Dietary walnut oil modulates liver steatosis in the obese Zucker rat.

PURPOSE: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. We aimed to clarify the impact of dietary walnut oil versus animal fat on hepatic steatosis, representing the initial step of multistage pathogenesis of NAFLD, in Zucker obese rats. METHODS: Zucker lean ad libitum (a.l.), Zucker obese a.l. or Zucker obese pair fed (p.f.) to the lean received isocaloric diets containing 8% walnut oil (W8), W14 or 14% lard (L14) (n = 10/group). Body weight, clinical serology, liver weight, lipid content and fatty acid composition and hepatic lipid metabolism-related transcripts were evaluated. RESULTS: Compared to lean, Zucker obese a.l. and p.f. showed hepatic triacylglyceride (TAG) accumulation. In Zucker obese p.f., W14 compared to W8 and L14 reduced liver lipids, TAG as well as hepatic omega-6 (n-6)/n-3 ratio and SCD activity index [(C18:0 + C18:1)/C18:0 ratio] paralleled by decreased lipoprotein lipase mRNA in obese p.f. and elevated microsomal triglyceride transfer protein mRNA in lean and obese. Further, W14 elevated the fasting blood TAG and reduced cholesterol levels in obese. CONCLUSIONS: In our model, consumption of W14 inhibited hepatic lipid accumulation along with modulated hepatic gene expression implicated in hepatic fatty acid influx or lipoprotein assembly. These results provide first indication that dietary lipids from walnut oil are modulators of hepatic steatosis as the initial step of progressive NAFLD pathogenesis.

Dietary fat quality in regular fat diets has minor effects on biomarkers of inflammation in obese Zucker rats.

PURPOSE: Adipose tissue-associated chronic inflammation is involved in the pathogenesis of obesity-related diseases. Dietary fatty acids are known to influence inflammatory processes. The aim of this study was to investigate, whether diets with regular fat contents but variable fat qualities affect adipose tissue-associated inflammation through the fatty acid composition of mesenteric adipose tissue (MAT). METHODS: Obese Zucker rats were fed diets containing 7 % wt:wt rapeseed oil, corn oil, or lard for 10 weeks. Fatty acid composition and endocrine function regarding adipokines and cytokines of MAT, number of total CD3(+) T cells, and cytokine secretion of mesenteric lymph node (MLN)-derived lymphocytes were determined. Local effects in MAT and MLN were compared to systemic effects assessed in serum and peripheral blood mononuclear cells. RESULTS: Fatty acid composition of MAT reflected dietary fatty acid intake, without affecting endocrine function. Feeding the lard diet for 10 weeks increased the serum adiponectin and TNF-α secretion of blood lymphocytes, whereas CD3(+) T cells in blood were decreased. No effects were seen for the secretion of adipokines and cytokines from MAT, the amount of T cells in MLN, and cytokine secretion of MLN lymphocytes. CONCLUSIONS: In conclusion, feeding obese rats a diet with regular fat content but variable fat sources for 10 weeks, changed the fatty acid composition of MAT but not its secretory properties or MLN functions. Although the local immune system was not influenced, lard-feeding induced minor changes in systemic immune function.

LC-MS/MS quantification of sulforaphane and indole-3-carbinol metabolites in human plasma and urine after dietary intake of selenium-fortified broccoli.

This study aimed at developing a sensitive LC-MS/MS method for the quantification of sulforaphane (SFN) and indole-3-carbinol metabolites in plasma and urine after dietary intake of regular and selenium-fertilized broccoli using stable isotope dilution analysis. In a three-armed, placebo-controlled, randomized human intervention study with 76 healthy volunteers, 200 g of regular (485 µg of total glucosinolates and <0.01 µg of selenium per gram fresh weight) or selenium-fertilized broccoli (589 µg of total glucosinolates and 0.25 µg of selenium per gram fresh weight) was administered daily for 4 weeks. Glucoraphanin and glucobrassicin metabolites quantified in plasma and urine were SFN-glutathione, SFN-cysteine, SFN-cysteinylglycine, SFN-acetylcysteine, and indole-3-carboxaldehyde, indole-3-carboxylic acid, and ascorbigen, respectively. Dietary intake of selenium-fertilized broccoli increased serum selenium concentration analyzed by means of atomic absorption spectroscopy by up to 25% (p < 0.001), but affected neither glucosinolate concentrations in broccoli nor their metabolite concentrations in plasma and urine compared to regular broccoli.

