Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography

A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (+/-5.6%) in breast tissue homogenate and 100% (+/-14.1%) in urine and serum for all three compounds. Equol (less than 1 micromol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.     

Absorption of isoflavones in humans: effects of food matrix and processing

If soy isoflavones are to be effective in preventing or treating a range of diseases, they must be bioavailable, and thus understanding factors which may alter their bioavailability needs to be elucidated. However, to date there is little information on whether the pharmacokinetic profile following ingestion of a defined dose is influenced by the food matrix in which the isoflavone is given or by the processing method used. Three different foods (cookies, chocolate bars and juice) were prepared, and their isoflavone contents were determined. We compared the urinary and serum concentrations of daidzein, genistein and equol following the consumption of three different foods, each of which contained 50 mg of isoflavones. After the technological processing of the different test foods, differences in aglycone levels were observed. The plasma levels of the isoflavone precursor daidzein were not altered by food matrix. Urinary daidzein recovery was similar for all three foods ingested with total urinary output of 33-34% of ingested dose. Peak genistein concentrations were attained in serum earlier following consumption of a liquid matrix rather than a solid matrix, although there was a lower total urinary recovery of genistein following ingestion of juice than that of the two other foods.     

Growth performance, carcass characteristics and bioavailability of isoflavones in pigs fed soy bean based diets

A growth trial with 38 weaners (castrated male pigs) was designed to compare the growth performance and carcass quality of pigs fed diets containing either soy bean meal or soy protein concentrate in a pair-feeding design. Soy bean meal (SBM) and soy protein concentrate (SPC) differed in isoflavone (daidzein plus genistein) content (782 microg/g in SBM and 125 microg/g in SPC, respectively). During the experiment, all pigs were fed four-phases-diets characterized by decreasing protein concentrations with increasing age (weaner I, weaner II, grower, finisher diets). Rations of control and experimental groups were isoenergetic, isonitrogenous, and isoaminogen. The weanling pigs with an initial live weight of 8.4 +/- 1.1 kg were allotted to flat deck boxes. During the growing/finishing period (days 70-170 of age), the pigs were housed in single boxes. Both, the weaning and the grower/finishing performances (daily body weight gain, feed intake, feed conversion ratio) were similar in both groups. No differences were found between the groups in carcass composition (percentages of cuts, tissues, and protein/fat), and meat quality of pigs. Moreover, the IGF-1R mRNA expression in longissimus muscle was not influenced by the kind of soy product. However, circulating levels of isoflavones were clearly different between pigs fed SBM (genistein 239 +/- 44; daidzein 162 +/- 42; equol 12 +/- 4 ng/ml plasma) and animals fed SPC (genistein 22 +/- 9 and daidzein 8 +/- 3, and equol 10 +/- 3 ng/ml plasma). The results confirm the expected differences in the bioavailability of soy isoflavones, yet, there were no significant differences in performance of pigs fed either soy bean meal or soy protein concentrate.     

Growth performance,carcass characteristics and bioavailability of isoflavones in pigs fed soy bean based diets

A growth trial with 38 weaners (castrated male pigs) was designed to compare the growth performance and carcass quality of pigs fed diets containing either soy bean meal or soy protein concentrate in a pair-feeding design. Soy bean meal (SBM) and soy protein concentrate (SPC) differed in isoflavone (daidzein plus genistein) content (782?μg/g in SBM and 125?μg/g in SPC, respectively). During the experiment, all pigs were fed four-phases-diets characterized by decreasing protein concentrations with increasing age (weaner I, weaner II, grower, finisher diets). Rations of control and experimental groups were isoenergetic, isonitrogenous, and isoaminogen. The weanling pigs with an initial live weight of 8.4?±?1.1?kg were allotted to flat deck boxes. During the growing/finishing period (days 70?–?170 of age), the pigs were housed in single boxes. Both, the weaning and the grower/finishing performances (daily body weight gain, feed intake, feed conversion ratio) were similar in both groups. No differences were found between the groups in carcass composition (percentages of cuts, tissues, and protein/fat), and meat quality of pigs. Moreover, the IGF-1R mRNA expression in longissimus muscle was not influenced by the kind of soy product. However, circulating levels of isoflavones were clearly different between pigs fed SBM (genistein 239?±?44; daidzein 162?±?42; equol 12?±?4?ng/ml plasma) and animals fed SPC (genistein 22?±?9 and daidzein 8?±?3, and equol 10?±?3?ng/ml plasma). The results confirm the expected differences in the bioavailability of soy isoflavones, yet, there were no significant differences in performance of pigs fed either soy bean meal or soy protein concentrate.     

