Reduction of leptin secretion by soy isoflavonoids in murine adipocytes in vitro

Daidzein and genistein are the main aglycones of soy isoflavonoid, and have many useful activities in vitro and in vivo. However, equol, a metabolite of daidzein in vivo, has attracted attention due to its stronger activity than that of the naturally occurring isoflavonoids. We subjected the soy isoflavonoids, including the naturally occurring (S)-equol, to mouse adipocytes, and compared the inhibitory activity on the leptin secretion. Equol, daidzein and genistein inhibited the leptin secretion, whereas O-desmethylangolensin had a lower activity. The inhibitory activity of the isoflavones was not affected by the addition of an iNOS inhibitor and an estrogen.     

Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions

Only about one third of humans possess a microbiota capable of transforming the dietary isoflavone daidzein into equol. Little is known about the dietary and physiological factors determining this ecological feature. In this study, the in vitro metabolism of daidzein by faecal samples from four human individuals was investigated. One culture produced the metabolites dihydrodaidzein and O-desmethylangolensin, another produced dihydrodaidzein and equol. From the latter, a stable and transferable mixed culture transforming daidzein into equol was obtained. Molecular fingerprinting analysis (denaturing gradient gel electrophoresis) showed the presence of four bacterial species of which only the first three strains could be brought into pure culture. These strains were identified as Lactobacillus mucosae EPI2, Enterococcus faecium EPI1 and Finegoldia magna EPI3, and did not produce equol in pure culture. The fourth species was tentatively identified as Veillonella sp strain EP. It was found that hydrogen gas in particular, but also butyrate and propionate, which are all colonic fermentation products from poorly digestible carbohydrates, stimulated equol production by the mixed culture. However, when fructo-oligosaccharides were added, equol production was inhibited. Furthermore, the equol-producing capacity of the isolated culture was maintained upon its addition to a faecal culture originating from a non-equol-producing individual.     

The role of diet in the metabolism of daidzein by human faecal microbiota sampled from Italian volunteers

The intestinal microbial transformation of daidzein into equol is subject to a wide inter-individual variability. The aim of this study was to investigate in vitro this transformation and to evaluate possible correlations between individual diet and equol production. The transformation of daidzein was investigated in anaerobic batch cultures inoculated with mixed fecal bacteria from 90 volunteers. The daidzein metabolism was monitored by liquid chromatography-mass spectrometry, and a chiral column was used to distinguish equol and dihydrodaidzein enantiomers. The obtained results show that daidzein was unchanged (≈27%) or degraded to equol (≈28%), O-desmethylangolensin (≈12%) or dihydrodaidzein (≈31%). Furthermore, some subjects (≈2%) are able to produce both equol and O-desmethylangolensin. Bacteria represent sub-dominant populations (10⁵–10⁹ cell/g wet faeces) in “slow” equol producers, while higher counts of equol-producing microorganisms (10¹⁰–10¹¹ cell/g wet faeces) were found in “quick” equol producers. The in vitro test to evaluate equol-producing status is quick and not invasive, and the obtained results are comparable with those reported in vivo. Indeed, the only enantiomer present in the batch cultures containing equol was the S-form. No significant correlations between equol production, BMI, age and sex were found. It seems that the equol-producer group consumed less fibre, vegetables and cereals, and more lipids from animal sources.     

Quantitative analysis of urinary daidzein and equol by gas chromatography after solid-phase extraction and high-performance liquid chromatography

Daidzein and its main metabolite equol are isoflavone phytoestrogens. Several studies have suggested that intake of an isoflavone-rich diet may prevent hormone-related cancer and estrogen-related disorders (cardiovascular disease, osteoporosis and menopausal symptoms). To better understand the role of isoflavones in preventing such severe disease, several methods have been developed to measure these compounds in biological fluids. However, the analytical procedures to measure isoflavones are often time-consuming and require highly skilled technicians. In this paper we describe a method for urinary daidzein and equol measurement that combines solid phase extraction and HPLC purification before gas chromatographic determination. The specificity of the method was confirmed by the gas chromatography-mass spectrometry technique. The mean recovery of daidzein and equol was 94.6% and 97.0%, respectively. The repeatability of the method was in the range of 2.0-7.4% for daidzein and 1.3-4.9% for equol. A linear relationship between observed and expected values was found in the dilution (r2=0.9983 for daidzein; r2=0.9982 for equol) and addition (r2=0.9984 for daidzein; r2=0.9989 for equol) assays. The method is suitable to measure changes in the urinary excretion of isoflavones and to investigate urinary isoflavonoids as biomarkers of isoflavone exposure.     

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.     

