A UPLC-MS/MS Based Rapid, Sensitive, and Non-Enzymatic Methodology for Quantitation of Dietary Isoflavones in Biological Fluids.

Dietary isoflavones, a type of phytoestrogens, have gained importance owing to their health-promoting benefits. However, the beneficial effects of isoflavones are mediated by smaller metabolites produced with the help of gut bacteria that are known to metabolize these phytoestrogenic compounds into Daidzein and Genistein and biologically active molecules such as S-Equol. Identifying and measuring these phytoestrogens and their metabolites is an important step towards understanding the significance of diet and gut microbiota in human health and diseases. We have overcome the reported difficulties in quantitation of these isoflavones and developed a simplified, sensitive, non-enzymatic, and sulfatases-free extraction methodology. We have subsequently used this method to quantify these metabolites in the urine of mice using UPLC-MS/MS. The extraction and quantitation method was validated for precision, linearity, accuracy, recoveries, limit of detection (LOD), and limit of quantification (LOQ). Linear calibration curves for Daidzein, Genistein, and S-Equol were set up by performing linear regression analysis and checked using the correlation coefficient (r2 > 0.995). LOQs for Daidzein, Genistein, and S-Equol were 2, 4, and 2 ng/mL, respectively. This UPLC-MS/MS swift method is suitable for quantifying isoflavones and the microbial-derived metabolite S-Equol in mice urine and is particularly useful for large numbers of samples.

Urinary phytoestrogens and the risk of uterine leiomyomata in US women.

BACKGROUND: Uterine leiomyomata (UL) is a common gynecological disease in women. Studied on the relationship between single metabolites of urinary phytoestrogens and UL, especially for the combined effects of mixed metabolites on UL still are insufficient. METHODS: In this cross-sectional study, we included 1,579 participants from the National Health and Nutrition Examination Survey. Urinary phytoestrogens were assessed by measuring urinary excretion of daidzein, genistein, equol, O-desmethylangolensin, enterodiol and enterolactone. The outcome was defined as UL. Weighted logistic regression was used to analyze the association between single metabolites of urinary phytoestrogens and UL. Notably, we adopted the weighted quantile sum (WQS) regression, Bayesian kernel machine regression (BKMR), and quantile g-computation (qgcomp) models, to investigate the combined effects of six mixed metabolites on UL. RESULTS: The prevalence of UL was approximately 12.92%. After adjusting age, race/ethnicity, marital status, drinking status, body mass index, waist circumference, menopausal status, ovary removed status, use of female hormones, hormones/hormone modifiers, total energy, daidzein, genistein, O-desmethylangolensin, enterodiol, and enterolactone, the association of equol with UL was significant [Odds ratio (OR) = 1.92, 95% confidence interval (CI): 1.09-3.38]. In the WQS model, mixed metabolites of urinary phytoestrogen had a positive association with UL (OR = 1.68, 95%CI: 1.12-2.51), with the highest weighted chemical of equol. In the gpcomp model, equol had the largest positive weight, followed by genistein and enterodiol. In the BKMR model, equol and enterodiol have positive correlation on UL risk, while enterolactone has negative correlation. CONCLUSION: Our results implied a positive association between the mixed metabolites of urinary phytoestrogen and UL. This study provides evidence that urinary phytoestrogen-metabolite mixture was closely related to the risk of female UL.

Association of urinary phytoestrogens with hormone-related cancers and cancer biomarkers: NHANES 1999-2010.

Phytoestrogens may have potential effects on hormone-related cancers (HRC) and cancer biomarkers, but the findings have been inconsistent so far. Participants from the National Health and Nutrition Examination Survey 1999-2010 with information on the levels of urinary phytoestrogens, serum cancer biomarkers and cancer history were included. Sampling-weighted logistic regression models examined the association between urinary phytoestrogens concentrations (creatinine-standardised and log-transformed) and HRC, followed by stratified analyses by race/ethnicity, age and menopausal status for different gender. Correlation analyses between phytoestrogens and cancer biomarkers were performed. Of the total 8844 participants, there were 373 with HRC. We observed total isoflavone and enterodiol excretion were positively associated with HRC, especially in non-Hispanic white female subpopulations (Ptrend < 0·05). Similar association also existed in the total isoflavones and enterodiol levels with breast cancer. Whereas the highest concentration of total isoflavones was significantly linked to a reduced prevalence of HRC (OR = 0·40, 95 % CI: 0·19, 0·84) in white males and of prostate cancer (OR = 0·40, 95 % CI: 0·18, 0·86). Among twenty-four participants with HRC, urinary equol concentration was positively correlated with CA15.3. Also, an inverse correlation of total prostate-specific antigens (PSA) and positive correlation of the PSA ratio with urinary enterolactone were detected in thirteen prostate cancer patients. Our findings indicated that higher concentrations of total isoflavones and enterodiol were positively associated with HRC. Urinary certain phytoestrogen excretion may affect serum cancer biomarker levels in cancer patients. But further prospective studies are needed to provide stronger evidence.

