A Non-Probiotic Fermented Soy Product Reduces Total and LDL Cholesterol: A Randomized Controlled Crossover Trial.

Traditional Asian fermented soy food products are associated with reduced cardiovascular disease risk in prospective studies, but few randomized controlled trials have been conducted in at-risk populations. The aim of this study was to investigate the effect of a commercial non-probiotic fermented soy product on blood lipids in adults with cardiovascular risk biomarkers. In a randomized, crossover, intervention study, 27 men and women (aged 29-75 y) exhibiting at least two risk factors, consumed two packets (12.5 g each) daily of a fermented powdered soy product, or an isoenergic control powder made from germinated brown rice for 12 weeks each. The consumption of the fermented soy product resulted in a significantly greater mean change from baseline (compared to the germinated rice, all p < 0.05) in total cholesterol of -0.23 mmol/L (CI: -0.40, -0.06) compared with 0.14 mmol/L (CI: -0.03, 0.31), respectively; and low density lipoprotein (LDL) cholesterol -0.18 mmol/L (CI: -0.32, -0.04) compared with 0.04 mmol/L (CI: -0.01, 0.018) respectively. This was accompanied by an increase in high density lipoprotein (HDL) cholesterol in the germinated rice group, a decrease in apolipoprotein B (ApoB) in the fermented soy group, and a between-treatment effect in apolipoprotein A1 (ApoA1); however, the ratio of the LDL:HDL and of Apo B:ApoA1 did not differ between the groups. The ratio of total cholesterol:LDL decreased in men in the fermented soy group (p < 0.001). Twenty-four-hour urine collection at the end of each treatment period resulted in an increased excretion expressed as a ratio in µmol/d between treatments of 10.93 (CI: 5.07, 23.54) for daidzein; 1.24 (CI: 1.14, 4.43) for genistein; and, 8.48 (CI: 4.28, 16.80) for glycitein, all p < 0.05. The fermented soy powder consumed by participants in this study without implementing other changes in their typical diets, decreased the total and LDL cholesterol, and may serve as a dietary strategy to manage blood lipids. The trial was registered at ClinicalTrials.gov as NCT03429920.

The Connection between Urinary Equol Levels and the Prevalence of Atopic Dermatitis.

BACKGROUND: Soy isoflavones and their metabolites such as equol have been associated with a reduced risk of hormone-sensitive tumors and metabolic syndromes. However, individual soy isoflavones and equol levels in atopic dermatitis remain uninvestigated. OBJECTIVE: The aim of this study is to compare the levels of urinary daidzein, genistein, and equol between atopic dermatitis patients and normal subjects and to examine the correlation between equol concentration and the severity of clinical symptoms. METHODS: A cross-sectional study was conducted at Akita University Hospital and Aso Iizuka Hospital in Japan. Fifty patients with confirmed atopic dermatitis diagnosis and 67 healthy controls were recruited. Daidzein, genistein, and equol in urine were measured by using a high-performance liquid chromatography-mass spectrometry system. RESULTS: Urinary equol levels were significantly lower in the atopic dermatitis patients than in the healthy controls (p = 0.002). The difference was particularly noticeable in young people (6-19 years, p < 0.001). No correlations were found between urinary equol levels and the severity of clinical symptoms and laboratory data in the atopic dermatitis patients. CONCLUSION: Equol levels in childhood might be involved in the development of atopic dermatitis.

Simultaneous determination of formononetin, biochanin A and their active metabolites in human breast milk, saliva and urine using salting-out assisted liquid-liquid extraction and ultra high performance liquid chromatography-electrospray ionization tandem mass spectrum.