雌马酚产生细菌及其雌马酚合成代谢机制

大豆食品中通常富含染料木素和大豆苷元等异黄酮素,人和动物肠道中的某些细菌具有将异黄酮素代谢转化为S-雌马酚的能力。到目前为止,S-雌马酚被认为是一种具有潜在健康调节作用的化合物。啮齿类动物均具备产雌马酚的能力,但不同人群之间存在差异,产雌马酚细菌是否存在可能是造成这种差异的重要原因;不同产雌马酚细菌的代谢机制可能不同,并影响机体最终产雌马酚的能力。本文对已知的各种产雌马酚细菌及其细菌的雌马酚合成机制进行综述,以期为进一步了解雌马酚产生个体差异、雌马酚代谢转化效率、体外雌马酚的发酵生产,以及临床产雌马酚细菌的应用等提供理论参考。     

Comparative Activities of the S-Enantiomer and Racemic Forms of Equol on Bone Fragility in Ovariectomized Mice

We compared the effects of the S-enantiomer and racemic forms of equol on bone using ovariectomized (OVX) mice. Femoral bone mineral density and bone strength decreased in the OVX mice, but not in OVX mice administered 0.5 mg/d S-equol. This, however, did not hold for racemic equol. Serum and urine S-equol concentrations were higher in the mice administered S-equol than in those administered racemic equol. These results suggest that the inhibitory effects of S-equol on bone fragility in OVX mice are greater than those of racemic equol.     

Equol suppresses inflammatory response and bone erosion due to rheumatoid arthritis in mice

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune inflammatory disease. Typical pathological findings of RA include persistent synovitis and bone degradation in the peripheral joints. Equol, a metabolite of the major soybean isoflavone daidzein, shows superior bioactivity than other isoflavones. We investigated the effects of equol administration on inflammatory response and bone erosion in mice with collagen-induced arthritis (CIA). The severity of arthritis symptoms was significantly low in the equol-administered CIA mice. In addition, equol administration improved the CIA-induced bone mineral density decline. In the inflamed area of CIA mice, equol administration suppressed the expression of interleukin-6 and its receptor. Furthermore, equol reduced the expression of genes associated with bone formation inhibition, osteoclast and immature osteoblast specificity and cartilage destruction. These results suggest that equol suppresses RA development and RA-induced bone erosion by regulating inflammation and bone metabolism.     

Antioxidant effect of a phytoestrogen equol on cultured muscle cells of embryonic broilers

Previous studies have shown that the in ovo injection of equol can markedly improve the water-holding capacity of muscles of broilers chickens at 7 wk of age through promotion of the antioxidant status. We aimed to investigate directly the antioxidant effects of equol on muscle cells in broilers. Muscle cells were separated from leg muscle of embryos on the 11th day of incubation and treated with equol and H₂O₂, either alone or together. Cells were pretreated with medium containing 1, 10, or 100 μM equol for 1 h prior to the addition of 1 mM H₂O₂ for a further 1 h. Photomicrographs of cells were obtained. Cell viability, malondialdehyde (MDA) content, and L-lactate dehydrogenase (LDH) activity in the cell supernatant, as well as intracellular total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities were determined. Treatment with 1 mM H₂O₂ caused serious damage to cells, indicated by comets with no clear head region but a very apparent tail of DNA fragments. Pretreatment with low (1 μM) but not high concentrations of equol (10 μM) inhibited cell damage, while 100 μM equol caused more serious damage than H₂O₂ alone. Pretreatment with 1 μM equol had no effect on cell viability, while pretreatment with 10 and 100 μM equol significantly decreased cell viability in a dose-dependent manner. Compared with H₂O₂ alone, pretreatment with low-dosage equol markedly decreased LDH activity and MDA production in the supernatant, significantly increased intracellular T-SOD activity (P < 0.05) and tended to increase intracellular GSH-Px activity (0.05 < P < 0.1). Pretreatment with high-dosage equol (10 and 100 μM) significantly enhanced LDH activity, but had no effect on MDA content, T-SOD or GSH-Px activity induced by H₂O_2, except for an obvious increase in GSH-Px activity caused by 10 μM equol. These results indicate that equol at low dosage can prevent skeletal muscle cell damage induced by H₂O₂, while pretreatment with high-dosage equol shows a synergistic effect with H₂O₂ in inducing cell damage.     