Associations of Urinary Phytoestrogen Concentrations with Nonalcoholic Fatty Liver Disease among Adults.

Phytoestrogens can alleviate some pathological processes related to nonalcoholic fatty liver disease (NAFLD). However, there are limited and contradictory studies on the relationships between phytoestrogens (especially single phytoestrogen) and NAFLD. The purpose of this study was to explore the relationships between urinary phytoestrogen concentrations and NAFLD in American adults. This cross-sectional study used the data of the National Health and Nutrition Examination Survey from 1999 to 2010, and 2294 adults were finally enrolled in this study. The concentrations of phytoestrogens were measured in urine samples, and urinary phytoestrogens were divided into tertiles according to the concentration distributions. The diagnosis of NAFLD was determined by the United States fatty liver index. The main analysis used a multivariate logistic regression model. The fully adjusted models included gender, age, race, education, marriage, poverty, body mass index, waist circumference, smoking, diabetes, hypertension, total cholesterol, high-density lipoprotein cholesterol, triglycerides, and other five phytoestrogens. In the fully adjusted model, the urinary enterolactone (ENL) concentration was negatively correlated with NAFLD (OR of Tertile 3 : 0.48, 95% CI 0.25-0.94). When stratified by age and gender, the urinary ENL concentration was negatively correlated with NAFLD in males aged 40-59 years (OR of Tertile 3 : 0.08, 95% CI 0.01-0.82), while the urinary equol concentration was positively correlated with NAFLD in such population (OR of Tertile 3 : 4.27, 95% CI 1.02-17.85). In addition, a negative correlation between enterodiol (END) concentration and NAFLD was observed in males aged 60 years or over (OR of Tertile 2 : 0.18, 95% CI 0.05-0.69). Collectively, in middle-aged males, urinary ENL may be associated with a lower risk of NAFLD, while urinary equol may be related to a higher risk. In addition, urinary END has a possible relationship with a reduced risk of NAFLD in elder males. Definitely, clinical randomized controlled trials are needed to further verify the conclusions.

Associations of Urinary Phytoestrogen Concentrations with Sleep Disorders and Sleep Duration among Adults.

Current evidence on the relationship of phytoestrogens with sleep is limited and contradictory. In particular, studies on individual phytoestrogens and sleep have not been reported. Thus, this study aimed to appraise the associations of individual phytoestrogens with sleep disorders and sleep duration. This cross-sectional study comprising 4830 adults utilized data from the National Health and Nutrition Examination Survey 2005-2010. Phytoestrogens were tested in urine specimens. Sleep disorders and sleep duration were based on a self-reported doctor's diagnosis and usual sleep duration. The main analyses utilized logistic and multinomial logistic regression models and a restricted cubic spline. In the fully adjusted model, compared with tertile 1 (lowest), the odds ratios (95% confidence intervals (CIs)) of sleep disorders for the highest tertile of urinary concentrations of enterolactone, enterodiol, and O-desmethylangolensin were 0.64 (0.41-1.00), 1.54 (1.07-2.21), and 1.89 (1.26-2.85), respectively. Linear inverse, approximatively linear positive, and inverted L-shaped concentration-response relationships were found between enterolactone, enterodiol, and O-desmethylangolensin and sleep disorders, respectively. Compared with normal sleep (7-8 h/night), the relative risk ratio (RRR) (95% CI) of very short sleep for enterolactone was 0.56 (0.36-0.86), and the RRR (95% CI) of long sleep risk for genistein was 0.62 (0.39-0.99). Furthermore, negative associations of genistein with sleep disorders and enterolactone with long sleep risk, as well as positive associations of enterodiol with both long and very short sleep, were observed in the stratified analysis by age or gender. Finally, a notable finding was that urinary O-desmethylangolensin concentration was positively related to sleep disorders in both females aged 40-59 years and non-Hispanic Whites but inversely associated with sleep disorders in both females aged 60 years or over and other Hispanics. Our findings suggested that enterolactone and genistein might be beneficial for preventing sleep disorders or non-normal sleep duration among adults, and enterodiol might be adverse toward this goal. However, the association of O-desmethylangolensin with sleep disorders might be discrepant in different races and females of different ages.