Urinary equol excretion in relation to 2-hydroxyestrone and 16alpha-hydroxyestrone concentrations: an observational study of young to middle-aged women

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.     

Circulating isoflavonoid levels in CD-1 mice: effect of oral versus subcutaneous delivery and frequency of administration

The CD-1 mouse is a commonly used animal model to understand the biological effects of early-life exposure to soy isoflavones in infants. Most studies using CD-1 mice have administered isoflavones by daily subcutaneous injection, while infants receive oral feeds every few hours. The study objectives were to compare the total serum levels of genistein (GEN), daidzein (DAI) and the DAI metabolites equol and O-desmethyl-angolensin (O-DMA), after subcutaneous injection and oral dosing and to determine if frequency of oral administration results in different circulating levels of isoflavones using the CD-1 mouse model. From postnatal days 1 to 5, pups randomly received corn oil or soy isoflavones (total daily dose, 0.010 mg DAI+0.025 mg GEN) by subcutaneous injection once a day, orally once a day or orally every 4 hours. On postnatal day 5, 1 h posttreatment, mice were killed and serum was collected. Mice treated with soy isoflavones had higher (P<.05) serum GEN (female: 1895–3391 ng/ml and male: 483–578 ng/ml) and DAI (female: 850–1580 ng/ml and male: 248–322 ng/ml) concentrations versus control (5–20 ng/ml) mice, regardless of route or frequency of administration, and were similar among dosing strategies. Total serum concentrations of GEN and DAI were higher (P<.05) among females (GEN: 2714 ± 393 ng/ml and DAI: 1205 ± 164 ng/ml) than males (GEN: 521 ± 439 ng/ml and DAI: 288 ± 184 ng/ml) across treatment groups. Serum equol and O-DMA concentrations were negligible (<3 ng/ml) across groups. In conclusion, different routes of delivery and frequency of administration resulted in similar total serum levels of GEN, DAI¸ equol or O-DMA.     

Dietary isoflavones differentially induce gene expression changes in lymphocytes from postmenopausal women who form equol as compared with those who do not

Human and animal studies suggest that dietary soy isoflavones reduce cancer risk, ameliorate postmenopausal syndrome and decrease bone resorption in postmenopausal women. The capacity to form the metabolite equol from daidzein is suggested as an important modulator of response to isoflavones; this capacity depends on gut colonization with appropriate bacteria. We administered a dietary supplement containing high-dose purified soy isoflavones (genistein, 558 mg/day; daidzein, 296 mg/day; and glycitein, 44 mg/day) to 30 postmenopausal women for 84 days and collected peripheral lymphocytes at timed intervals. Using microarray analysis, we determined whether changes in gene expression associated with this treatment support existing hypotheses as to isoflavones' mechanisms of action. Expression of a large number of genes was altered by isoflavone treatment, including induction of genes associated with cyclic adenosine 3',5'-monophosphate (cAMP) signaling and cell differentiation and decreased expression of genes associated with cyclin-dependent kinase activity and cell division. We report that isoflavone treatment in subjects who have the capacity to produce equol differentially affects gene expression as compared with nonproducers, supporting the plausibility of the importance of equol production. In general, isoflavones had a stronger effect on some putative estrogen-responsive genes in equol producers than in nonproducers. Our study suggests that, in humans, isoflavone changes are related to increased cell differentiation, increased cAMP signaling and G-protein-coupled protein metabolism and increased steroid hormone receptor activity and have some estrogen agonist effects; equol-production status is likely to be an important modulator of responses to isoflavones.     