Anthocyanin-rich blackberry extract suppresses the DNA-damaging properties of topoisomerase I and II poisons in colon carcinoma cells.

In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 µM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 µM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 µM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.

Influence of ultra-high pressure homogenisation on antioxidant capacity, polyphenol and vitamin content of clear apple juice.

Ultra-high pressure homogenisation (UHPH) is a recently developed technology and is still under study to evaluate its effect on different aspects of its application to food products. The aim of this research work was to evaluate the effect of UHPH treatments on quality characteristics of apple juice such as antioxidant capacity, polyphenol composition, vitamin C and provitamin A contents, in comparison with raw (R) and pasteurised (PA) apple juice. Several UHPH treatments that include combinations of pressure (100, 200 and 300MPa) and inlet temperatures (4 and 20°C) were assayed. Apple juice was pasteurised at 90°C for 4min. Antioxidant capacity was analysed using the oxygen radical antioxidant capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP) assay while total phenolic content was determined by the Folin-Ciocalteau assay. According to the FRAP and DPPH assays, UHPH processing did not change apple juice antioxidant capacity. However, significant differences were detected between samples analysed by TEAC and ORAC assays. In spite of these differences, high correlation values were found between the four antioxidant capacity assays, and also with total polyphenol content. The analysis and quantification of individual phenols by HPLC/DAD analytical technique reflects that UHPH-treatment prevented degradation of these compounds. Vitamin C concentrations did not change in UHPH treated samples, retaining the same value as in raw juice. However, significant losses were observed for provitamin A content, but lower than in PA samples. UHPH-treatments at 300MPa can be an alternative to thermal treatment in order to preserve apple juice quality.

The production of aflatoxin B1 or G 1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression.

The influence of varying combinations of water activity (aw) and temperature on growth, aflatoxin biosynthesis and aflR/aflS expression of Aspergillus parasiticus was analysed in the ranges 17-42°C and 0.90-0.99 aw. Optimum growth was at 35°C. At each temperature studied, growth increased from 0.90 to 0.99 aw. Temperatures of 17 and 42°C only supported marginal growth. The external conditions had a differential effect on aflatoxin B1 or G1 biosynthesis. The temperature optima of aflatoxin B1 and G1 were not at the temperature which supported optimal growth (35°C) but either below (aflatoxin G1, 20-30°C) or above (aflatoxin B1, 37°C). Interestingly, the expression of the two regulatory genes aflR and aflS showed an expression profile which corresponded to the biosynthesis profile of either B1 (aflR) or G1 (aflS). The ratios of the expression data between aflS:aflR were calculated. High ratios at a range between 17 and 30°C corresponded with the production profile of aflatoxin G1 biosynthesis. A low ratio was observed at >30°C, which was related to aflatoxin B1 biosynthesis. The results revealed that the temperature was the key parameter for aflatoxin B1, whereas it was water activity for G1 biosynthesis. These differences in regulation may be attributed to variable conditions of the ecological niche in which these species occur.

No effect of the farming system (organic/conventional) on the bioavailability of apple (Malus domestica Bork., cultivar Golden Delicious) polyphenols in healthy men: a comparative study.

