Isoflavone conjugates are underestimated in tissues using enzymatic hydrolysis

Many health effects of soy foods are attributed to isoflavones. Isoflavones upon absorption present as free form, glucuronide, and sulfate conjugates in blood, urine, and bile. Little is known about the molecular forms and the relative concentrations of soy isoflavones in target organs. Acid hydrolysis or enzymatic hydrolysis (glucuronidases and sulfatases) was used to study isoflavone contents in the heart, brain, epididymis, fat, lung, testis, liver, pituitary gland, prostate gland, mammary glands, uterus, and kidney from rats fed diets made with soy protein isolate. The heart had the lowest isoflavone contents (undetectable), and the kidney had the highest (1.8 +/- 0.6 nmol/g total genistein; 3.0 +/- 1.1 nmol/g total daidzein). Acid hydrolysis released 20-60% more aglycon in tissues than enzymatic digestion (p < 0.05), and both hydrolysis methods gave the same level of isoflavones in serum. Approximately 28-44% of the total isoflavone content within the liver was unconjugated aglycon, and the remainder was conjugated mainly as glucuronide. The subcellular distribution of total isoflavones was 55-60% cytosolic and 13-16% in each of the nuclear, mitochondrial, and microsomal fractions. These results demonstrated that (1) soy isoflavones distribute in a wide variety of tissues as aglycon and conjugates and (2) the concentrations of isoflavone aglycons, which are thought to be the bioactive molecules, are in the 0.2-0.25 nmol/g range, far below the concentrations required for most in vitro effects of genistein or daidzein.     

Antioxidant activity of isoflavones and their major metabolites using different in vitro assays

Isoflavone phytoestrogens found mainly in soybeans and clover are widely studied phytochemicals. Genistein and daidzein, the major isoflavones found in soy, have received the most attention. However, they undergo extensive metabolism in the intestine and the liver, which might affect their biological properties, e.g. their antioxidant capacities. Furthermore, the biological activities of other naturally occurring isoflavones, for instance, glycitein from soy or biochanin A from red clover, have not yet been studied in detail. The aim of this study was to investigate the antioxidant activities of six naturally occurring isoflavones and their corresponding oxidative and bacterial metabolites. The oxygen radical absorbance capacity assay as well as the in vitro oxidation of low density lipoproteins with the conjugated diene and the thiobarbituric acid reacting substances formation as end points were used. The oxidative metabolites of genistein and daidzein as well as equol exhibited the highest antioxidant activities in all three assays. With few exceptions, they were more effective than the positive controls quercetin and ascorbic acid. Formononetin, the 4'-O-methyl ether of daidzein, showed the lowest antioxidant property. Because the antioxidant efficacy of isoflavones as effective antioxidants is evident at concentrations well within the range found in the plasma of subjects consuming soy products, this biological activity could be of physiological relevance.     

Soy isoflavones and other isoflavonoids activate the human bitter taste receptors hTAS2R14 and hTAS2R39

The aim of this study was to identify the bitter receptor(s) that recognize the bitter taste of the soy isoflavone genistein. Screening of all 25 human bitter receptors revealed genistein as agonist of hTAS2R14 and hTAS2R39. Genistein displayed threshold values of 4 and 8 μM on hTAS2R14 and hTAS2R39 and EC(50) values of 29 and 49 μM, respectively. In addition, the behavior of structurally similar isoflavonoids was investigated. Although the two receptors are not closely related, the results for hTAS2R14 and hTAS2R39 were similar toward most isoflavonoid aglycones. By trend, threshold values were slightly lower on hTAS2R14. Glucosylation of isoflavones seemed to inhibit activation of hTAS2R14, whereas four of five glucosylated isoflavones were agonists of hTAS2R39, namely, glycitin, genistin, acetylgenistin, and malonylgenistin. A total of three hydroxyl substitutions of the A- and B-rings of the isoflavonoids seemed to be more favorable for receptor activation than fewer hydroxyl groups. The concentration of the trihydroxylated genistein in several soy foods exceeds the determined bitter receptor threshold values, whereas those of other soy isoflavones are around or below their respective threshold value. Despite its low concentration, genistein might be one of the main contributors to the bitterness of soy products. Furthermore, the bioactive isoflavonoids equol and coumestrol activated both receptors, indicating that their sensory impact should be considered when used as food ingredients.     