Detection of the isoflavone aglycones genistein and daidzein in urine using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry

An improved method of detection of the isoflavone aglycones, genistein and daidzein, is reported using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry (SPME–HPLC–ESI-MS). Extraction of the isoflavonoids from urine using SPME with a Carbowax–templated resin fiber coating allows rapid preconcentration of the analytes without the usual sample preparation required by other methods. Detection of the analytes is accomplished by HPLC–ESI-MS. Analysis of spiked samples of urine resulted in a linear range of 0.25 to 250 ng/ml for daidzein and 0.27 to 27.0 ng/ml for genistein. Limits of detection of daidzein and genistein were measured at 25.4 pg/ml for daidzein and 2.70 pg/ml for genistein. Daidzein and genistein were detected in urine following consumption of a soy drink.     

Effects of isoflavone supplements vs. soy foods on blood concentrations of genistein and daidzein in adults

The objective of this investigation was to examine the pharmacokinetics of isoflavone concentrations over a 24-h period among healthy adults consuming either soy foods or soy isoflavone tablets at different doses. This randomized, cross-over trial was conducted with 12 generally healthy adults. The three phases of the intervention included isoflavone tablets at (1) 144 mg/day or (2) 288 mg/day and (3) soy foods designed to provide a calculated 96 mg isoflavones/day (doses in aglycone equivalents). Doses were spread out over three meals per day. After 6 days on each study phase, plasma isoflavone concentrations were determined on the seventh day at 0, 4, 8, 10, 12 and 24 h. Average levels of total isoflavone concentrations at 8, 10 and 12 h were >4 micromol/L for the soy food phase and for the higher dose tablet phase. Genistein concentrations were higher overall in the soy food vs. both the lower and the higher dose supplement phases of the study (P<.05). When comparing plasma concentrations for the two doses of tablets, saturation appeared more evident for genistein than for daidzein at the higher dose level. In conclusion, we observed important differences in the pharmacokinetics of genistein and daidzein contrasting the sources and doses of isoflavones when administered three times daily, including a possible advantage for increasing serum concentrations of isoflavones from consuming soy foods relative to isoflavone supplements.     

Soybean isoflavonoids and their metabolic products inhibit in vitro lipoprotein oxidation in serum

Isoflavonoids are compounds present in many legumes, but are derived in the human diet mainly from soybeans and various soybean-based food products. The major isoflavonoids occurring in soy are the glycosides of genistein and daidzein. The metabolic products of genistein metabolism in humans have not been clearly shown. The two main products of daidzein metabolism in humans appear to be equol and O-desmethylangolensin. Increasing evidence suggests that oxidative modification to low-density lipoprotein is involved in atherogenesis, and that natural antioxidants that prevent or inhibit oxidative damage to low-density lipoprotein may beneficially influence atherogenesis. In the present experiments, the effects of genistein and daidzein, and the daidzein metabolites equol and O-desmethylangolensin on Cu²⁺-induced oxidation of lipoproteins in serum were examined. Three concentrations of each compound (0.1 μM, 1 μM, 10 μM) were tested for antioxidant activity in six individual serum samples. All compounds tested inhibited lipoprotein oxidation. The minimum concentration for significant inhibition was 1 μM for genistein and daidzein (P < 0.05), and 0.1 μM equol and O-desmethylangolensin (P < 0.05). Equol and O-desmethylangolensin were more potent inhibitors of in vitro lipoprotein oxidation in serum than the two major dietary isoflavonoids. This study has demonstrated that soybean isoflavonoids and metabolic products of daidzein metabolism inhibit lipoprotein oxidation in vitro. Human intervention studies are needed to determine if these compounds can influence oxidation in vivo.     