Gender Differences in Phytoestrogens and the Relationship with Speed of Processing in Older Adults: A Cross-Sectional Analysis of NHANES, 1999-2002.

Sex hormone changes in adults are known to play a part in aging, including cognitive aging. Dietary intake of phytoestrogens can mimic estrogenic effects on brain function. Since sex hormones differ between genders, it is important to examine gender differences in the phytoestrogen-cognition association. Therefore, the goal of this study is to examine the relationship between urinary phytoestrogens and speed of processing (SOP) and the variation of the association between genders in older adults. Participants were drawn from the 1999-2002 National Health and Nutrition Examination Survey and included 354 individuals aged 65-85 years old. General linear models (GLMs) were used to test for significant gender differences in the relationship between phytoestrogens and SOP. Results from the GLMs showed significant gender differences in the relationship between genistein and SOP. Higher levels of genistein were associated with better SOP in women. This relationship was reversed in men: higher genistein levels were associated with worse performance. Results indicate that there are distinct gender differences in the relationship between genistein and SOP. These results emphasize the importance of considering gender differences when devising dietary and pharmacologic interventions that target phytoestrogens to improve brain health.

Association of Urinary Phytoestrogens With Pelvic Organ Prolapse and Fecal Incontinence Symptoms in Postmenopausal Women.

OBJECTIVES: The aim of this study was to investigate the association between urinary phytoestrogen levels with symptoms of pelvic organ prolapse (POP) and fecal incontinence (FI) in postmenopausal women. METHODS: This is a cross-sectional study using the National Health and Nutrition Examination Survey database from 2005 to 2010 including postmenopausal women 40 years or older who either had both ovaries removed and/or had no period in the past year due to hysterectomy and/or menopause. Urinary concentrations of 6 phytoestrogens were measured. Pelvic organ prolapse symptoms were defined as a positive response to the question, "Do you see or feel a bulge in the vaginal area?" Fecal incontinence was defined as leakage of mucus, liquid, or solid stool occurring at least monthly. Using appropriate sample weights, prevalence estimates and 95% confidence intervals were calculated. Logistic regression was performed assessing associations between pelvic floor symptoms and log-transformed phytoestrogen levels adjusting for appropriate covariates. RESULTS: Participants included 1341 postmenopausal women with phytoestrogen data; 1213 with or without POP symptoms and 1221 with or without FI symptoms. Multivariable analysis revealed no association of urinary phytoestrogen levels with POP symptoms. Higher urinary O-desmethylangolensin level was associated with decreased odds of FI symptoms in postmenopausal women (adjusted odds ratio, 0.92; 95% confidence interval, 0.86-0.99). CONCLUSIONS: Increased urinary O-desmethylangolensin level was associated with lower odds of FI. Future research may be warranted to further investigate the potential of specific phytoestrogens as mediators of FI, as well as the role of phytoestrogens on POP symptoms.

Optimal cut-off value for equol-producing status in women: The Japan Nurses' Health Study urinary isoflavone concentration survey.

Equol is one of the most active soy isoflavones. When the association between soy food intake in daily life and health outcomes is examined in epidemiological studies, it is important to define the equol-producing status of each individual. However, few studies have assessed equol-producing status without a soy challenge test. To determine a robust cutoff criterion for equol producer classification in observational studies, we conducted a urinary isoflavone concentration survey in daily life among women. Furthermore, we examined the association between eating habits regarding soy foods and equol-producing status. A total of 4,412 participants were included in the analyses. Urinary isoflavones were analyzed using a high-performance liquid chromatography method. We examined the distribution of the log10 equol/daidzein ratios, finding a mixture of two normal distributions, corresponding to equol producer and non-producer subpopulations. Applying a finite mixture model, we estimated the means, standard deviations, and mixing proportions of these two distributions. The estimation was carried out using the SAS NLIN procedure. The optimal cutoff point for the log10 equol/daidzein ratio in the study population was determined to be -1.42, according to the estimated parameters of the mixture distribution. Based on this criterion, 1,830 (41.5%) of the participants were identified as equol producers. Compared with non-consumers of soy foods, consumers of soy foods had significantly higher odds of being equol producers. Using log10-transformed equol/daidzein ratios ≥ -1.42 to define equol producers among Japanese women is reasonable and suitable for determining equol-producing status in epidemiological studies. We found that soy food eating habits were associated with equol-producing status. Further investigation is required to evaluate associations between equol-producing status in daily life and health outcomes. The results of this study suggest the best cutoff point to use in the definition of equol-producing status in daily life.

