (0 to 1.00), medium (1.01 to 2.00), or high (>2.00) groups according to their estrogen metabolite ratio., Results: Fifty-one percent (25 of 49) of the cases had a low 2/16 ratio compared to 31% (15 of 49) in the control group while 20% (10 of 49) of the control group had a high 2/16 ratio as compared to 8% (4 of 49) in the case group (P value < 0.05)., Conclusions: Increased 16alpha-hydroxyestrone activity compared to 2-hydroxyestrone activity appears to be associated with proliferative thyroid disease., Significance: Further study of estrogen metabolites in relation to proliferative thyroid disease is warranted and may lead to implications for new treatment modalities for proliferative thyroid disease., Ebm Rating: B-3b.

Background: Vegetarians are generally deficient in long-chain n-3 fatty acids. Long-chain n-3 fatty acids have a beneficial effect on plasma lipid levels, and some studies showed that they had breast cancer suppression effect. One of the biomarkers of breast cancer risk is the ratio of urinary 2-hydroxyestrone (2-OHE(1)) to 16alpha-hydroxyestrone (16alpha-OHE(1))., Objective: To investigate the effect of docosahexaenoic acid (DHA, 22:6n-3) supplementation on blood lipids, estrogen metabolism and oxidative stress in vegetarians., Design: Single-blind, randomized, placebo-controlled trial., Interventions: Twenty-seven postmenopausal vegetarian women were recruited. After a 2-week run-in period with 6 g placebo corn oil, the subjects were subsequently randomized to receive either 6 g corn oil (n=13) or 6 g DHA-rich algae oil (2.14 g of DHA/day) (n=14) for 6 weeks. Two subjects in corn oil group withdrew before completion., Main Outcome Measures: Plasma lipids, urinary 2-OHE(1) and 16alpha-OHE(1), urinary F(2)-isoprostanes and plasma alpha-tocopherol., Results: Plasma LDL-DHA and EPA level increased significantly by DHA supplementation. DHA decreased plasma cholesterol (C) levels (P=0.04), but did not influence the levels of plasma TG, LDL-C and HDL-C, alpha-tocopherol, urinary F(2)-isoprostanes, 2-OHE(1), 16alpha-OHE(1) and ratio of 2-OHE(1) to 16alpha-OHE(1) as compared to corn oil., Conclusion: DHA supplementation at a dose of 2.14 g/day for 42 days decreases plasma cholesterol but neither does it show beneficial effects on estrogen metabolism, nor does it induce deleterious effects on the observed in vivo antioxidant or oxidative stress marker in postmenopausal vegetarian women., Sponsorship: A grant (# DOH89-TD-1062) from Department of Health, Executive Yuan, Taiwan.

Background: Specific pathways involved in estrogen metabolism may play a role in the etiology of breast cancer. We used data from a large population-based case-control study to assess the association of the urinary estrogen metabolites 2-hydroxyestrone (2-OHE1), 16alpha-hydroxyestrone (16-OHE1), and their ratio (2/16) with both invasive and in situ breast cancer., Methods: Study participants from the Long Island Breast Cancer Study Project provided a spot urine specimen and completed a comprehensive interviewer-administered questionnaire. Women who used exogenous hormones or who took tamoxifen in the 6 months before urine collection were excluded from the analysis, leaving 269 invasive cases, 158 in situ cases, and 326 controls. Unconditional logistic regression was used to obtain adjusted odds ratios (ORs) for invasive and in situ breast cancer, separately, in relation to tertiles of the individual metabolites (standardized for creatinine) and the 2/16 ratio, stratified by menopausal status., Results: The OR for invasive breast cancer was inversely associated with the 2/16 ratio among premenopausal women (OR = 0.50 for extreme tertiles; 95% confidence interval = 0.25-1.01). ORs ranged from 0.32 to 0.60 when women were stratified by whether cases had received chemotherapy within 6 months before urine collection and by estrogen receptor status. In postmenopausal women, there was a slight reduction in the odds ratio for invasive cancer with high levels of the 2/16 ratio (OR = 0.78; 95% confidence interval = 0.46-1.33). Neither the individual metabolites nor the ratio were associated with in situ breast cancer., Conclusion: These data provide support for the hypothesis that the 2/16 ratio is associated with reduced breast cancer risk. The most consistent associations were observed with invasive cancer in premenopausal women.