Characterization of isoflavones and their conjugates in female rat urine using LC/MS/MS

Isoflavone phytoestrogens found in soybeans are the most widely studied phytochemicals in human diets and soy infant formulas. The health benefits of the isoflavones daidzein and genistein have been reported, and concerns about potential adverse effects have also been raised. However, the results of direct analysis of isoflavones and their metabolites in biological fluids after consumption of soy-containing diets are scarce. This study describes an LC/MS/MS method for the analysis of isoflavones and their metabolites in the urine of female rats fed diets made with soy protein isolate. Five isoflavones (daidzein, genistein, glycitein, dihydrodaidzein, and O-desmethylangolensin) were identified by comparison with authentic standards. Seventeen conjugates of isoflavones were characterized in the urine, the most unusual being genistein 5-glucuronide and four glucuronide conjugates of reductive metabolites of daidzein. The application of LC/MS/MS to analyze isoflavone metabolites is simple and sensitive, and appears to be an excellent method for determining the bioavailability and metabolism of food phytochemistry.     

Estrogenic activity of glycitein, a soy isoflavone

Glycitein (4',7-dihydroxy-6-methoxyisoflavone) accounts for 5-10% of the total isoflavones in soy food products. The biological activity of this compound has not been reported to date, although numerous studies have been performed with the other soy isoflavones, daidzein and genistein. Glycitein was isolated from soy germ to 99% purity. Weaning female B6D2F1 mice were dosed with glycitein (3 mg/day), genistein (3 mg/day), and diethylstilbestrol (DES) (0.03 microg/day) in 5% Tween 80 by gavage for 4 days. A control group received an equal volume of 5% Tween 80 solution daily. The uterine weight increased 150% with glycitein (p < 0.001), 50% with genistein (p < 0. 001), and 60% with DES (p < 0.001) compared with the control group. DES, 17beta-estradiol, and three isoflavones (daidzein, genistein, and glycitein) were examined for their competitive binding abilities with 17beta-((3)H)estradiol to the estrogen receptor proteins of the B6D2F1 mouse uterine cytosol. The concentrations of each compound required to displace 50% of the ((3)H)estradiol at 5 nM in the competitive binding assay were 1.15 nM DES, 1.09 nM 17beta-estradiol, 0.22 microM genistein, 4.00 microM daidzein, and 3.94 microM glycitein. These data indicated that glycitein has weak estrogenic activity, comparable to that of the other soy isoflavones but much lower than that of DES and 17beta-estradiol.     

Effects of dose and glycosylation on the transfer of genistein into the eggs of the Japanese quail (Coturnix japonica)

Soy isoflavones have been associated with several beneficial effects of soy in human diets. However, most soy is consumed by livestock in the Western countries. It is possible that isoflavones could be transferred and/or accumulated into animal products, which could become additional sources of dietary isoflavones for humans. Our objectives were to determine whether dietary isoflavone genistein could be transferred and/or accumulated into the eggs of Japanese quail (Coturnix japonica) and how the supplementation dosage and glycosylation of the isoflavone would affect this transfer. Adult reproductive female Japanese quail were randomly assigned to treatment groups that received encapsulated 50 or 100 mg genistein or 80 mg genistin per day (four quail per treatment) for 5 days. A control group (two quail) received placebo capsules. Eggs were collected prior to treatment and then daily for 15 days. The egg, separated into yolk and white, and pulverized quail diet were extracted in 80% methanol for 2 h and either centrifuged or filtered before evaporation of the solvent. The extracts were redissolved in 16% acetonitrile for high-performance liquid chromatography (HPLC) analyses. Genistein and genistein metabolites were detected in the egg yolks of treated quail. Trace concentrations of genistein were detected in the control group, due to the presence of genistein derivatives in the diet. Neither genistein nor its metabolites were found in egg white. Levels of genistein in the eggs increased significantly from the 3rd day of supplementation and reached the maximum about 2 days after the supplementation stopped. The higher dose of genistein supplementation resulted in higher genistein concentrations in egg yolks. Glycosylation decreased the transfer and accumulation of genistein into the egg yolks.     