Absolute oral bioavailability and disposition kinetics of puerarin in female rats.

BACKGROUND: Pueraria candollei var. mirifica is a medicinal plant that is promoted as a "Champion Product" by the Government of Thailand. This plant has been reported to relieve postmenopausal symptoms, prevent and reverse bone loss, inhibit the growth of breast cancer, and alleviate cardiovascular diseases in preclinical and clinical studies. However, there is little information on the oral bioavailability and tissue distribution of puerarin with respect to its pharmacodynamic activities. Therefore, the aim of this study was to determine the pharmacokinetics of puerarin, including absorption, distribution, metabolism, and elimination, in rats. Moreover, this is the first study to examine the tissue distribution of puerarin in the hippocampus, femur, tibia, and mammary gland. METHODS: Adult female rats were administered puerarin at 1 mg/kg intravenously or 5 and 10 mg/kg orally. Blood, tissue, urine, and feces were collected and analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: Puerarin reached a maximum concentration in the blood of 140-230 µg/L within 1 h of oral dosing, and had an absolute oral bioavailability of approximately 7%. Following intravenous administration, puerarin was widely distributed in several tissues, including the hippocampus, heart, lung, stomach, liver, mammary gland, kidney, spleen, femur, and tibia. Approximately 50% of the intravenous dose was excreted as glucuronide metabolites via the urinary route. CONCLUSIONS: The absolute oral bioavailability of puerarin was approximately 7% at doses of 5 and 10 mg/kg. Puerarin was widely distributed to several organs related to the diseases of aging, including the hippocampus, femur, tibia, and mammary gland. Glucuronides were the major metabolites of puerarin and were mainly excreted in the urine. These results are useful for the development of puerarin and Pueraria candollei var. mirifica as phytopharmaceutical products.

Rapid Screening and Identification of Daidzein Metabolites in Rats Based on UHPLC-LTQ-Orbitrap Mass Spectrometry Coupled with Data-Mining Technologies.

Daidzein, the main bioactive soy isoflavone in Nature, has been found to possess many biological functions. It has been investigated in particular as a phytoestrogen owing to the similarity of its structure with that of the human hormone estrogen. Due to the lack of comprehensive studies on daidzein metabolism, further research is still required to clarify its in vivo metabolic fate and intermediate processes. In this study, an efficient strategy was established using UHPLC-LTQ-Orbitrap mass spectrometry to profile the metabolism of daidzein in rats. Meanwhile, multiple data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filtering (MMDF), neutral loss fragment (NLF), and diagnostic product ion (DPI) were utilized to investigate daidzein metabolites from the HR-ESI-MS¹ to ESI-MSn stage in both positive and negative ion modes. Consequently, 59 metabolites, including prototype compounds, were positively or tentatively elucidated based on reference standards, accurate mass measurements, mass fragmentation behaviors, chromatographic retention times, and corresponding calculated ClogP values. As a result, dehydration, hydrogenation, methylation, dimethylation, glucuronidation, glucosylation, sulfonation, ring-cleavage, and their composite reactions were ascertained to interpret its in vivo biotransformation. Overall, our results not only revealed the potential pharmacodynamics forms of daidzein, but also aid in establishing a practical strategy for rapid screening and identifying metabolites of natural compounds.

Phytoestrogen Concentrations in Human Urine as Biomarkers for Dietary Phytoestrogen Intake in Mexican Women.