Catechol estrogen quinones (CEQ) derived from oxidation of the catechol estrogens 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) can conjugate with glutathione (GSH), a reaction that prevents damage to DNA and can provide biomarkers of exposure to CEQs. Monoclonal antibodies (MAb) to 4-OHE1(E2)-2-N-acetylcysteine [4-OHE1(E2)-2-NAcCys] were developed and characterized by immunological and spectroscopic studies. The NAcCys conjugate is the hydrolytic product of the corresponding conjugate with GSH, followed by N-acetylation of cysteine. MAbs were produced by immunizing mice with 4-OHE1(E2)-2-NAcCys attached to an appropriate linker that was conjugated to keyhole limpet hemocyanin (KLH). Hybridoma cell lines were screened using 4-OHE1(E2)-2-NAcCys conjugated to ovalbumin (OA). There is no immunological cross-reactivity between KLH and OA. Hence, positive hybridoma cell lines secreting antibody against 4-OHE1(E2)-2-NAcCys could be rapidly identified using OA-4-OHE1(E2)-2-NAcCys. An affinity column was developed and used to purify MAb against 4-OHE1(E2)-2-NAcCys. The purified MAb was immobilized on an agarose bead column. This column was used to capture and preconcentrate the hapten of interest out of urine samples. A number of structurally related standards were used to estimate the selectivity and specificity of the chosen MAb. Capillary electrophoresis (CE) with field-amplified sample stacking in absorbance detection mode and laser-induced low temperature luminescence measurements were used to identify and quantitate the 4-OHE1(E2)-2-NAcCys conjugates and related compounds released from the affinity column. Femtomole detection limits have been demonstrated. Future prospects in clinical diagnostics for testing human exposure to CEQ by urine analysis are briefly addressed.

Background: It has been suggested that a low level of the 2-hydroxyestrogen metabolites (2-OHE) and a high level of 16alpha-hydroxyestrone (16alpha-OHE1) are associated with an enhanced risk of breast cancer. We examined the association between the metabolite levels and breast cancer in a nested case-control study, which also addressed hormone replacement therapy (HRT) and estrogen receptor status of the tumors., Methods: 24,697 postmenopausal Danish women were enrolled in the "Diet, Cancer and Health" cohort. During follow-up, 426 breast cancer cases were identified and controls were matched by age at diagnosis, baseline age, and HRT use. The concentrations of 2-OHE and 16alpha-OHE1 in spot urine were measured by an enzyme immunoassay. Incidence rate ratios (IRR) and 95% confidence intervals (95% CI) were estimated for total and estrogen receptor-specific breast cancer and were stratified according to HRT use., Results: A higher incidence of estrogen receptor-positive breast cancer with an enhanced 2-OHE level was observed among current HRT users, IRR per doubling = 1.30 (95% CI, 1.02-1.66), whereas no association was seen among nonusers of HRT, IRR per doubling = 1.00 (95% CI, 0.69-1.45). The association between estrogen receptor-positive breast cancer and the 16alpha-OHE1 metabolite level was in the opposite direction but slightly weaker and statistically insignificant. For estrogen receptor-negative breast cancer, no significant associations were seen., Conclusions: The risk of breast cancer, in particular the estrogen receptor-positive type, was enhanced among postmenopausal women using estradiol-based HRT and among those who had a high 2-OHE concentration.

Objectives: Estrogen is metabolized in the body through two mutually exclusive pathways yielding metabolites with different biological activities: the low estrogenic 2-hydroxyestrone (2-OHE1) and the highly estrogenic 16alpha-hydroxyestrone (16alpha-OHE1). The ratio of these metabolites (2/16) may be predictive of risk for developing breast cancer. Early evidence has demonstrated that exercise may alter estrogen metabolism to favor the weak estrogen, 2-OHE1., Methods: Seventy-seven eumenorrheic females completed physical activity logs for two weeks prior to providing a luteal phase urine sample. Concentrations of 2-OHE1 and 16alpha-OHE1 were measured and the 2/16 ratio computed. Hierarchical regression, controlling for age and body mass index (BMI), was used to determine relationships between estrogen metabolites and daily physical activity., Results: Regression analyses indicated significant positive relationships between physical activity and 2-OHE1 and the 2/16 ratio (p < 0.05) that appears to be independent of BMI. 16alpha-OHE1 was not significantly related to physical activity., Conclusion: These results indicate that physical activity may modulate estrogen metabolism to favor the weak estrogen, 2-OHE1, thus producing a higher 2/16 ratio. This alteration in estrogen metabolism may represent one of the mechanisms by which increased physical activity reduces breast cancer risk.