Phytoestrogen content of foods of animal origin: dairy products, eggs, meat, fish, and seafood

Dietary phytoestrogens may be involved in the occurrence of chronic diseases. Reliable information on the phytoestrogen content in foods is required to assess dietary exposure and disease risk in epidemiological studies. However, existing analyses have focused on only one class of these compounds in plant-based foods, and there is only little information on foods of animal origin, leading to an underestimation of intake. This is the first comprehensive study of phytoestrogen content in animal food. We have determined the phytoestrogen content (isoflavones: biochanin A, daidzein, formononetin, genistein, and glycitein; lignans: secoisolariciresinol and matairesinol; coumestrol; equol; enterolactone; and enterodiol) in 115 foods of animal origin (including milk and milk-products, eggs, meat, fish, and seafood) and vegetarian substitutes using liquid chromatography-mass spectrometry (LC-MS) with (13)C-labeled internal standards. Phytoestrogens were detected in all foods analyzed; the average content was 20 microg/100 g of wet weight (isoflavones, 6 microg/100 g; lignans, 6 microg/100 g; equol, 3 microg/100 g; and enterolignans, 6 microg/100 g). In infant soy formula, 19 221 microg/100 g phytoestrogens were detected (compared to 59 microg/100 g in non-soy formula). Our study shows that all foods analyzed contained phytoestrogens and most foods (except for fish, seafood, and butter) contained mammalian phytoestrogens (enterolignans and equol). This is the first comprehensive study of phytoestrogen content of foods of animal origin and will allow for a more accurate estimation of exposure to dietary phytoestrogens.     

Antioxidant properties of soybean isoflavone extract and tofu in vitro and in vivo

Isoflavones in soybean were extracted in the crude form using 80% food-grade ethanol at 80 degrees C for 6 h and followed by concentration and dehydration. The soy extract contained isoflavones primarily in the forms of glucosides. In vitro antioxidant activities of the soy extract containing 20-500 ppm isoflavones were conducted using a Rancimat method. The results showed that soy isoflavone extract had strong in vitro antioxidant activity. There was a dose-dependent response for the in vitro antioxidant activity at the lower concentrations but not at the higher concentrations. In vivo antioxidant property was determined by measuring the antioxidant enzymes, superoxide dismutase, and catalase in various organs of rats that were fed with diets containing partially oxidized oil and various levels of isoflavones for up to 24 weeks. Neither short-term (8 weeks) feeding nor low isoflavone content (50 ppm) induced changes in superoxide dismutase or catalase activities in rats. Only diets containing high isoflavone contents (150 and 250 ppm) showed obvious elevated enzymatic levels in various organs. In addition, a laboratory-prepared tofu containing approximately 50 ppm isoflavones had better effects than the soy extract with the 250 ppm isoflavone group, which indicated that molecules other than isoflavones may have a synergistic effect on in vivo antioxidant enzyme inductions of tofu.     

Experimental determination of octanol-water partition coefficients of quercetin and related flavonoids

Octanol-water partition coefficient (log P) values were determined for flavonoids from the flavone, flavonol, flavanone, and isoflavonoid subclasses. Each flavonoid was dissolved in an octanol-water system and allowed to equilibrate, and then both fractions were analyzed by high-performance liquid chromatography. log P was calculated as log[ratio of the concentration in the octanol phase to the concentration in the aqueous phase at pH 7.4]. The aglycons were more lipophilic than any conjugate. The conjugate moiety had a more significant effect on log P than the aglycon moiety. Quercetin was the least lipophilic aglycon (log P = 1.82 +/- 0.32) and, together with kaempferol (log P = 3.11 +/-0.54), gave the most variable results. The isoflavones genistein and daidzein and the isoflavone metabolite equol gave relatively high log P values (3.04 +/- 0.02, 2.51 +/- 0.06, and 3.20 +/- 0.13, respectively), while glycitein had an unexpectedly low value of 1.97 +/- 0.05. The conjugation characteristics and hydroxylation pattern were the most important determinants of log P in general, and log P was highly variable within the flavonoid subclass. The results are discussed in terms of further understanding of the in vivo fate of the flavonoids as important dietary bioactives.     

Bioavailability of genistein and its glycoside genistin as measured in the portal vein of freely moving unanesthetized rats

The present study describes an in vivo bioavailability experiment for genistein and its glycoside genistin, either as pure compounds or from a soy protein isolate extract, using freely moving unanesthetized rats with a cannulation in the portal vein. The results show that genistein is readily bioavailable, being observed in portal vein plasma at the first point of detection at 15 min after dosing. The AUC(0-24h) values for total genistein and its conjugates were 54, 24, and 13 microM h for genistein, genistin, and an enriched protein soy extract, respectively. These results indicate that the bioavailability of genistein is higher for the aglycon than for its glycoside. Genistin is partly absorbed in its glycosidic form. It is concluded that bioavailability studies based on portal vein plasma levels contribute to insight into the role of the intestine and liver in deglycosylation and uptake characteristics of glycosylated flavonoids.     