Isoflavonoid phytoestrogens, referred as "dietary estrogens" are widely distributed in the plant kingdom. Formononetin, biochanin A and their active metabolites daidzein and genistein are known to be the most potent among other isoflavonoid phytoestrogens. Thus there is a growing need to determine accurately their concentration in different biological fluids. In the present work, a sensitive analytical method was developed for the quantitative determination of these compounds in human breast milk, saliva and urine. The glycoside conjugates of these compounds were enzymatically hydrolysis prior to salting-out assisted liquid-liquid extraction. Quantitative analysis was done by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The obtained results showed high correlation coefficients (r2 > 0.998) for the linear range established for formononetine, biochanin A, daidzein and genistein. The limits of detection (LODs) and low limits of quantitation (LLOQs) were in the ranges of 0.05-1.0 ng/mL and 1.0-4.0 ng/mL for all analytes in human biological fluids, respectively. The average recoveries ranged from 83.29% to 115.24% for the analytes with relative standard deviation (n = 5) values from 1.84% to 9.75% in samples. Both intra-day and inter-day precisions and accuracy were found to be within 12.53% and ± 12.92% respectively. Under different conditions of stability, the concentrations for four isoflavonoid phytoestrogens deviated within ±12.87% of norminal values. The developed method was successfully validated and applied to human breast milk, saliva and urine. The average concentrations of daidzein and genistein found in breast milk, saliva and urine samples ranged from 0 to 104.2 µg/kg, 18.17 to 786.0 µg/kg, 0 to 10974 µg/kg, respectively. Their presence in breast milk samples shows exposure of breast-fed baby to isoflavones. It also allows for the rapid screening of human biological fluids when testing for formononetin, biochanin A, daidzein and genistein production status in human.

Urinary equol, but not daidzein and genistein, was inversely associated with the risk of type 2 diabetes in Chinese adults.

PURPOSE: Many studies have examined the association of isoflavone intake with type 2 diabetes (T2D), and produced inconsistent results. Few studies, however, explored the association using objective biomarkers (particular for daidzein metabolite-equol) of isoflavones. We aimed to explore the association of urinary equol, daidzein and genistein concentrations with T2D and examine the mediating roles of high-sensitivity C-reactive protein (hsCRP) and retinol binding protein 4 (RBP4). METHODS: This prospective study included 2818 subjects. Urinary concentrations of equol, daidzein and genistein were measured by high-performance liquid chromatography-tandem mass spectrometry. The associations between urinary isoflavones and T2D incidence were evaluated by cox proportional hazards model. RESULTS: After adjustment for covariates, urinary equol except daidzein and genistein was inversely associated with T2D incidence. In comparison with the first tertile, multivariable adjusted hazard ratios (HRs) and 95% confidence intervals (95% CIs) for T2D incidence in the second and third tertile of equol concentration were 0.52 (0.37, 0.73) and 0.72 (0.53, 0.97), respectively. In stratified analyses by sex, the HR (95% CI) of men in the second vs. first tertile of equol was 0.29 (0.14, 0.58). Equivalent estimation in women was 0.67 (0.45, 1.01). Neither women nor men in the third tertile showed significant difference of T2D incidence compared with the first tertile. In path analyses, there was no evidence of mediating effects of hsCRP and RBP4 on the "equol-T2D" relationship. CONCLUSIONS: Urinary equol was favorably associated with a decreased T2D incidence in Chinese adults. The equol-T2D relationship might not be mediated by hsCRP and RBP4. TRIAL REGISTRATION: This study has been registered at http://www.clinicaltrials.gov as NCT03179657.

Daidzein and genistein have differential effects in decreasing whole body bone mineral density but had no effect on hip and spine density in premenopausal women: A 2-year randomized, double-blind, placebo-controlled study.

Soy isoflavones are potentially beneficial phytoestrogens, but their tissue-selective effects in women are poorly understood. We tested the hypothesis that soy isoflavones affect bone mineral density (BMD), which may be influenced by individual differences in isoflavone metabolism and serum calcium levels. Ninety-nine healthy premenopausal women were randomized to isoflavones (136.6 mg aglycone equivalence) and 98 to placebo for 5 days per week for up to 2 years. BMD, serum calcium, and urinary excretion of daidzein and genistein were measured before and during treatment. In 129 adherent subjects, we found that isoflavone exposure, determined by urinary excretion levels, but not by dose assignment, interacted with serum calcium in affecting whole body BMD, but not hip and spine BMD. The regression coefficient was -0.042 for genistein excretion (GE) and 0.091 for the interaction between GE and serum calcium (all P < .05). Daidzein excretion had similar but marginal effect. Genistein significantly decreased whole body BMD only at low normal serum calcium levels but increased whole body BMD at higher serum calcium levels. Comparing maximum to minimum GE, mean changes in whole body BMD were +0.033 and -0.113 g/cm2 at serum calcium levels of 10 and 8.15 mg/dL, respectively. These associations were not evident by intention-to-treat analysis, which could not model for inter-individual differences in isoflavone metabolism. In summary, soy isoflavones decrease whole body BMD only when serum calcium is low. Isoflavones are dietary substances that may influence calcium homeostasis by releasing calcium from bone while sparing the common fracture risk sites hip and spine.

The application of the supported liquid membrane and molecularly imprinted polymers as solid acceptor phase for selective extraction of biochanin A from urine.