Unlabelled: Chronic physical activity may alter estrogen metabolism, a proposed biomarker of breast cancer risk, by causing a shift toward higher 2-OHE1 and lower 16alpha-OHE1 levels., Purpose: To investigate the association between an objective indicator of chronic exercise, aerobic fitness, and 2-OHE1 and 16alpha-OHE1 in premenopausal women., Methods: Women with high aerobic fitness (N=17; VO2max>or=48 mL.kg.min-1) were compared with women with average aerobic fitness (N=13; VO2max

Some epidemiologic studies reported an association between a low ratio of urinary 2-hydroxyestrogens (2-hydroxyestradiol + 2-hydroxyestrone) to 16alpha-hydroxyestrone (2:16OHE(1)) and increased breast cancer risk. Some studies show that soy consumption increases this ratio, and it is suggested that this effect may reduce breast cancer risk. We hypothesized that consumption of probiotic bacteria would alter fecal bacteria and enzymes involved in soy isoflavone metabolism, thereby increasing isoflavone bioavailability and enhancing the beneficial effects of soy on estrogen metabolism. Breast cancer survivors (n = 20) and controls (n = 20) were given 4 treatments for 6 wk each, separated by 2-wk washout periods, in a randomized, crossover design: soy protein (26.6 +/- 4.5 g protein/d containing 44.4 +/- 7.5 mg isoflavones/d); soy protein + probiotics (10(9) colony-forming units Lactobacillus acidophilus DDS(R)+1 & Bifidobacterium longum, 15-30 mg fructooligosaccharide/d); milk protein (26.6 +/- 4.5 g protein/d); and milk protein + probiotics. Survivors tended to have a lower baseline urine 2:16OHE(1) ratio than controls (P = 0.10). In the group as a whole, soy consumption tended to increase urinary 2-hydroxyestrogens (P = 0.07) and 16alpha-hydroxyestrone (P = 0.11) but had no effect on the urinary 2:16OHE(1) ratio. When subjects were divided into groups by plasma concentrations and urinary levels of the daidzein metabolite equol, soy increased urinary 2-hydroxyestrogens (P = 0.01) and the 2:16OHE(1) ratio (P = 0.04) only in subjects with high plasma equol concentrations. None of these results were influenced by probiotic consumption. These results are consistent with studies that found lower urine 2:16OHE(1) ratios in women with breast cancer and suggest that soy consumption increases this ratio only in women who are equol producers.

Cytochrome P450 (CYP) is a multigene family of enzymes involved in important life functions; some of these genes are inducible and are implicated in the oxidative metabolic activation and detoxification of many endogenous and exogenous compounds. CYP1B1 codes for an enzyme that catalyses the production of a 2- and 4-hydroxyl group in estrone and estradiol, while CYP1A1 catalyzes the 2-hydroxylation of estradiol in endometrium. The two genes were evaluated in a cohort of 150 subjects: African-American women had significantly lower 2-hydroxyl estrone/16-hydroxyl estrone (2-OHE1/16-OHE1) urinary metabolite ratios than Caucasian women (2.06+/-1.05 vs. 1.43+/-0.56; p=0.0002). A common polymorphism in the CYP1B1 gene (leucine to valineat codon 432) was associated with changes in urinary estrogen levels: both Caucasian and African-American women carrying the variant allele showed higher urinary metabolite ratios than women with the wild-type allele. No effect of the CYP1A1 MspI was observed. The 4-OHE1/2-OHE1 ratio was lower in subjects carrying the variant allele (Leu). The percentage change in 2-OHE1/16-OHE1 urinary ratio after indole treatment was significant in both Caucasian and African-American women carrying the wild-type CYP1B1 genotype, although it was more evident in African-Americans than in Caucasians. These results suggest that the Leu/Val CYP1B1 polymorphism may modify estradiol metabolism.