Effect of extrusion on isoflavone content and antiproliferative bioactivity of soy/corn mixtures

The present studies were conducted to determine changes in the quantities of select isoflavones and in the bioactivity (ability to inhibit proliferation of human breast cancer cell lines) of extracts from blends of soy protein and cornmeal during extrusion processing. The extrusion of samples resulted in an average 24% decrease in the concentration of total isoflavones for all samples. Although the amounts of specific genistein-derived and daidzein-derived forms changed following extrusion, the content of the aglycones genistein and daidzein per g sample generally did not change. The extrusion of samples generally resulted in decreased antiproliferative action toward breast cancer cells, although antiproliferative activity was not eliminated. Therefore, extrusion of soy protein/cornmeal-containing foods are likely to retain a considerable portion of their isoflavone content and some of the health benefits associated with soy.     

Phytoestrogens modulate binding response of estrogen receptors alpha and beta to the estrogen response element

Binding of estrogen receptor (ER) to estrogen response element (ERE) induces gene activation and is an important step in estrogen-induced biological effects. Here, we investigated the effects of some dietary phytoestrogens such as the isoflavones genistein and daidzein, its metabolite equol, and the coumestane coumestrol on the binding rate of ERalpha and ERbeta to ERE by a nonradioactive real-time method, the Biacore Technology. ERalpha and ERbeta were able to bind to ERE immobilized on the surface of a sensor chip even in the absence of estrogens. 17beta-Estradiol and phytoestrogens induced an increase in ER binding to ERE in a concentration-dependent manner. 17beta-Estradiol was a more potent activator of binding than the phytoestrogens studied. The concentrations of 17beta-estradiol inducing an increase in the binding response of ERalpha and ERbeta to ERE by 50% (EC(50)) as compared to unliganded ER were 0.03 and 0.01 microM, respectively. Regarding the efficacy of activation of ERalpha, from the most to the least effective compound, the sequence and the EC(50) were as follows: 17beta-estradiol (0.03 microM) > coumestrol (0.2 microM) > equol (3.5 microM) > genistein (15 microM) > daidzein (>300 microM) and for ERbeta 17beta-estradiol (0.01 microM) > coumestrol (0.025 microM) > genistein (0.03 microM) > daidzein (0.35 microM) > equol (0.4 microM). The ratios EC(50)alpha/EC(50)beta were calculated to be for 17beta-estradiol, 3; coumestrol, 8; equol, 8.8; genistein, 500; daidzein > 850. These ratios indicate that genistein and daidzein preferentially activate the binding of ERbeta to ERE. The endogenous hormone 17beta-estradiol as well as coumestrol and daidzein metabolite equol activate the binding of ERbeta to ERE only slightly more effectively than the binding of ERalpha to ERE. Thus, the effect of daidzein can be changed from a specific activator of ERbeta to an activator of both ER isotypes alpha and beta in humans who are able to convert daidzein to equol. While the results of the measurements with ERalpha were in line with the binding affinities of compounds tested for ER, there was a distinct difference between our results and the binding affinities of phytoestrogens for the ERbeta. This leads to the conclusion that phytoestrogens differ not only in their binding affinities for the ER, but also in their potential to increase the rate of receptor binding to the ERE.     

Extraction and purification of isoflavones from soybeans and characterization of their estrogenic activities

Soybean isoflavones have multiple beneficial health effects especially on estrogen-deficient diseases such as menopausal symptoms. In this study, isoflavones were produced from soybean flour, and the extraction and purification parameters were optimized to give a high yield of total isoflavones, about 0.62 mg of aglycones/g of soybean flour, which is >2 times the initial yield. HPLC analysis and MTT cell proliferation assay using MCF-7 cells revealed that the product thus obtained not only contained a high content of isoflavone aglycones but also had estrogenic activity. MTT data also revealed that both genistein and daidzein exhibited estrogenic effects at lower concentrations and antiproliferative effects at higher concentrations, and 1 microM genistein and 10 microM daidzein exerted significant estrogenic activities, which were not more than that of the endogenous level of 17beta-estradiol (E2). The production method developed can be used as a guideline for manufacturing soy isoflavones, and the MTT assay was demonstrated to be suitable for quality control on isoflavone products. The results on the estrogenic properties of isoflavones can be used as reference data for their effective and safe usages in estrogenic therapy.     