BACKGROUND: The organic food sales have been increasing during the recent years. It has been hypothesised that organically grown fruits are healthier based on their higher content of phytochemicals. However, data on the bioavailability of phytochemicals from organically or conventionally produced plant foods are scarce. METHODS: Two human intervention studies were performed to compare the bioavailability of polyphenols in healthy men after ingestion of apples from different farming systems. The administered apples were grown organically and conventionally under defined conditions and characterised regarding their polyphenol content and antioxidant capacity. No significant differences in the polyphenol content and the antioxidant capacity from the organic and conventional farming system were observed. RESULTS: In the short-term intervention study, six men consumed either organically or conventionally produced apples in a randomized cross-over study. After intake of 1 kg apples, phloretin (C (max) 13 + or - 5 nmol/l, t (max) 1.7 + or - 1.2 h) and coumaric acid (C (max )35 + or - 12 nmol/l, t (max) 3.0 + or - 0.8 h) plasma concentrations increased significantly (P < 0.0001) in both intervention groups, without differences between the two farming systems. In the long-term intervention study, 43 healthy volunteers consumed organically or conventionally produced apples (500 g/day; 4 weeks) or no apples in a double-blind, randomized intervention study. In this study, 24 h after the last dosing regime, the apple intake did not result in increasing polyphenol concentrations in plasma and urine compared to the control group suggesting no accumulation of apple polyphenols or degradation products in humans. CONCLUSION: Our study suggests that the two farming systems (organic/conventional) do not result in differences in the bioavailability of apple polyphenols.

Influence of the production method on phytochemical concentrations in whole wheat (Triticum aestivum L.): a comparative study.

The present study was performed to evaluate the concentrations of phytochemicals (carotenoids and phenolic acids) in wheat varieties grown under comparable organic and conventional conditions over three years as part of a long-term field trial. Phenolic acids of the hydroxybenzoic acid and hydroxycinnamic acid group were quantified by HPLC-DAD after extraction as free, soluble conjugated, and bound forms. Furthermore, the carotenoids lutein and zeaxanthin were determined by HPLC-DAD. There were no statistically significant differences between the two farming systems (sum of carotenoids (microg/g) for 2003, 0.91 +/- 0.55 organic vs 0.96 +/- 0.34 conventional; for 2005, 1.61 +/- 0.22 organic vs 1.33 +/- 0.19 conventional; for 2006, 0.87 +/- 0.33 organic vs 0.83 +/- 0.11 conventional; sum of phenolic acids (microg/g) for 2003, 448.4 +/- 151.1 organic vs 327.3 +/- 232.8 conventional; for 2005, 502.8 +/- 168.3 organic vs 484.4 +/- 111.2 conventional; for 2006, 659.1 +/- 112.5 organic vs 945.9 +/- 353.6 conventional). However, statistically significant year-to-year differences up to 55% were observed. Taken together, these results indicate that climate factors have a greater impact on the phytochemical concentrations in whole wheat than the production method (organic/conventional).

Role of plasma lipoproteins in the transport of the soyabean isoflavones daidzein and daidzein-7-O-beta-D-glucoside.

Isoflavone intake is associated with various properties beneficial to human health which are related to their antioxidant activity, for example, to their ability to increase LDL oxidation resistance. However, the distribution of isoflavones among plasma lipoproteins has not yet been elucidated in vivo. Therefore, the objective of the present study was to investigate the association between daidzein (DAI) and lipoproteins in human plasma upon administration of the aglycone and glucoside form. Five men aged 22-30 years participated in a randomised, double-blind study in cross-over design. After ingestion of DAI and daidzein-7-O-beta-D-glucoside (DG) (1 mg DAI aglycone equivalents/kg body weight) blood samples were drawn before isoflavone administration as well as 1, 2, 3, 4.5, 6, 8, 10, 12, 24 and 48 h post-dose. Concentrations of DAI in the different lipoprotein fractions (chylomicrons, VLDL, LDL, HDL) and in the non-lipoprotein fraction were analysed using isotope dilution capillary GC/MS. The lipoprotein fraction profiles were similar for all subjects and resembled those obtained for plasma in our previously published study. The lipoprotein distribution based on the area under the concentration-time profiles from 0 h to infinity in the different fractions were irrespective of the administered form: non-lipoprotein fraction (53%) > LDL (20%) > HDL (14%) > VLDL (9.5%) > chylomicrons (2.5%). Of DAI present in plasma, 47 % was associated to lipoproteins. Concentrations in the different lipoprotein fractions as well as in the non-lipoprotein fraction were always higher after the ingestion of DG than of DAI. Taken together, these results demonstrate an association between isoflavones and plasma lipoproteins in vivo.