Introduction: Estrogen metabolites have been linked to risk of breast cancer, and we were interested in whether they are associated with prostate specific antigen (PSA) and other factors associated with prostate cancer. African-American (AA) men in South Carolina have among the highest prostate cancer rates in the world, and thus provide an ideal population in which to investigate this hypothesis., Methods: We recruited AA men attending prostate cancer screenings in and around Columbia, South Carolina. Because very few men had elevated PSAs, we restricted our study to the 77 men whose PSA was below the cutpoint used by the screening program to indicate need for diagnostic workup. These men provided spot urine samples and answered demographic and lifestyle questions including self-reported body weight, height, exercise, tobacco use, medications, cancer history and age. Levels of urinary 2-hydroxyestrone (2-OHE1) and 16alpha-hydroxyestrone (16alpha-OHE1), and their ratio (2/16) and blood PSA levels were determined., Results: After adjusting for a statistically significant interaction between age and BMI, we found a reduction of 14.2% in 2-OHE1 for each 1.0 ng/ml increase in PSA (p=0.05). For obese AA men only (BMI> or =30 kg/m2), 2-OHE1 increased by 36% for each decade of age (p=0.009)., Conclusions: Estrogen metabolites may be related to PSA level in AA men. Older men with BMIs greater than 30 kg/m2 had an unexpected increase in 2-OHE1, suggesting a dysregulation of this estrogen metabolism pathway. Further studies of estrogen metabolites may provide insights into prostate cancer risk factors.

Equol and O-desmethylangolensin (O-DMA) are products of bacterial metabolism of daidzein, an isoflavone in soybeans; thus, the presence or absence of equol and/or O-DMA in urine is a marker of particular intestinal bacteria profiles. Plasma hormone concentrations may be lower in pre-menopausal women who harbor the bacteria capable of producing equol (equol producers) compared to women who do not (equol non-producers). We evaluated concentrations of serum hormones, sex hormone-binding globulin (SHBG), and urinary 2-hydroxyestrone (2-OH E(1)) and 16alpha-hydroxyestrone (16alpha-OH E(1)) in relation to equol-producer and O-DMA-producer phenotypes in 89 post-menopausal women. Follicle stimulating hormone (FSH) was 23% greater in O-DMA-producers compared to non-producers (P = 0.04). No significant differences in serum estrogens, androgens, metabolic hormones, or SHBG were observed in relation to either daidzein-metabolizing phenotype. Compared with non-producers within each phenotype, age-adjusted 2-OH E(1):16alpha-OH E(1) was 27% greater (P = 0.06) in equol-producers and 9% greater (P > 0.10) in O-DMA-producers, and 2-OH E(1) concentrations were 24% greater in equol producers (P = 0.07) and 42% greater in O-DMA producers (P = 0.02). No significant differences in 16alpha-OH E(1) were observed in relation to either phenotype. These results suggest that interindividual variability in intestinal bacteria may be related to differences in products of hormone metabolism in post-menopausal women.

Objective: Recent clinical studies indicate that an increase in D-ring estradiol metabolites over A-ring metabolites may be a risk factor for breast cancer. The present work was aimed to investigate the effect of oral contraceptives (OC) on the endogenous estradiol metabolism in premenopausal women., Methods: Two studies were conducted, firstly comparing 2 different progestins, i.e. norethisterone and dienogest, each in combination with a constant ethinyl estradiol dosage (study A) and secondly comparing a single progestin, i.e. levonorgestrel in 2 ethinyl estradiol/progestin dosage combinations (study B). The main A- and D-ring metabolites, i.e. 2-OHE1 and 16-OHE1, were measured by enzyme immunoassay in 8-h night-urine collected before and after 3 cycles of OC administration., Results: In study A, i.e. ethinyl estradiol plus dienogest or norethisterone acetate, the ratios of 16-OHE1 to 2-OHE1 before administration were 0.62 and 0.68, and after 3 months 0.31 and 0.54, respectively. The ratio after ethinyl estradiol and dienogest was significantly lower after treatment. In study B, i.e. ethinyl estradiol plus levonorgestrel (0.03 mg/0.15 mg and 0.02 mg/0.1 mg), the ratios before treatment were 0.71 and 0.75 for the higher and the lower dosages, respectively, which changed not significantly to 0.73 and 0.71 after 3 cycles., Conclusion: OCs containing norethisterone acetate, dienogest or levonorgestrel did not have a negative effect on estradiol metabolism, i.e. they did not elicit a higher D-ring metabolism, which is considered to increase breast cancer risk.