An efficient sample clean-up and preconcentration procedure for phytoestrogens analysis in urine has been developed. It was based on a combination of solid phase extraction with hollow-fiber supported liquid membrane and molecularly imprinted beads (MIPs-HF-SLM-SPE). The molecularly imprinted polymers (MIPs) were synthesized by precipitation polymerization technique with biochanin A (BCA) as a template, giving narrowly dispersed microspheres with a regular shape. As the functional monomer, (dimethylamino)ethyl methacrylate (-DEM) turned out to be better than methacrylic acid (MAA) to get the best-imprinted effects. The MIPs used as sorbents in the MIPs-HF-SLM-SPE extraction process exhibited excellent binding selectivity for BCA, in comparison to non-imprinted polymers as well as its structural analogs (genistein and daidzein). Finally, the developed method was used to detect BCA in urine. Under optimal extraction conditions, the recovery of BCA in urine samples (using 4.5 mL sample spiked with 10 µg L-1) was over 41%, with a coefficient of variation (CV) < 6.6% (n = 5). The detection limit (LOD) and quantification limit (LOQ) for BCA analysis in urine were 0.41 and 1.36 µg L-1, respectively.

The association between urinary genistein levels and mortality among adults in the United States.

BACKGROUND: Current research on the relationship between phytoestrogens and mortality has been inconclusive. We explored the relationship between genistein, a phytoestrogen, and mortality in a large cohort representative of the United States population. METHODS: Data were analyzed from the National Health and Nutrition Examination Survey (NHANES) from 1999-2010. Normalized urinary genistein (nUG) was analyzed as a log-transformed continuous variable and in quartiles. Mortality data were obtained from the National Death Index and matched to the NHANES participants. Survival analyses were conducted using the Kaplan-Meier analysis. Cox proportional hazard models were constructed for all-cause and cause-specific mortality without and with adjustment for potential confounding variables. RESULTS: Of 11,497 participants, 944 died during the 64,443 person-years follow-up. The all-cause mortality rate was significantly lower in the lowest quartile compared to the highest quartile (incidence rate ratio = 2.14, 95%CI = 1.76 to 2.60). Compared to the lowest quartile, the highest quartile had significantly higher adjusted all-cause (HR = 1.57, 95%CI = 1.23 to 2.00), cardiovascular (HR = 1.67, 95%CI = 1.04 to 2.68), and other-cause (HR = 1.85, 95%CI = 1.33 to 2.57) mortality. CONCLUSION: We found that high urinary genistein levels were associated with increased risk of all-cause, cardiovascular, and other-cause mortality. This is contrary to popular opinion on the health benefits of genistein and needs further research.

The relationship between urinary genistein levels and serum alanine aminotransferase levels in adults in the USA: National Health and Nutrition Examination Survey 1999-2010.

BACKGROUND: Genistein, a phytoestrogen with similarities to female sex hormones, has been shown to protect against oxidative stress and fibrosis in nonalcoholic fatty liver injury in animal studies. However, few studies have examined genistein's effects on liver function in humans. PARTICIPANTS AND METHODS: We analyzed data from the National Health and Nutrition Examination Survey from 1999 to 2010. Individuals younger than 21 years, with viral hepatitis, or with serum alanine aminotransferase (ALT) at the extremes of distribution (5% on each extreme) were excluded. Urinary genistein was normalized by urinary creatinine levels. The relationship between normalized urinary genistein (nUG) and serum ALT was examined using linear regression models with and without adjustment for potential confounders, and the differential effect of sex was examined using an interaction term. RESULTS: Of the 9864 participants, 52% were female, 50% were White, 24% were elderly, 36% had hypertension, 12% had diabetes, and 8.1% were heavy alcohol drinkers. Serum ALT was significantly lower in the highest quartile compared with the lowest quartile of nUG (22.3 vs. 23.5 U/l; P<0.001). In adjusted models, individuals in the highest quartile had 0.75 U/l lower ALT levels than those in the lowest quartile (P=0.02). We found a significant difference in ALT levels between the lowest and highest quartiles of nUG in males, but not in females (difference in differences=1.77 U/l, interaction P=0.04). CONCLUSION: We found a statistically significant association between higher nUG and lower serum ALT in males, but not in females. The sex-specific role of genistein in mitigating liver disease merits further study.

Soybean soluble polysaccharide enhances absorption of soybean genistein in mice.