There has been substantial interest in phytoestrogens, because of their potential effect in reducing cancer and heart disease risk. Measuring concentrations of phytoestrogens in urine is an alternative method for conducting epidemiological studies. Our objective was to evaluate the urinary excretion of phytoestrogens as biomarkers for dietary phytoestrogen intake in Mexican women. Participants were 100 healthy women from 25 to 80 years of age. A food frequency questionnaire (FFQ) and a 24 h recall were used to estimate habitual and recent intakes of isoflavones, lignans, flavonols, coumestrol, resveratrol, naringenin, and luteolin. Urinary concentrations were measured by liquid chromatography (HPLC) coupled to mass spectrometry (MS) using the electrospray ionization interface (ESI) and diode array detector (DAD) (HPLC-DAD-ESI-MS). Spearman correlation coefficients were used to evaluate associations between dietary intake and urine concentrations. The habitual consumption (FFQ) of total phytoestrogens was 37.56 mg/day. In urine, the higher compounds were naringenin (60.1 µg/L) and enterolactone (41.7 µg/L). Recent intakes (24 h recall) of isoflavones (r = 0.460, p < 0.001), lignans (r = 0.550, p < 0.0001), flavonoids (r = 0.240, p < 0.05), and total phytoestrogens (r = 0.410, p < 0.001) were correlated to their urinary levels. Total phytoestrogen intakes estimated by the FFQ showed higher correlations to urinary levels (r = 0.730, p < 0.0001). Urinary phytoestrogens may be useful as biomarkers of phytoestrogen intake, and as a tool for evaluating the relationship of intake and disease risk in Mexican women.

Association between Urinary Phytoestrogens and C-reactive Protein in the Continuous National Health and Nutrition Examination Survey.

OBJECTIVE: A reduced risk of some cancers and cardiovascular disease associated with phytoestrogen intake may be mediated through its effect on serum C-reactive protein (CRP; an inflammation biomarker). Therefore, this study examined the associations between urinary phytoestrogens and serum CRP. METHODS: Urinary phytoestrogen and serum CRP data obtained from 6009 participants aged ≥ 40 years in the continuous National Health and Nutrition Examination Survey during 1999-2010 were analyzed. RESULTS: After adjustment for confounders, urinary concentrations of total and all individual phytoestrogens were inversely associated with serum concentrations of CRP (all p < 0.004). The largest reductions in serum CRP (mg/L) per interquartile range increase in urinary phytoestrogens (ng/mL) were observed for total phytoestrogens (ß = -0.18; 95% confidence interval [CI], -0.22, -0.15), total lignan (ß = -0.15; 95% CI, -0.18, -0.12), and enterolactone (ß = -0.15; 95% CI, -0.19, -0.12). A decreased risk of having high CRP concentrations (≥3.0 mg/L) for quartile 4 vs quartile 1 was also found for total phytoestrogens (OR = 0.63; 95% CI, 0.53, 0.73), total lignan (OR = 0.64; 95% CI, 0.54, 0.75), and enterolactone (OR = 0.59; 95% CI, 0.51, 0.69). CONCLUSION: Urinary total and individual phytoestrogens were significantly inversely associated with serum CRP in a nationally representative sample of the U.S.

Association between Dietary Share of Ultra-Processed Foods and Urinary Concentrations of Phytoestrogens in the US.

The aim of this study was to examine the relationship between dietary contribution of ultra-processed foods and urinary phytoestrogen concentrations in the US. Participants from cross-sectional 2009-2010 National Health and Nutrition Examination Survey aged 6+ years, selected to measure urinary phytoestrogens and with one 24-h dietary recall were evaluated (2692 participants). Food items were classified according to NOVA (a name, not an acronym), a four-group food classification based on the extent and purpose of industrial food processing. Ultra-processed foods are formulations manufactured using several ingredients and a series of processes (hence "ultra-processed"). Most of their ingredients are lower-cost industrial sources of dietary energy and nutrients, with additives used for the purpose of imitating sensorial qualities of minimally processed foods or of culinary preparations of these foods. Studied phytoestrogens included lignans (enterolactone and enterodiol) and isoflavones (genistein, daidzein, O-desmethylangolensin and equol). Gaussian regression was used to compare average urinary phytoestrogen concentrations (normalized by creatinine) across quintiles of energy share of ultra-processed foods. Models incorporated survey sample weights and were adjusted for age, sex, race/ethnicity, family income, and education, among other factors. Adjusted enterodiol geometric means decreased monotonically from 60.6 in the lowest quintile to 35.1 µg/g creatinine in the highest, while adjusted enterolactone geometric means dropped from 281.1 to 200.1 across the same quintiles, respectively. No significant linear trend was observed in the association between these quintiles and isoflavone concentrations. This finding reinforces the existing evidence regarding the negative impact of ultra-processed food consumption on the overall quality of the diet and expands it to include non-nutrients such as lignans.