Three-year comparison of the polyphenol contents and antioxidant capacities in organically and conventionally produced apples ( Malus domestica Bork. Cultivar 'Golden Delicious').

The present study was performed to evaluate the polyphenol content and antioxidant capacity of apples (cv. ;Golden Delicious') grown under defined organic and conventional conditions. Apples were harvested at five comparable commercial farms over the course of three years (2004-2006). In 2005 and 2006 the antioxidant capacity was 15% higher (p < 0.05) in organically produced apples than in conventionally produced fruits. In 2005 significantly higher polyphenol concentrations were found in the organically grown apples. In 2004 and 2006 no significant differences were observed (2004, 304 +/- 68 microg/g organic vs 284 +/- 69 microg/g conventional, p = 0.18; 2005, 302 +/- 58 micro/g organic vs 253 +/- 41 microg/g conventional, p = 0.002; 2006, 402 +/- 100 microg/g organic vs 365 +/- 58 microg/g conventional, p = 0.17). Year-to-year variations in the antioxidant capacity and the polyphenol content of up to 20% were more significant than the production method found within one year. Finally, flavanols and flavonols were major determinants of the antioxidant capacities in these apples. Overall, the production method had a smaller impact on the variation in the polyphenol content and antioxidant capacity of apples than the yearly climate.

Bioavailability and nutritional effects of carotenoids from organically and conventionally produced carrots in healthy men.

It has been hypothesised that organically grown vegetables are healthier than conventionally produced ones due to a higher content of phytochemicals. However, few data from controlled human studies exist. The aim of the present study was to compare the carotenoid content and antioxidant capacity of organically and conventionally produced carrots under defined conditions. Furthermore, a human intervention study was conducted to compare bioavailability, plasma antioxidant capacity, endogenous DNA strand breaks and immune parameters in healthy men ingesting carrots from different agricultural systems. Thirty-six volunteers consumed either organically or conventionally produced blanched carrots (200 g/d; 2 weeks) or no carrots in a double-blind, randomised intervention study. No statistically significant differences were observed in the total carotenoid contents (121 (sd 7) microg/g organic v. 116 (sd 13) microg/g conventional) and the antioxidant capacities (0.43 (sd 0.08) mumol Trolox equivalents/g organic v. 0.32 (sd 0.07) mumol Trolox equivalents/g conventional) of the carrots administered to the volunteers. Intake of organically or conventionally produced carrots significantly increased (P < 0.001) plasma alpha- and beta-carotene concentrations in both intervention groups without differences in plasma carotenoid concentrations. Consumption of carrots did not lead to significant changes in the plasma antioxidant status, endogenous DNA strand breaks and parameters of the immune system in all groups. Therefore, these results indicate that the agricultural system has neither an effect on the carotenoid content, the antioxidant capacity of carrots, nor on the bioavailability of carotenoids and on antioxidant, antigenotoxic and immunological effects as assessed in a human intervention study.

A strain of Fusarium kyushuense is able to produce aflatoxin B1 and G 1.