This study was designed to probe the promoting effects of soybean soluble polysaccharide (SSPS) on bioavailability of genistein in mice and the underlying molecular mechanism. Male Kunming mice (n=8) were administered intragastrically with either saline, SSPS (5mg/kgbw), genistein (100mg/kgbw), or SSPS (5 or 50mg/kgbw) together with genistein (100mg/kgbw) for consecutive 28days. UPLC-qTOF/MS analysis showed that co-administration of SSPS and genistein in mice caused significant elevation in the urinary levels of genistein and its metabolites (p<0.05). Furthermore, the fecal excretion of genistein was also enhanced by co-administration of SSPS. However, the feces level of dihydrogenistein, a characteristic metabolite of genistein degraded by gut microorganism, was dose-dependently decreased by the combined treatment of SSPS. Additionally, co-treatment of SSPS with genistein also decreased the small intestinal levels of uridinediphosphate-glucuronosyltransferase (UGT), sulfotransferase (SULT), P-glycoprotein (P-gp), multidrug resistance-associated protein-1 (MRP1), and multidrug resistance-associated protein-2 (MRP2) in mice. These findings suggest that the inhibition of SSPS against small intestinal first-pass metabolism of genistein is involved in the promoting effect of genistein bioavailability in mice.

Novel effects of phytoestrogenic soy isoflavones on serum calcium and chloride in premenopausal women: A 2-year double-blind, randomized, placebo-controlled study.

BACKGROUND: Soy phytoestrogens are potential alternatives to postmenopausal hormone replacement therapy (HRT). Adverse effects of HRT such as myocardial infarction, stroke, and pulmonary embolism are mediated by calcium-induced signaling. OBJECTIVE: To determine whether soy isoflavones affect serum calcium in healthy female subjects. DESIGN: In a double-blind trial, 197 premenopausal women were randomly assigned to either isoflavone (N = 99) or placebo pills (N = 98) 5 days per week for up to 2 years, plus prenatal vitamins. Isoflavone pills contained 60 mg genistein, 60 mg daidzein and 16.6 mg glycitein (expressed as aglycone equivalents). All pills contained 15 mg riboflavin as an adherence marker. Blood chemistries and urinary daidzein, genistein and riboflavin were measured multiple times during the luteal phase before and during treatment. RESULTS: Analysis of the adherent population (N = 83 per group), revealed significantly strong associations between urinary levels of isoflavones and serum concentrations of calcium (regression coefficients 0.082 for daidzein and 0.229 for genistein, all P < 0.01) and chloride (regression coefficient, -1.537 for genistein, P < 0.0001), mediated in part by albumin. The effects amounted to mean changes of +0.24 mg/dL for calcium and -1.45 mEq/L for chloride, with each visit for subjects excreting the most vs. the least amounts of isoflavones. These associations were not evident in the intention-to-treat analysis (N = 197) that did not assess expected variations in isoflavone levels within and between subjects from metabolism and adherence. CONCLUSIONS: These novel and strong effects of soy isoflavones on calcium homeostasis have important implications for long term effects of these natural substances on cardiovascular diseases.

Adding Soy Protein to Milk Enhances the Effect of Resistance Training on Muscle Strength in Postmenopausal Women.

Resistance training (RT) and high-quality protein ingestion improves muscle mass (MM) and strength (MS). However, no study has evaluated the effect of ingesting milk plus soy protein (SOY) on MM and MS in postmenopausal women (PW). Thus, the aim of this study was to evaluate the effects of adding SOY to milk on MM and MS after 16 weeks of RT. Thirty-two PW were randomized and allocated into two groups: placebo and RT (PL+RT, n = 16) and SOY and RT (SOY+RT, n = 16). The SOY+RT received 25 g of SOY while the PL+RT received 25 g of maltodextrin (placebo). All supplements were given in the form of a chocolate-flavored powder added to 200 mL of milk. The RT protocol consisted of eight total body exercises at 70% of one repetition maximum (1RM), three sets of 8-12 repetitions, 2-3 times/week. No differences were found in the baseline measures between groups (age, menopause status, anthropometric and nutrition patterns), except for protein intake, which was higher in the SOY+RT. Both groups increased the MM (bioimpedance) showing no difference between groups (PL+RT = 1.5 kg; SOY+RT = 1.1 kg). For MS, the SOY+RT showed a larger (p < .05) increase in 1RM of bench press (PL+RT = 6.7 kg; SOY+RT = 12.5 kg), knee extension (PL+RT = 3.7 kg; SOY+RT = 6.7 kg), total load (PL+RT = 15.1 kg; SOY+RT = 24.2 kg), and the total load exercises/MM (PL+RT = 0.3 kg; SOY+RT = 0.9 kg). These results suggest that adding SOY to milk combined with 16 weeks of RT resulted in more significant increases in MS in PW.