Exposure to phytoestrogens in utero and age at menarche in a contemporary British cohort.

Phytoestrogens are estrogenic compounds that occur naturally in plants. Phytoestrogens can cross the placenta, and animal studies have found associations between in utero exposure to phytoestrogens and markers of early puberty. We investigated the association between in utero exposure to phytoestrogens and early menarche (defined as <11.5 years of age at onset) using data from a nested case-control study within the Avon Longitudinal Study of Parents and Children, a longitudinal study involving families living in the South West of England. Concentrations of six phytoestrogens were measured in maternal urine samples collected during pregnancy. Logistic regression was used to explore associations between tertiles of phytoestrogen concentrations and menarche status, with adjustment for maternal age at menarche, maternal education, pre-pregnancy body mass index (BMI), child birth order, duration of breastfeeding, and gestational age at sample collection. Among 367 mother-daughter dyads, maternal median (interquartile range) creatinine-corrected concentrations (in µg/g creatinine) were: genistein 62.1 (27.1-160.9), daidzein 184.8 (88.8-383.7), equol 4.3 (2.8-9.0), O-desmethylangolensin (O-DMA) 13.0 (4.4-34.5), enterodiol 76.1 (39.1-135.8), and enterolactone 911.7 (448.1-1558.0). In analyses comparing those in the highest tertile relative to those in the lowest tertile of in utero phytoestrogen exposure, higher enterodiol levels were inversely associated with early menarche (odds ratio (OR)=0.47; 95% confidence interval (CI): 0.26-0.83), while higher O-DMA levels were associated with early menarche (OR=1.89; 95% CI: 1.04-3.42). These findings suggest that in utero exposure to phytoestrogens may be associated with earlier age at menarche, though the direction of association differs across phytoestrogens.

Urinary Phytoestrogen Concentrations Are Not Associated with Incident Endometriosis in Premenopausal Women.

BACKGROUND: Phytoestrogens have been associated with subtle hormonal changes, but their effects on endometriosis are largely unknown. OBJECTIVE: We assessed the association between urinary concentrations of phytoestrogens and incident endometriosis. METHODS: We included an operative sample of 495 premenopausal women aged 18-44 y undergoing laparoscopies and laparotomies at 14 clinical sites between 2007 and 2009 and a general population sample of 131 women from the same geographic area who were matched on age and menstruation status. Endometriosis in the surgical sample was assessed by surgical visualization (clinical gold standard), whereas disease in the general population sample was assessed with the use of a pelvic MRI. Urine concentrations of genistein, daidzen, O-desmethylangolensin, equol, enterodiol, and enterolactone were measured at baseline. Poisson regression with robust error variance was used to estimate the risk of an endometriosis diagnosis for each sample after adjusting for age and body mass index (in kg/m2). Separate models were run for each phytoestrogen. RESULTS: Overall geometric mean urine concentrations of phytoestrogens were as follows: genistein [88 nmol/L (95% CI: 72, 108 nmol/L)], daidzein [194 nmol/L (95% CI: 160, 236 nmol/L)], O-desmethylangolensin [4 nmol/L (95% CI: 3, 6 nmol/L)], equol [4 nmol/L (95% CI: 4, 6 nmol/L)], enterodiol [29 nmol/L (95% CI: 22, 38 nmol/L)], and enterolactone [355 nmol/L (95% CI: 395, 544 nmol/L)]. Geometric mean concentrations of phytoestrogens did not significantly differ by endometriosis status in either sample. Adjusted RRs for endometriosis ranged from 0.87 to 1.09 for the 6 phytoestrogens measured, with all CIs including a value ≥1. Phytoestrogens were not associated with the severity of endometriosis when restricting the analysis to women with moderate-to-severe disease per the revised American Society for Reproductive Medicine criteria. Furthermore, no associations were observed between self-reported high soy intake and endometriosis. CONCLUSIONS: Despite endometriosis being an estrogen-dependent disease, we found no evidence that urinary phytoestrogens were associated with a higher risk of an endometriosis diagnosis in either a sample of premenopausal women or in a surgical sample.