Objective: In patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), 17beta-estradiol was thought to play a dual pro- and antiinflammatory role depending on its concentration or probably conversion to downstream mitogenic 16 alpha-hydroxyestrone or naturally occurring antiestrogens such as 2-hydroxyestrone. We compared renal excretion of these 2 types of estrogens in healthy subjects and patients with RA and SLE., Methods: In a prospective study with 30 patients with RA, 32 with SLE, and 54 healthy subjects, we measured urinary levels of 16 alpha-hydroxyestrone and 2-hydroxyestrogens by enzyme immunoassay. We studied renal excretion to estimate the time-integral of hormone production., Results: Urinary concentration and total urinary loss of 2-hydroxyestrogens was 10 times higher in healthy subjects compared to patients with either SLE or RA irrespective of prior prednisolone treatment or sex. The urinary concentration and loss of 16 alpha-hydroxyestrone did not differ between healthy subjects and patients with RA/SLE. The ratio of urinary 16 alpha-hydroxyestrone/2-hydroxyestrogens was more than 20 times higher in RA and SLE than healthy subjects irrespective of prior glucocorticoid treatment or sex., Conclusion: This study in RA and SLE patients clearly demonstrates a large shift to mitogenic estrogens in relation to endogenous antiestrogens. Both steroids are converted from the precursor 17beta-estradiol and estrone. In patients with RA and SLE, the magnitude of conversion to the mitogenic 16 alpha-hydroxyestrone is greatly upregulated, which likely contributes to maintenance of the proliferative state in these diseases.

Dietary indoles, present in Brassica plants such as cabbage, broccoli, and Brussels sprouts, have been shown to provide potential protection against hormone-dependent cancers. 3,3'-Diindolylmethane (DIM) is under study as one of the main protective indole metabolites. Postmenopausal women aged 50-70 yr from Marin County, California, with a history of early-stage breast cancer, were screened for interest and eligibility in this pilot study on the effect of absorbable DIM (BioResponse-DIM) supplements on urinary hormone metabolites. The treatment group received daily DIM (108 mg DIM/day) supplements for 30 days, and the control group received a placebo capsule daily for 30 days. Urinary metabolite analysis included 2-hydroxyestrone (2-OHE1), 16-alpha hydroxyestrone (16alpha-OHE1), DIM, estrone (El), estradiol(E2), estriol (E3), 6beta-hydroxycortisol (6beta-OHC), and cortisol in the first morning urine sample before intervention and 31 days after intervention. Nineteen women completed the study,for a total of 10 in the treatment group and 9 in the placebo group. DIM-treated subjects, relative to placebo, showed a significant increase in levels of2-OHE1 (P=0. 020), DIM (P =0. 045), and cortisol (P = 0.039), and a nonsignificant increase of 47% in the 2-OHE1/16alpha-OHE1 ratio from 1.46 to 2.14 (P=0.059). In this pilot study, DIM increased the 2-hydroxylation of estrogen urinary metabolites.

Estradiol can be metabolized to substances eliciting different, partly opposite effects even at low concentrations as shown in own investigations, e. g., regarding (anti)angiogenic actions. Specific anticancerogenic effects are ascribed to 2-hydroxyestrone and particularly 2-methoxyestradiol. In contrast, 16alpha-hydroxyestrone and the 4-hydroxyestrogens may be genotoxic under certain circumstances. Furthermore there are indications that endogenous production of proliferation-stimulating metabolites is raised in some cancers. Especially the urinary excretion of 2-hydroxyestrone to 16alpha-hydroxyestrone was investigated showing in own and other clinical studies a lower ratio in postmenopausal women with breast cancer. Research is ongoing inasfar the determination of estradiol metabolites also in blood or directly in the breast tissue by means of sensitive laboratory methods may allow predictive statements. However, it has be to consider that estradiol metabolism can be influenced by external factors such as nutrition, smoking, sports and drugs such as L-thyroxine and H2-antagonists. We were able to demonstrate that estradiol metabolism during estradiol treatment depends on the application mode and might be differently influenced by addition of the various progestins. Whether the investigation of gene polymorphism of enzymes, which are involved in estradiol metabolism, may be helpful for the assessment or treatment of risk patients, to our opinion needs further research.