Relationship of equol production between children aged 5-7 years and their mothers.

PURPOSE: The factors responsible for the production of isoflavone metabolites have not yet been identified. We aimed to examine the relationships of equol production between mother and child in a birth cohort in Japan. METHODS: Subjects were a part of the participants in a longitudinal study on pregnant women and their offspring. When children were 5-7 years old, mothers and children were asked to reply to a questionnaire on lifestyles and a 3-day child's dietary record. Mothers and children were given a bar-shaped soy snack (Soyjoy®) daily on two consecutive days (soy challenge). The snack contained 14 mg of overall soy isoflavones as the sum of aglycones and the glucosides for mothers and 7.5 mg for children. On the morning of day 0 and 3, they were asked to mail their first-void urines. Urinary isoflavone metabolites of 159 mother-child pairs were measured by a high-performance liquid chromatography method. RESULTS: Equol producers were 35.5 % among mothers and 13.8 % among children. Equol producer status of a child was neither associated with dietary intake nor with urinary levels of daidzein and genistein. After multiple adjustments for potential confounders, the estimated relative risk of equol producer was 2.75 (95 % confidence interval 1.00, 7.52) among children whose mother was an equol producer, compared with children whose mother was a non-producer. CONCLUSION: Child's equol production was associated with the mother's equol producer status. The effects of maternal factors on child's equol production should be studied further.

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.

A combination of soy isoflavones and cello-oligosaccharides changes equol/O-desmethylangolensin production ratio and attenuates bone fragility in ovariectomized mice.

We examined the cooperative effects of isoflavones and cello-oligosaccharides on daidzein metabolism and bone fragility in ovariectomized mice. Cello-oligosaccharides increased urinary equol and decreased O-desmethylangolensin. A combination of isoflavones and cello-oligosaccharides attenuated decreases in bone breaking force and stiffness caused by ovariectomy. Combination treatment with isofalvones and cello-oligosaccharides increases urinary equol/O-desmethylangolensin production ratio and prevents ovariectomy-induced abnormalities in bone strength.

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.

Trapping Methylglyoxal by Genistein and Its Metabolites in Mice.

Increasing evidence supports dicarbonyl stress such as methylglyoxal (MGO) as one of the major pathogenic links between hyperglycemia and diabetic complications. In vitro studies have shown that dietary flavonoids can inhibit the formation of advanced glycation end products (AGEs) by trapping MGO. However, whether flavonoids can trap MGO in vivo and whether biotransformation limits the trapping capacity of flavonoids remain virtually unknown. In this study, we investigated whether genistein (GEN), the major soy isoflavone, could trap MGO in mice by promoting the formation of MGO adducts of GEN and its metabolites. Two different mouse studies were conducted. In the acute study, a single dose of MGO and GEN were administered to mice via oral gavage. In the chronic study, MGO was given to mice in drinking water for 1 month and then GEN was given to mice for 4 consecutive days via oral gavage. Two mono-MGO adducts of GEN and six mono-MGO adducts of GEN phase I and microbial metabolites were identified in mouse urine samples from these studies using liquid chromatography/electrospray ionization tandem mass spectrometry. The structures of these MGO adducts were confirmed by analyzing their MS(n) (n = 1-4) spectra as well as by comparing them with the tandem mass spectra of authentic standards. All of the MGO adducts presented in their phase II conjugated forms in mouse urine samples in the acute and chronic studies. To our knowledge, this is the first in vivo evidence to demonstrate the trapping efficacy of GEN in mice and to show that the metabolites of GEN remain bioactive.

Soy isoflavone metabolism in cats compared with other species: urinary metabolite concentrations and glucuronidation by liver microsomes.

1. Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here, we evaluated species' differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species. 2. Significant concentrations of conjugated (but not unconjugated) genistein, daidzein and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein, were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine. 3. ß-Glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats. 4. Glucuronidation rates of genistein, daidzein and equol by cat livers were consistently ranked within the lowest 3 out of 13 species' livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species. 5. Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species.

Urinary Phytoestrogens Are Associated with Subtle Indicators of Semen Quality among Male Partners of Couples Desiring Pregnancy.