Exposure to endocrine disrupting chemicals among residents of a rural vegetarian/vegan community.

BACKGROUND & AIMS: Endocrine-disrupting chemicals (EDCs) are increasingly thought to be involved in the rising prevalence of disorders such as obesity, diabetes, and some hormone-dependent cancers. Several lines of evidence have indicated that vegetarian and vegan diets may offer some protection from such diseases. We hypothesized that exposure to selected EDCs among residents of the unique vegetarian/vegan community of Amirim would be lower than what has recently been reported for the omnivorous population in the first Israel Biomonitoring Study (IBMS). METHODS: We studied 42 Amirim residents (29 vegetarians/13 vegans; 24 women/18men, aged 50.7±13.7y). Subjects answered detailed lifestyle, and multipass, memory-based 24-hr dietary recall questionnaires. Concentrations of bisphenol A (BPA), 11 phthalate metabolites, and the isoflavone phytoestrogens (genistein and daidzein) were determined by GC or LC tandem mass-spectrometry on a spot urine sample. The results were compared to those obtained following the same methodology in the Jewish subgroup of the IBMS (n=184). RESULTS: While a vegetarian/vegan nutritional pattern had no effect on exposure to BPA, it seemed to confer a modest protection (~21%) from exposure to high molecular weight phthalates. Furthermore, the summed metabolites of the high molecular weight phthalate DiNP were 36% lower in vegans compared to vegetarians (P<0.05). In contrast, Amirim residents exhibited a level of exposure to isoflavone phytoestrogens about an order of magnitude higher than in the IBMS (P<0.001). CONCLUSIONS: In Israel, a country whose inhabitants demonstrate exposure to EDCs comparable to that of the US and Canada, a voluntary lifestyle of vegetarianism and preference for organic food has a modest, but possibly valuable, impact on exposure to phthalates, while it is associated with a very steep increase in the exposure to phytoestrogens. Major reduction in exposure to EDCs will require regulatory actions.

Urine and serum biomonitoring of exposure to environmental estrogens II: Soy isoflavones and zearalenone in pregnant women.

UNLABELLED: Urine and serum biomonitoring was used to measure internal exposure to selected dietary estrogens in a cohort of 30 pregnant women. Exposure was measured over a period comprising one-half day in the field (6 h) and one day in a clinic (24 h). Biomonitoring of the dietary phytoestrogens genistein (GEN), daidzein (DDZ) and equol (EQ), as well as the mycoestrogen, zearalenone (ZEN) and its congeners, was conducted using UPLC-MS/MS. Biomonitoring revealed evidence of internal exposure to naturally occurring dietary estrogens during pregnancy. Urinary concentrations of total GEN, DDZ and EQ were similar to levels reported for general adult U.S. POPULATION: Measurable concentrations of total (parent and metabolites) GEN, DDZ and EQ were present in 240, 207 and 2 of 270 serum samples, respectively. Six out of 30 subjects had measurable concentrations of unconjugated GEN and/or DDZ in serum between 0.6 and 7.1 nM. Urine to serum total isoflavone ratios for GEN, DDZ and EQ were 13, 47, and 180, respectively. ZEN and its reductive metabolite, α-zearalenol (α-ZEL), were present in pregnant women (11 out of 30 subjects) as conjugates at levels near the limit of quantification. The average total urinary concentration was 0.10 µg/L for ZEN and 0.11 µg/L for α-ZEL.

The relationship between phytoestrogens and speed of processing.

OBJECTIVE: This study examined the cross-sectional association between phytoestrogens and speed of processing. We hypothesized that higher levels of phytoestrogens would be related to better cognitive performance among older women. METHODS: Participants were from the National Health and Nutrition Examination Survey and consisted of 200 older women (M = 74.4 y). Stepwise regressions examined indicators of speed of processing, measured by the Digit Symbol Substitution Test. Isoflavones, lignans, and individual phytoestrogens were added to the regression after including covariates of age, education, race, smoking, and creatinine. Isoflavones were further broken into quartiles among the sample to further evaluate the nature of the curvilinear association. RESULTS: Results showed a relationship between cognition and lignans, explaining 3.8% of the variance after including the covariates, indicating fewer lignans were associated with better speed of processing (P < 0.001). A significant curvilinear relationship with isoflavones explained 1.3% additional variance (P < 0.001). The moderate-high, low-moderate, and the lowest quartile of isoflavones were associated with better cognition, whereas the highest amount was associated with worse speed of processing. Among the individual phytoestrogens, only enterodiol accounted for 4.4% additional variance after taking into account covariates and indicated a negative association with cognition (P = 0.03). CONCLUSIONS: Results suggest that moderate levels of isoflavones, but not lignans, may be associated with better speed of processing. Caution must remain for high isoflavone amounts due to the negative association with cognition. Given the results, phytoestrogens have the potential to be an intervention target for older females' cognition. To become a viable intervention, further research is needed.