A strain of F. kyushuense is able to produce aflatoxin B1 and G1 under certain growth conditions. This was ultimately demonstrated by LC-MS analysis. By using a microarray covering oligonucleotides specific for the aflatoxin pathway genes of A. flavus, the expression of homologues of nearly the complete aflatoxin biosynthesis gene cluster was detected in F. kyushuense under producing conditions, e.g., after growth in liquid YES medium. In contrast, after growth of F. kyushuense on solid YES medium, only a very low transcription level of one of the homologues (nor-1) could be observed by microarray and Real Time PCR demonstrating differential gene expression. The cross-hybridization of the F. kyushuense mRNA to the A. flavus aflatoxin biosynthesis gene oligonucleotides of the array indicates homology between the sequences. PCR analyses with primers deduced from the nor-1 gene sequence of the aflatoxin biosynthesis gene cluster from A. flavus revealed the presence of a homologue of this gene in F. kyushuense. Sequencing of PCR fragments of the nor-1 homologue revealed a sequence similarity of >90% to the nor-1 gene of A. flavus.

Pharmacokinetics of the soybean isoflavone daidzein in its aglycone and glucoside form: a randomized, double-blind, crossover study.

BACKGROUND: There are conflicting results in the literature on the bioavailability of isoflavones in the aglycone and the glucoside forms. OBJECTIVE: The objective was to investigate the pharmacokinetics of the soy isoflavone daidzein (DAI) on oral administration of both the aglycone and glucoside form in a human intervention study. In addition, the pharmacokinetics of the bacterial and oxidative metabolites of DAI was assessed. DESIGN: Seven German men aged 22-30 y participated in a randomized, double-blind study in a crossover design. After ingestion of pure DAI or pure daidzein-7-O-beta-d-glucoside (DG) (1 mg DAI aglycone equivalent/kg body weight), blood samples were drawn before isoflavone administration and 1, 2, 3, 4.5, 6, 8, 10, 12, 24, and 48 h after the dose. Urine was collected before and 0-6, 6-12, and 12-24 h after the intake of the isoflavones. The concentrations of DAI and its major bacterial and oxidative metabolites in plasma and urine were measured with isotope dilution capillary gas chromatography-mass spectrometry. RESULTS: The systemic bioavailability (area under the curve; AUC(inf)), the maximal plasma concentration (C(max)), and the cumulative recovery of DAI in urine after administration of DG were 3-6 times greater than after the ingestion of DAI. Except for equol, which was formed by only one volunteer, all other quantified metabolites exhibited 2-12 times greater AUC(inf), C(max), and urinary recoveries after consumption of DG. CONCLUSION: Our results show that DG exhibits a greater bioavailability than its aglycone when ingested in an isolated form.

Bioavailability of astaxanthin stereoisomers from wild (Oncorhynchus spp.) and aquacultured (Salmo salar) salmon in healthy men: a randomised, double-blind study.

The objective of the present study was to investigate the bioavailability and the configurational isomer distribution of the carotenoid astaxanthin (AST) in human plasma after ingestion of wild (Oncorhynchus spp.) and aquacultured (Salmo salar) salmon. In a randomised and double-blind trial, twenty-eight healthy men consumed 250 g wild or aquacultured salmon daily for 4 weeks which provided 5 mug AST/g salmon flesh. The plasma AST concentrations as well as the isomer distribution were measured by HPLC using a reversed and a chiral stationary phase. After 6 d of intervention with salmon, plasma AST concentrations reached a plateau of 39 nmol/l after consumption of wild salmon and of 52 nmol/l after administration of aquacultured salmon. At days 3, 6, 10 and 14 -- but not at day 28 -- the AST concentrations in human plasma were significantly greater after ingestion of aquacultured salmon. After administration of wild salmon, the (3S,3'S) isomer predominated in plasma (80 %), whereas after intake of aquacultured salmon the meso form (3R,3'S) prevailed (48 %). Therefore, the AST isomer pattern in human plasma resembles that of the ingested salmon. However, after consumption of both wild and aquacultured salmon for 28 d the relative proportion of the (3S,3'S) isomer was slightly higher and the (3R,3'R) form lower in human plasma compared with the isomer distribution in salmon flesh. A selective process of isomer absorption could be responsible for the observed differences in the relative proportions of the (3S,3'S) and (3R,3'R) isomers in human plasma compared with salmon flesh.