BACKGROUND: Phytoestrogens have been associated with subtle hormonal changes, although effects on male fecundity are largely unknown. OBJECTIVE: We evaluated associations between male urinary phytoestrogen (isoflavone and lignan) concentrations and semen quality. METHODS: This study was a prospective cohort study of 501 male partners of couples desiring pregnancy and discontinuing contraception. Each participant provided up to 2 semen samples that were analyzed for 35 semen quality endpoints the following day. Linear mixed-effects models were used to estimate associations between baseline urinary phytoestrogen concentrations and semen quality parameters, adjusted for age, body mass index (BMI), research site, and serum lipid and cotinine concentrations. RESULTS: Most associations between urinary phytoestrogens and semen quality parameters were null. However, select individual phytoestrogens were associated with semen quality parameters, with associations dependent on the class of phytoestrogens and modified by BMI. Specifically, genistein and daidzein were associated with a lower percentage of normal sperm and increased abnormalities in semen morphology, with reduced associations observed as BMI increased (P < 0.05) [percentages (95% CIs) of normal morphology by WHO traditional criteria: genistein, main effect: -5.61% (-9.42%, -1.79%); interaction: 0.19% (0.06%, 0.31%) per log unit increase; daidzein, main effect: -5.35% (-9.36%, -1.34%); interaction: 0.18% (0.05%, 0.32%) per log unit increase]. Enterolactone was associated with fewer abnormalities in semen morphometry and morphology and decreased DNA fragmentation, with reduced associations observed as BMI increased (P < 0.05) [percentages (95% CIs) of abnormalities in the neck and midpiece: enterolactone, main effect: -3.35% (-6.51%, -0.19%); interaction: 0.11% (0.01%, 0.21%) per log unit increase]. CONCLUSIONS: These results suggest that male urinary phytoestrogen concentrations characteristic of the US population may be associated with subtle indicators of male fecundity and semen quality but were not associated with couple fecundity.

A novel UHPLC method for the rapid and simultaneous determination of daidzein, genistein and equol in human urine.

This work reports on a novel method involving reverse-phased ultra-high performance liquid chromatography (UHPLC) plus a spectrophotometric photodiode array/fluorescence (FLR) detection system for determining the concentration of equol and major soy isoflavones (daidzein and genistein) in human urine. The proposed method was validated in terms of its linearity, sensitivity, accuracy (recovery) and precision (intra- and inter-day repeatability). The isoflavone profiles of urine samples from a group of menopausal women following oral soy isoflavone supplementation were determined and compared. Screening for equol-producer status was accomplished with high sensitivity (detection limit of the FLR detector 2.93nM). The method involves a short chromatographic run time compared to conventional HPLC methods while allowing for the simultaneous and reliable quantification of daidzein, genistein and equol in human urine. It also allows for the rapid screening of multiple urine samples when testing for equol production status and checking patient adherence to isoflavone treatment regimens.

Urinary isoflavonoids and risk of type 2 diabetes: a prospective investigation in US women.

To examine the association between urinary excretion of isoflavonoids and risk of type 2 diabetes (T2D), we conducted a nested case-control study among 1111 T2D pairs identified during 1995-2008 in the Nurses' Health Study (NHS) and NHSII, who were free of diabetes, CVD and cancer at urine sample collection. Urinary excretion of daidzein and genistein, as well as their metabolites O-desmethylangolensin (O-DMA), dihydrogenistein (DHGE) and dihydrodaidzein (DHDE) was assayed using liquid chromatography MS. Self-reported T2D incident cases were confirmed using a validated questionnaire. Higher urinary excretion of daidzein and genistein was associated with a lower risk of T2D in the combined cohorts. Comparing extreme tertiles of the urinary markers, the OR of T2D were 0·71 (95 % CI 0·55, 0·93) for daidzein and 0·74 (95 % CI 0·56, 0·97) for genistein, although the test for linear trend was not significant for genistein (P trend=0·03 and 0·15, respectively). DMA, DHDE and DHGE were non-significantly associated with a lower T2D risk. The inverse association of daidzein with T2D risk was stronger among post-menopausal women who did not use hormone replacement therapy (P interaction=0·001): the OR was 0·58 (95 % CI 0·34, 0·97) comparing extreme tertiles among these women. In conclusion, urinary excretion of isoflavones was associated with a lower T2D risk in US women, especially among post-menopausal women who did not use hormone. Further research is warranted to replicate these observations among western populations with similarly low overall isoflavone intake.