Approximately one-third to one-half of individuals harbor the colonic bacteria that are capable of metabolizing the soy isoflavone daidzein to equol. Results of prior studies suggest beneficial effects of producing equol in relation to breast cancer risk, potentially through effects on endogenous hormones. High urinary excretion of 2-hydroxyestrone (2-OH E(1)) relative to 16alpha-hydroxyestrone (16alpha-OH E(1)) has been associated with a reduced risk of breast cancer. In this pilot study we examined associations between urinary excretion of equol and 2-OH E(1), 16alpha-OH E(1), and their ratio, and investigated whether excretion of these estrogen metabolites differed between two samples collected 48h apart. Isoflavones (genistein, daidzein, O-desmethylangolensin (ODMA), and equol) were measured in two overnight urines from 126 women. Excretion of 2-OH E(1) and 16alpha-OH E(1) were measured in the first overnight urine from all 126 women and in the second overnight urine from 30 of these women; there were no significant differences between samples collected 48h apart in excretion of 2-OH E(1) or 16alpha-OH E(1) (P=0.75 and 0.17, respectively). Among all women, correlations between total isoflavone excretion (sum of genistein, daidzein, ODMA, and equol) and estrogen metabolites were non-significant (P>0.05). Among women with detectable levels of equol, total isoflavone excretion was significantly positively correlated with 16alpha-OH E(1) (r=0.32, P=0.02), but was not correlated with 2-OH E(1) or 2-OH E(1):16alpha-OH E(1) ratio (r=0.21, P=0.14, and r=-0.05, P=0.70, respectively). Equol excretion (adjusted for other isoflavone excretion) was significantly positively correlated with 2-OH E(1):16alpha-OH E(1) ratio (r=0.38, P=0.005), but was not correlated with 2-OH E(1) or 16alpha-OH E(1) (r=0.15, P=0.29, and r=-0.17, P=0.24, respectively). The finding that equol excretion, but not total isoflavone excretion, correlated positively with the 2-OH E(1):16alpha-OH E(1) ratio suggests that the colonic bacterial profile associated with equol production may be involved in estrogen metabolism, and may therefore possibly influence breast cancer risk.

Whether postmenopausal hormone-replacement therapy (HRT) increases the risk of breast cancer remains controversial, despite numerous epidemiological studies. We approached the question from a biochemical rather than an epidemiological direction - we hypothesized that if estrogen administration increases the risk of breast cancer, it should also alter a known estrogen biomarker of risk towards what has been observed in patients who already have breast cancer. The specific biomarker we studied was the ratio of the urinary excretion of two principal estradiol metabolites, 2-hydroxyestrone and 16 alpha-hydroxyestrone, which is markedly decreased in women with breast cancer and women with familial risk for breast cancer. We studied 34 healthy postmenopausal women not on HRT and 19 women on HRT (Premarin 0.625 mg daily plus Provera, 2.5 mg daily, in women with a uterus and Premarin alone in women without a uterus); treatment duration ranged from 3 months to 15 years. We also studied four women with recently diagnosed, untreated breast cancer. The women with breast cancer showed a significantly lower 2-hydroxyestrone to 16 alpha-hydroxyestrone ratio than control women on HRT (1.35 +/- 0.13 vs. 2.71 +/- 0.84; p < 0.0001). There was no significant difference in the metabolite ratio between healthy women on HRT and women not on HRT (2.82 +/- 0.92 vs. 2.71 +/- 0.84). There was no significant difference between women receiving Premarin alone and women receiving Premarin plus Provera (2.46 +/- 0.84 vs. 3.13 +/- 0.90), and neither differed significantly from women not on HRT (2.71 +/- 0.84). The finding that the ratio of women on HRT was not decreased to or toward the ratio in women with breast cancer can be interpreted, we believe, as a suggestive item of biochemical evidence that HRT is not a risk for breast cancer.

Cytochrome P450 isoenzymes are known to contribute to estrone metabolism. The authors hypothesized that fluoxetine, a known inhibitor of multiple P450 isoenzymes including 3A4, 2C9, and 2D6, would affect estrone metabolism, altering the 2-hydroxyestrone:16-alpha-hydroxyestrone (2OHE1:16OHE1) ratio. In this preliminary study, four of eight recruited women with regular menstrual cycles, aged 21-37 years, completed a 24-hour urine collection prior to initiation of fluoxetine therapy and after at least 5 weeks of antidepressant treatment. In three of the four women who were nonsmokers, the 2OHE1:16OHE1 ratio was significantly higher after 5 weeks of fluoxetine therapy (pretreatment, 2.08 +/- 0.11; posttreatment, 3.50 +/- 0.46; paired Student's t-test P = 4.72, P = 0.021).