Genotoxic activity of four metabolites of the soy isoflavone daidzein

Products containing phytoestrogens are increasingly promoted as the “natural” alternative to estrogen replacement therapy. In the present study, we have used the in vitro micronucleus assay in L5178Y mouse lymphoma cells to investigate the genotoxic potential of the isoflavone daidzein, and of four daidzein metabolites known to be formed in humans. Whereas no induction of micronuclei was observed with daidzein up to the limit of solubility (100 μM), all four daidzein metabolites, i.e. equol (2.3-fold induction at 100 μM), O-desmethylangolensin (6.2-fold induction at 10 μM), 4′,6,7-isoflavone (6.7-fold induction at 100 μM) and 3′,4′,7-isoflavone (8.2-fold induction at 100 μM) induced micronuclei in a concentration-dependent manner. Thus, both reductive and oxidative metabolites of the soy isoflavone daidzein exhibit genotoxic potential in vitro.     

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.     

172名上海地区成人饮食及尿雌马酚和代谢产物的调查研究

目的确定上海地区成人雌马酚代谢表型及雌马酚的生理范围;把握由于大豆异黄酮负荷而产生的雌马酚产生者比例;调查雌马酚表型和食物摄取频率及有关激素间的关系。方法应用现状调查方法,筛选出172名居住在上海市区健康成年男女。填写问卷获得研究对象日常饮食频率,检测研究对象血清获得血液激素浓度,采用HPLC法分析负荷大豆异黄酮前后尿中雌马酚等大豆异黄酮24 h排泄量,统计产雌马酚者比例及其与摄食频率和激素的关系。结果负荷前雌马酚生理范围0~33.74μmol/24 h,产雌马酚者比例为30.2%,负荷大豆异黄酮后比例提高至53.5%。产雌马酚者与非产雌马酚者之间日常食品摄取频率的差别无统计学意义(P>0.05)。产Eq者血中游离雌二醇的浓度较非产Eq者低(P<0.05)。结论在通常膳食条件下,约有1/3上海成人尿液中能检测到雌马酚,但负荷大豆异黄酮后,约有1/2能产生雌马酚。     

Biomarkers of legume intake in human intervention and observational studies: a systematic review

There is a growing interest in assessing dietary intake more accurately across different population groups, and biomarkers have emerged as a complementary tool to replace traditional dietary assessment methods. The purpose of this study was to conduct a systematic review of the literature available and evaluate the applicability and validity of biomarkers of legume intake reported across various observational and intervention studies. A systematic search in PubMed, Scopus, and ISI Web of Knowledge identified 44 studies which met the inclusion criteria for the review. Results from observational studies focused on soy or soy-based foods and demonstrated positive correlations between soy intake and urinary, plasma or serum isoflavonoid levels in different population groups. Similarly, intervention studies demonstrated increased genistein and daidzein levels in urine and plasma following soy intake. Both genistein and daidzein exhibited dose-response relationships. Other isoflavonoid levels such as O-desmethylangolensin (O-DMA) and equol were also reported to increase following soy consumption. Using a developed scoring system, genistein and daidzein can be considered as promising candidate markers for soy consumption. Furthermore, genistein and daidzein also served as good estimates of soy intake as evidenced from long-term exposure studies marking their status as validated biomarkers. On the contrary, only few studies indicated proposed biomarkers for pulses intake, with pipecolic acid and S-methylcysteine reported as markers reflecting dry bean consumption, unsaturated aliphatic, hydroxyl-dicarboxylic acid related to green beans intake and trigonelline reported as marker of peas consumption. However, data regarding criteria such as specificity, dose-response and time-response relationship, reliability, and feasibility to evaluate the validity of these markers is lacking. In conclusion, despite many studies suggesting proposed biomarkers for soy, there is a lack of information on markers of other different subtypes of legumes. Further discovery and validation studies are needed in order to identify reliable biomarkers of legume intake.     

Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase,an Enzyme Involved in the Metabolism of Daidzein,from Equol-Producing Lactococcus Strain 20-92

Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.Isoflavones are flavonoids present in various plants and are known to be abundant in soybeans and legumes. These compounds have been called phytoestrogens because their chemical structure is similar to that of the female sex hormone, estrogen. Isoflavones have an ability to bind to estrogen receptors and show protection against or improvement in several sex hormone-dependent diseases, such as breast cancer, prostate cancer, menopausal disorder, lower bone density, and hypertension, due to their weak agonistic or antagonistic effects (1, 19, 27).Daidzein is one of the main soy isoflavonoids produced from daidzin by the glucosidase of intestinal bacteria (17). Equol is a metabolite produced from daidzein by the enterobacterial microflora (5). Recently, equol has attracted a great deal of attention because its estrogenic activity is more potent than that of other isoflavonoids, including daidzein (27). It is well known that individual variation exists in the ability of these enteric microflora to produce equol and that less than half the human population is capable of producing equol after ingesting soy isoflavones (3). Therefore, to increase the production of equol in the enteric environment of each individual, the development of probiotics using safe bacteria which have the ability to produce equol from daidzein is ongoing.Lactococcus strain 20-92 (Lactococcus 20-92; 30a) is an equol-producing lactic acid bacterium isolated from the feces of healthy humans by Uchiyama et al. (30). This bacterium is spherical and Gram positive and is a strain of L. garvieae. The application of Lactococcus 20-92 in probiotics is advantageous because L. garvieae is not pathogenic or toxic to humans.To date, other bacterial strains that are capable of transforming daidzein to dihydrodaidzein or equol have been isolated (9, 21, 22, 23, 29, 32, 36, 37). Daidzein is thought to be metabolized by human intestinal bacteria to equol or to O-desmethylangolensin via dihydrodaidzein and tetrahydrodaidzein (14, 15, 22, 32); however, neither the enzymes involved in the metabolism of daidzein to equol nor even the metabolic pathway has been clarified fully for equol-producing bacteria.In this study, we purified an enzyme from Lactococcus 20-92 that assisted in the conversion of daidzein to dihydrodaidzein. Furthermore, we cloned the L-DZNR gene and expressed the active recombinant enzyme in E. coli.     

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.     

Effect of Circulating Forms of Soy Isoflavones on the Oxidation of Low Density Lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.     

<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.     

Serum equol, bone mineral density and biomechanical bone strength differ among four mouse strains

The extent of conversion of daidzein to its metabolite, equol, by intestinal microflora may be a critical step that determines if a diet rich in daidzein protects against the deterioration of bone after estrogen withdrawal. The objective was to determine the extent that daidzein is converted to equol. In addition, bone mineral content (BMC), bone mineral density (BMD) and strength of femurs and lumbar vertebrae (LV) in four mouse strains were measured. Mice were ovariectomized and fed control diet (AIN93G) with or without daidzein (200 mg daidzein/kg diet) for 3 weeks, after which serum, femurs and LV were collected. Serum daidzein and equol were elevated in all mice fed daidzein. Among mice fed daidzein, the CD-1 and Swiss–Webster (SW) mice had higher (P<.001) serum equol than C57BL/6 (C57) and C3H mice. Differences due to mouse strain were observed for all bone outcomes. C57 mice had lower femur BMC (P<.001), BMD (P<.001) and peak load at femur midpoint (P<.001) and neck (P<.001) than other mouse strains. C57 mice also had a lower femur midpoint yield load (P<.001) and resilience (P<.001) than C3H mice. C57 mice had a lower LV1–4 BMC (P<.001) and BMD (P<.001) compared with all mouse strains and peak load of LV3 was lower than CD-1 and SW mice. Differences in serum equol, BMD and bone strength properties should be considered when selecting a mouse strain for investigating whether dietary strategies that include isoflavones preserve bone tissue after ovariectomy.     

Equol an isoflavonoid: potential for improved prostate health, <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>evidence

Background  

To determine: in vitro binding affinity of equol for 5alpha-dihydrotestosterone (5alpha-DHT), in vitro effects of equol treatment in human prostate cancer (LNCap) cells, and in vivo effects of equol on rat prostate weight and circulating levels of sex steroid hormones.     

Development of a high-throughput LC/APCI-MS method for the determination of thirteen phytoestrogens including gut microbial metabolites in human urine and serum

The investigation into the potential usefulness of phytoestrogens in the treatment of menopausal symptoms requires large-scale clinical trials that involve rapid, validated assays for the characterization and quantification of the phytoestrogenic precursors and their metabolites in biological matrices, as large interindividual differences in metabolism and bioavailability have been reported. Consequently, a new sensitive high-performance liquid chromatography-mass spectrometry method (HPLC–MS) for the quantitative determination of thirteen phytoestrogens including their most important gut microbial metabolites (genistein, daidzein, equol, dihydrodaidzein, O-desmethylangolensin, coumestrol, secoisolariciresinol, matairesinol, enterodiol, enterolactone, isoxanthohumol, xanthohumol and 8-prenylnaringenin) in human urine and serum within one single analytical run was developed. The method uses a simple sample preparation procedure consisting of enzymatic deconjugation followed by liquid–liquid extraction (LLE) or solid-phase extraction (SPE) for urine or serum, respectively. The phytoestrogens and their metabolites are detected with a single quadrupole mass spectrometer using atmospheric pressure chemical ionization (APCI), operating both in the positive and the negative mode. This bioanalytical method has been fully validated and proved to allow an accurate and precise quantification of the targeted phytoestrogens and their metabolites covering the lower parts-per-billion range for the measurement of relevant urine and serum levels following ingestion of phytoestrogen-rich dietary supplements.