Equol producer status, salivary estradiol profile and urinary excretion of isoflavones in Irish Caucasian women, following ingestion of soymilk

Equol production, isoflavone excretion, and the salivary estradiol profile among 36 females, native Irish Caucasian volunteers following ingestion of 200mL soymilk is reported. The soymilk contained daidzein (73+/-6.7mg) and genistein (86+/-10.2mg). Volunteers provided personal and family medical history. Dietary analysis revealed that all volunteers regularly consumed soy-based or soy-supplemented food products. The mean age, mean age at menarche, and body mass index of volunteers were 46.6+/-12.3 years, 13.1 years and 26.1, respectively. The average number of children per volunteer was 2.13. Twelve (34%) of the volunteers were found to be first-degree relatives of breast cancer patients. Following consumption of the soymilk, equol was detected in the urine of 18 (51%) of the volunteers. Mean urinary daidzein and genistein concentrations during the hours following soymilk ingestion were 13.5 and 16.7microg/mg creatinine, respectively, however, some volunteers excreted little (less than 4.0microg/mg) or no isoflavone. Salivary estradiol in most (24) volunteers had decreased from 51.5+/-28.67pmol/L pre-ingestion to 29.75+/-16.13pmol/L 5h after drinking the soymilk. However, the salivary estradiol in 12 subjects (34%) increased from 33.76+/-13.4pmol/L to 137.4+/-65.64pmol/L over the same period. Individuals whose salivary estradiol increased had significantly less children (1.58 (P<0.05)), were more likely to (a) return urine samples with low isoflavone content (50.3% compared to 25%), (b) to be equol producers (67% compared to 41.7%), and (c) to be first-degree relatives of breast cancer patients (41.7% compared to 25%). Volunteers who reported a first-degree link to breast cancer were more likely to have a higher body mass index (29.0 compared to 26.1 (P<0.05)), to be equol producers (75% compared to 51%), and to excrete isoflavones in low quantities only (60% compared to 50%). First-degree relatives also had fewer children (1.75 (P<0.05)). The results indicate a significant, distinctive variation in equol production, isoflavone excretion and salivary estradiol profile among individual volunteers following ingestion of soymilk.     

<Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> JCM 2771: Impact on Metabolism of Isoflavonoids in the Fecal Flora from a Male Equol Producer

Many beneficial effects of probiotics have been reported; however, few have focussed on the effects of Lactobacillus, a probiotic, on the bioconversion of isoflavonoids. We hypothesized that Lactobacillus rhamnosus will modify the metabolism of isoflavone. In an in vitro incubation, L. rhamnosus JCM 2771 produced daidzein from daidzin along with genistein. However, daidzin and genistein were not detected in the incubation solution of daidzein with L. rhamnosus. In the fecal suspension from a male equol producer with daidzein, equol was detected in the presence of a low or high concentration of L. rhamnosus. In the fecal incubation with daidzin, the equol concentration increased with an increasing concentration of L. rhamnosus JCM 2771. L. rhamnosus affected the equol production in the in vitro incubation of daidzein with fecal flora from a male equol producer. We demonstrated for the first time that L. rhamnosus JCM 2771 could produce genistein from daidzin and affect the equol production of fecal flora from a male equol producer in vitro.     

Circulating levels of isoflavones and markers of 5alpha-reductase activity are higher in Japanese compared with New Zealand males: what is the role of circulating steroids in prostate disease?

Epidemiological evidence implicates dietary isoflavone intake as protective against prostate disease. A putative mechanism is attenuated circulating androgen levels in male populations consuming an isoflavone rich diet. We investigated this hypothesis by collecting plasma from 60 Japanese and 60 New Zealand males aged between 21 and 31 years each consuming their traditional diets. We measured plasma testosterone, dihydrotestosterone (DHT), androstenedione, dehydroepiandrosterone sulfate (DHEAS), the combined levels of androsterone sulfate and epiandrosterone sulfate (AoS/epiAoS), sex hormone-binding globulin, and cortisol and corticosteroid-binding globulin as well as the isoflavones genistein and equol. Plasma genistein and equol levels were several times higher in Japanese males as would be expected from an isoflavone rich diet. However, androstenedione, DHEAS, calculated free testosterone and paradoxically markers of 5alpha-reductase, DHT and AoS/epiAoS were all also significantly higher in Japanese rather than the New Zealand male counterparts. All other comparisons were not significant. Plasma DHT and DHEAS correlated positively with plasma equol and plasma AoS/epiAoS correlated positively with genistein levels. Taken together the results suggest that, rather than reduced levels of steroidogenesis, Japanese males may have increased 5alpha-reductase activity and possibly altered 17beta OH steroid dehydrogenase activity. Significantly the positive association between isoflavones levels and 5alpha-steroids is counter-intuitive to isoflavone intake offering prostate protection, unless this is postulated to occur through other mechanisms.     