To evaluate the potential effect of estrogens in premenopausal female thyroid cancer, the concentrations of 14 estrogens were quantitatively determined in the urine of pre- and post-operative patients with thyroid papillary cancer (18 patients case, 26 approximately 54 years) and normal female subjects (20 cases, 31 approximately 52 years). The highly sensitive gas chromatography-mass spectrometry-selected ion-monitoring method was used for estrogens analysis. And an estrogen-oxidative metabolism and 16alpha-hydroxyestrone to 2-hydroxyestrone (16alpha-OH E1/2-OH E1) which is the two primary and competing site of estrogen-oxidation, were determined. Catechol estrogens, including 2-OH E1, were also increased without significant changes of the other estrogen metabolites in pre-operative patients with thyroid papillary cancer compared with normal subjects. The lowest mean value of 16alpha-OH E1/2-OH E1 was remarked in pre-operative patients, and it was significantly different from the ratio of post-operative cases. As a result, it is suggested that the increase of 2-hydroxylation in estrogen metabolism may have a significant association with female thyroid cancer.

Introduction: The products of estrogen metabolism may affect breast carcinogenesis. The 16alpha-hydroxyestrone (16-OHE) metabolite has a higher affinity for the estrogen receptor (ER) than the 2-hydroxyestrone (2-OHE) metabolite, while conjugated 2-OHE metabolite may inhibit angiogenesis. We investigated the association between the relative concentrations of these metabolites in urine (2-OHE/16-OHE) and breast cancer in a case-control study of Chinese women living in Shanghai., Methods: Incident breast cancer cases between 25 and 65 years of age (n=110) were identified from hospital or population tumor registries in Shanghai, China. Controls (n=110) were randomly selected from a complete registry of the Shanghai population, and individually matched to cases by menopausal status, age, and pre-treatment or post-treatment urine collection time. Urine samples were collected prior to any breast cancer treatment or surgery among 78 case-control pairs, while urine was collected after surgery, and perhaps other treatments, among 32 case-control pairs. A commercial enzyme-immunoassay kit was used to measure urinary estrogen metabolite concentrations. Conditional logistic regression was used to calculate odds ratios summarizing the 2-OHE/16-OHE and breast cancer association within subjects providing either pre-treatment or post-treatment urine samples., Results: Subjects with a higher urinary 2-OHE/16-OHE ratio were less likely to be diagnosed with breast cancer, but only when urine samples were collected prior to breast cancer treatment (OR(Tertile3(T3)versusTertile1(T1))=0.5, 95% CI (0.2, 1.1)). In contrast, a higher 2-OHE/16-OHE ratio was significantly associated with breast cancer among subjects providing urine specimens after treatment initiation (OR(T3versusT1)=8.7, 95% CI (1.6, 47.1)). This observed cross-over modification occurred within both pre-menopausal and post-menopausal women, and independent of body mass index or recent dietary intake., Conclusion: Cross-study differences in urine collection protocols may explain observed inconsistencies in the 2-OHE/16-OHE and breast cancer association. Our case-control analysis using pre-treatment urine samples suggested that a lower 2-OHE/16-OHE ratio was associated with an increased risk of pre-menopausal and post-menopausal breast cancer diagnosis among Chinese women.

Sex steroid concentrations in urine samples from post-menopausal women have been associated with risk of various chronic diseases. The basic requirement for the assessment of risk in such large-scale epidemiological studies is that subjects be ranked accurately by their average, long-term hormone levels. We examined the reproducibility over time of measurements of urinary testosterone (T), 5alpha-androstane-3alpha, 17beta-diol (ADIOL), estrone (E1), estradiol (E2), 2-hydroxy estrone and 2-hydroxy estradiol, (2(OH)-E), 16alpha-hydroxyestrone (16alpha(OH)-E1) and the ratio of 2(OH)-E and 16alpha(OH)-E1, in a representative sub-sample of post-menopausal women (n = 43) participating in an ongoing prospective cohort study. Women collected three first morning urine voids on different occasions, with average time difference between the first and the third urine sample of 5.1 years. T, ADIOL, E1 and E2 were measured by radio immunoassay after enzymatic hydrolysis, solid-phase extraction and HPLC purification of the samples, while 2(OH)-E and 16alpha(OH)-E1 were assayed by solid-phase enzyme immunoassay after enzymatic hydrolysis. Intra-class correlation co-efficients (ICCs) over time were very good for T (r = 0.85), acceptable for E2, E1 and ADIOL (r > 0.55), but low for 2(OH)-E, 16alpha(OH)-E1 and their ratio (r < 0.46). The adjustment for creatinine concentrations did not increase these correlations.