Isoflavone aglycon and glucoconjugate content of high- and low-soy U.K. foods used in nutritional studies

The isoflavone aglycon and glucoconjugate content of commercially prepared and "home-prepared" high- and low-soy foods selected for use in an on-going nutritional study were measured by LC-MS. The daidzin, daidzein, 6"-O-malonyldaidzin, 6"-O-acetyldaidzin, genistein, genistin, 6"-O-malonylgenistin, 6"-O-acetylgenistin, glycitin, glycitein, 6"-O-malonylglycitin, and 6"-O-acetylglycitin content are expressed in terms of individual isoflavones, total isoflavone equivalents, and milligrams of isoflavones per portion served. Soybeans (774 mg x kg(-1) total isoflavones) and soybean-containing foods had the highest isoflavone content of the foods examined. The low-soy foods all contained very low concentrations (<8 mg x kg(-1) total isoflavones) of the isoflavone aglycons and glucoconjugates. High- and low-soy 11 day rotating menus were constructed from the analyzed foods to deliver 100.0 and 0.5 mg of isoflavones per day, respectively.     

LC/UV/ESI-MS analysis of isoflavones in Edamame and Tofu soybeans

High-performance liquid chromatography coupled with ultraviolet and electrospray ionization mass spectrometry (HPLC/UV/ESI-MSD) was applied to the study of isoflavones in both Edamame and Tofu soy varieties, from which the immature fresh soybeans or the mature soybean seeds are consumed, respectively. Positive atmospheric pressure interface (API) MS and MS/MS were used to provide molecular mass information and led to the identification of a total 16 isoflavones, including three aglycones, three glycosides, two glycoside acetates, and eight glycoside malonates. The major isoflavones in soybean seeds were daidzein and genistein glycoside and their malonate conjugates. Trace levels of daidzein and genistein acetyl glycosides were found only in the mature dry soybean seeds. To facilitate quantitative analysis, acid hydrolysis during extraction of soy samples was selected to convert the various phytoestrogen conjugates into their respective isoflavone aglycones, allowing accurate quantitation of total phytoestrogens as aglycones. On the basis of HPLC combined with UV and MS detection, all three targeted soy isoflavone aglycones, daidzein, genistein and glycitein in hydrolyzed extracts were successfully quantified within 25 min with formononetin used as the internal standard. The standard curves of UV detection were fitted in the range of 14.16-29000 ng/mL for daidzein, 15.38-31500 ng/mL for genistein, and 11.72-24000 ng/mL for glycitein. For MS detection, the standard curves were established in the range of 3.54-1812.5 ng/mL for daidzein, 3.85-1968.75 ng/mL for genistein, and 2.93-1500 ng/mL for glycitein. Good linearities (r(2) > 0.999 for UV and r(2) > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and precision (RSD) were within 10% for UV detection and 15% for MS detection (n = 10). Using this method, the phytoestrogen levels of total isoflavone aglycones from 30 soybean seed varieties were then evaluated for confirmation of the technique. Total isoflavones ranged across the varieties from 0.02 to 0.12% in the Edamame varieties, which are harvested while the seeds are still immature, and from 0.16 to 0.25% in Tofu varieties, harvested when the seeds are physiologically mature. While the literature has focused on the isoflavone content of soy products and processing soy, this report provides a reliable analytical technique for screening of authenticated fresh immature Edamame soybeans and Tofu soybeans.     

Isoflavone glycitein diminished plasma cholesterol in female golden Syrian hamsters

The soybean isoflavones, daidzein, genistein, and glycitein, were hypothesized to act as cholesterol-lowering components, separate from soy protein. Pure synthetic daidzein, genistein, or glycitein (0.9 mmol/kg diet) or a casein-based control diet was fed to groups of 10 female Golden Syrian hamsters for 4 weeks. Hamsters fed glycitein had significantly lower plasma total (by 15%) and non-HDL (by 24%) cholesterol compared with those fed casein (P<0.05). Daidzein and genistein's effects on these lipids did not differ from the effects of either casein or glycitein. Plasma HDL cholesterol and triglyceride concentrations were not significantly affected by dietary treatments. The percentage of urinary recovery of the ingested dose of each isoflavone was glycitein>daidzein>genistein (33.2%, 4.6%, 2.2%, respectively), with the apparent absorption of glycitein significantly greater than that of the other isoflavones. These data suggest that glycitein's greater cholesterol-lowering effect was due to its greater bioavailability, as reflected in its urinary recovery compared with that of the other isoflavones.     