Enhanced bioavailability of soy isoflavones by complexation with beta-cyclodextrin in rats

In order to improve the solubility and bioavailability of a soy isoflavone extract (IFE), inclusion complexes (IFE-beta-CD) of the isoflavone extract with beta-cyclodextrin (beta-CD) were prepared and studied for their solubility and bioavailability. The aqueous solubility of the complexes of IFE with beta-CD (2.0 mg/ml) was about 26 times that of IFE itself (0.076 mg/ml). The same dosages of IFE and IFE-beta-CD were orally administered to SD rats (Sprague-Dawley) on an isoflavone glycoside (IFG) basis (daidzin, genistin and glycitin), and the plasma concentrations of daidzein, genistein and glycitein were measured over time to estimate the average AUC (area under the plasma concentration versus time curve) of the isoflavones. After the oral administration, the AUC values for daidzein, genistein and glycitein were 340, 11 and 28 microg x min/ml, respectively. In contrast, the respective AUC values after the administration of IFE-beta-CD were 430, 20 and 48 microg x min/ml. The bioavailability of daidzein in IFE-beta-CD was increased to 126% by the formation of inclusion complexes with beta-CD, compared with that in IFE. Furthermore, the bioavailability of genistein and glycitein in IFE-beta-CD formulation was significantly higher by up to 180% and 170%, respectively, compared with that of IFE p=0.008 and p=0.028, respectively). These results show that the absorption of IFE could be improved by the complexation of IFE with beta-CD (IFE-beta-CD).     

Soy isoflavone equol perpetuates dextran sulfate sodium-induced acute colitis in mice

The effects of the soy isoflavones, genistein, daidzein and equol, on experimental colitis were examined. Equol severely perpetrated dextran sulfate sodium (DSS)-induced colitis as evaluated by the weight loss. Production of the anti-inflammatory cytokine, IL-10, from T cells was decreased in the equol-treated mice. The results show that the soy isoflavone, equol, played an important role in the inflammatory response in the gastrointestinal tract.     

Determination of urinary phytoestrogens by HPLC-MS/MS: a comparison of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI)

A comparison of the analytical performance of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) for the quantitative determination of six urinary phytoestrogens (daidzein, O-desmethylangolensin, equol, enterodiol, enterolactone and genistein) by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is presented here. Both APCI and ESI were suitable for the analysis of these compounds; however, ESI did improve measurement imprecision and sensitivity in certain cases. Method imprecision (between-run coefficients of variation [CVs] from duplicate analysis of three quality control [QC] urine pools across 20 runs) was 5.6-12% for ESI, as opposed to 5.3-30% for APCI. At low concentrations (3-60 ng/mL, analyte dependent) imprecision was lower with ESI, whereas both techniques were generally commensurate at high concentrations (200-1000 ng/mL, analyte dependent). Method accuracy (spiked analyte recovery from the QC pools) was comparable between techniques: 86-114% for ESI; 95-105% for APCI. Limits of detection (LODs) were equivalent or better with ESI compared to APCI, with the most significant LOD improvement observed for equol (ESI: 0.3 ng/mL; APCI: 2.7 ng/mL). This translated into a substantial increase in equol detection frequency (% of sample results above LOD) within a random patient sample subset (98% for ESI, compared to 81% for APCI, n=378). Correlation (Pearson) and agreement (Deming regression, Bland-Altman bias) between ESI and APCI results in the patient subset was better in cases where imprecision and sensitivity was similar for both techniques (daidzein, enterolactone, genistein: r=0.993-0.998; slope=0.98-1.03; bias=-4.2 to -0.8%); correlation and/or agreement was poorer for analytes, where APCI imprecision and sensitivity were inferior (equol, O-desmethylangolensin, enterodiol). Baring significant factors arising from differences in ionization source design, these observations suggest that ESI is more appropriate for urinary biomonitoring of these compounds by LC-MS/MS.     

Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. β-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in β-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.     

Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. β-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in β-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.     

Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.