Effect of consumption of organically and conventionally produced apples on antioxidant activity and DNA damage in humans.

The present study was performed to compare the effects on antioxidant activity and on DNA damage of organic and conventionally produced apples grown under controlled conditions in human peripheral blood lymphocytes. Six healthy volunteers consumed either organically or conventionally grown apples (Golden Delicious, 1000 g) from two neighboring commercial farms in a double-blinded, randomized, cross-over study. The average content of total identified and quantified polyphenols in the organically and conventionally produced apples was 308 and 321 microg/g fresh weight, respectively. No statistically significant differences in the sum of phenolic compounds or in either of the polyphenol classes were found between the agricultural methods. Consumption of neither organically nor conventionally grown apples caused any changes in antioxidant capacity of low-density lipoproteins (lag time test), endogenous DNA strand breaks, Fpg protein-sensitive sites, or capacity to protect DNA against damage caused by hydrogen peroxide. However, a statistically significant decrease in the levels of endonuclease III sensitive sites and an increased capacity to protect DNA against damage induced by iron chloride were determined 24 h after consumption in both groups of either organic or conventionally grown apples, indicating the similar antigenotoxic potential of both organically and conventionally grown apples.

In vitro and in vivo metabolism of the soy isoflavone glycitein.

Glycitein (GLY) is a major isoflavone of soy germ which is used as a functional ingredient to enrich foods with isoflavones as well as a component of soy supplements. Since data on the metabolism of GLY are incomplete, the in vitro phase I metabolism of GLY using rat liver microsomes, human liver microsomes (HLM), as well as human fecal flora was investigated. Furthermore, the in vivo metabolism has been studied after administration of GLY to Sprague-Dawley rats. The identification of the formed metabolites has been elucidated using HPLC/DAD, HPLC/API-ES MS, GC/MS, as well as reference compounds. With respect to the phase I metabolism, GLY has been converted to ten metabolites by liver microsomes of Aroclor-induced Wistar rats with 8-hydroxy(OH)-GLY as the main metabolite. HLM converted GLY to six metabolites with 8-OH-GLY and 6-OH-daidzein (DAI) being the major products. No sex-related differences were observed in each system. Intestinal metabolism of GLY led to four metabolites with 6-OH-DAI as the main product. The in vivo metabolism in rat urine after single-dose administration of GLY resulted in the identification of three oxidative and two bacterial metabolites as well as the demethylation product 6-OH-DAI as the main metabolite.

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.

Structural elucidation of hydroxylated metabolites of the isoflavan equol by gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry.

Equol has, as have other isoflavonoids, recently gained considerable interest due to its possible health effects. However, detailed studies on the metabolism of equol are scarce. Therefore, we investigated the phase I metabolism of equol using liver microsomes from Aroclor-treated male Wistar rats as well as from a male human. The identification of the metabolites formed was elucidated using high performance liquid chromatography (HPLC) with diode array detection, HPLC/atmospheric pressure ionization electrospray mass spectrometry, and gas chromatography-mass spectrometry, as well as reference compounds. (+/-)-Equol was converted to 11 metabolites by the liver microsomes from Aroclor-pretreated rats comprising three aromatic monohydroxylated and four aliphatic monohydroxylated as well as four dihydroxylated products. The main metabolite was identified as 3'-hydroxy-equol. Using human liver microsomes, equol was converted to six metabolites with 3'-hydroxy- and 6-hydroxy-equol as main products. Furthermore, the aliphatic hydroxylated metabolite 4-hydroxyequol, which was recently detected in human urine after soy consumption, was formed. On the basis of these findings, it is suggested that phase I metabolism of equol is part of a complex biotransformation of the soy isoflavone daidzein in humans in vivo.