Stabilities of daidzin,glycitin, genistin,and generation of derivatives during heating

The soy isoflavones daidzin, glycitin, and genistin were purified from defatted soy flour using preparative-scale reverse-phase HPLC. The stabilities of the three isoflavones at different heating temperatures were investigated. Daidzin, glycitin, and genistin were lost at a rate of 26, 27, and 27% of their original concentration, respectively, after 3 min at 185 degrees C. At 215 degrees C, decreases of daidzin, glycitin, and genistin were 65, 98, and 74% after 3 min and 91, 99, and 94% after 15 min, respectively. The order of the thermal stabilities, from lowest to highest, was glycitin, genistin, and daidzin. Acetyl daidzin and acetyl genistin, daidzein, glycitein, and genistein were produced during heating at temperatures above 135 degrees C. The rate of binding of an acetyl group to form acetyl daidzin and acetyl genistin from daidzin and genistin was higher than the rate of loss of a glucoside group to form daidzein and genistein. However, acetyl daidzin and acetyl genistin decreased sharply at temperatures above 200 degrees C, while daidzein, glycitein, and genistein were relatively stable over 30 min. The stability of daidzein was higher than that of glycitein or genistein.     

Heat and pH effects on the conjugated forms of genistin and daidzin isoflavones

Isoflavones occur primarily as glycosides (namely, malonyl-, acetyl-, and non-conjugated beta-glycosides) and a small percentage as the bioactive aglycon. The different chemical structures of isoflavones can dictate their stability during processing. Therefore, our objective was to determine the effects of pH and thermal treatments on conjugated isoflavones with regard to interconversions and loss. Conjugated daidzin and genistin were heated at 25, 80, and 100 degrees C under neutral, acidic, and basic conditions. Changes in isoflavone derivatives were monitored using high-performance liquid chromatography. Along with interconversions, considerable loss in total known isoflavone derivatives was noted for each isoflavone, especially under elevated pH and temperature. The malonylglycosides showed more stability than acetylglycosides, especially under acidic conditions. Overall, loss in isoflavone derivatives was significantly higher for daidzin than for genistin glycoside forms. Our results highlighted the significance of chemical structure with regard to stability, which is a key factor in determining soy processing conditions.     

Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo.

The soy isoflavones daidzein and genistein are found in high concentrations in human plasma and urine after soy consumption. However, in vitro and in vivo data regarding the oxidative metabolism of isoflavones in humans are scarce. Therefore, we have studied the oxidative metabolites of these compounds formed in human liver microsomes and excreted in urine of male and female humans ingesting soy products for 2 days. Human liver microsomes transformed the soy isoflavone daidzein to three monohydroxylated and three dihydroxylated metabolites according to GC/MS analysis. On the basis of a previous study with rat liver microsomes and with the help of reference substances, these metabolites were identified as 6,7,4'-trihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone, 7,8,4'-trihydroxyisoflavone, 7,8,3',4'-tetrahydroxyisoflavone, 6,7,8,4'-tetrahydroxyisoflavone, and 6,7,3',4'-tetrahydroxyisoflavone. Significant amounts of the same metabolites except 6,7,8,4'-tetrahydroxyisoflavone were also found in urine of female and male volunteers after soy intake. Genistein was metabolized by human liver microsomes to six hydroxylation products. The main metabolites were the three aromatic monohydroxylated products 5,6,7,4'-tetrahydroxyisoflavone, 5,7,8,4'-tetrahydroxyisoflavone and 5,7,3',4'-tetrahydroxyisoflavone. The aliphatic monohydroxylated metabolite 2,5,7,4'-tetrahydroxyisoflavone and two aromatic dihydroxylated metabolites, 5,7,8,3',4'-pentahydroxyisoflavone and 5,6,7,3',4'-pentahydroxyisoflavone, were formed in trace amounts. The same hydroxylated genistein metabolites except the aliphatic hydroxylated one could also be detected in human urine samples. Methylated forms of the catechol metabolites, which were generated by incubations with catechol-O-methyltransferase in vitro could be detected only in trace amounts in the urine samples. This implies that this reaction does not play a major role in the biotransformation of the hydroxylated daidzein and genistein metabolites in vivo. Most of these oxidative metabolites are described as human in vivo metabolites for the first time. Their biological significance remains to be established.