Urine phyto-oestrogen metabolites are not significantly associated with risk of type 2 diabetes: the Singapore Chinese health study.

We evaluated the relationship between urine concentrations of phyto-oestrogens (isoflavones and lignans) and risk of incident type 2 diabetes in middle-aged and elderly Chinese residing in Singapore. Urine metabolites of isoflavones and lignans were assayed by HPLC among 564 diabetes cases and 564 matched controls in a case-control study nested within the Singapore Chinese Health Study cohort. Participants were free of diagnosed diabetes, CVD and cancer at morning urine collections during 1999-2004. Cases were participants who reported to have physician-diagnosed diabetes at follow-up visits during 2006-2010, whereas controls were randomly selected among those who remained free of diabetes and were matched to the index cases by age, sex, dialect group and date of urine collection. Conditional logistic regression models were used to calculate OR and 95 % CI with adjustment for potential confounders. The mean age of the participants at the time of urine collection was 59·8 years, and the average interval between urine collection and diabetes diagnosis was 4·0 years. The multivariate-adjusted OR for diabetes were 1·00 (reference), 0·76 (95 % CI 0·52, 1·11), 0·78 (95 % CI 0·53, 1·14) and 0·79 (95 % CI 0·54, 1·15) across quartiles of urine isoflavones (P for trend=0·54), and were 1·00 (reference), 0·87 (95 % CI 0·60, 1·27), 1·10 (95 % CI 0·77, 1·56) and 0·93 (95 % CI 0·63, 1·37) for lignans (P for trend=0·93). The results were similar in men and women, as well as for individual metabolites of isoflavones (genistein, daidzein, glycitin and equol) or lignans (enterodiol and enterolactone). The present study did not find a significant association between urine phyto-oestrogen metabolites and risk of type 2 diabetes in Chinese adults.

Urinary phytoestrogens and cancer, cardiovascular, and all-cause mortality in the continuous National Health and Nutrition Examination Survey.

PURPOSE: Experimental studies suggest that phytoestrogen intake alters cancer and cardiovascular risk. This study investigated the associations of urinary phytoestrogens with total cancer (n = 79), cardiovascular (n = 108), and all-cause (n = 290) mortality among 5179 participants in the continuous National Health and Nutrition Examination Survey (1999-2004). METHODS: Urinary phytoestrogens were measured using high-performance liquid chromatography with tandem mass spectrometric detection. Survival analysis was performed to evaluate hazard ratios (HRs) and 95 % confidence intervals (CIs) for each of the three outcomes in relation to urinary phytoestrogens. RESULTS: After adjustment for confounders, higher urinary concentrations of total enterolignans were associated with a reduced risk of death from cardiovascular disease (HR for tertile 3 vs. tertile 1 0.48; 95 % CI 0.24, 0.97), whereas higher urinary concentrations of total isoflavones (HR for tertile 3 vs. tertile 1 2.14; 95 % CI 1.03, 4.47) and daidzein (HR for tertile 3 vs. tertile 1 2.05; 95 % CI 1.02, 4.11) were associated with an increased risk. A reduction in all-cause mortality was observed for elevated urinary concentrations of total enterolignans (HR for tertile 3 vs. tertile 1 0.65; 95 % CI 0.43, 0.96) and enterolactone (HR for tertile 3 vs. tertile 1 0.65; 95 % CI 0.44, 0.97). CONCLUSIONS: Some urinary phytoestrogens were associated with cardiovascular and all-cause mortality in a representative sample of the US population. This is one of the first studies that used urinary phytoestrogens as biomarkers of their dietary intake to evaluate the effect of these bioactive compounds on the risk of death from cancer and cardiovascular disease.