Phytoestrogens Daidzein Research Articles

Effects of the Phytoestrogens Genistein and Daidzein on BRCA2 Tumor Suppressor Gene Expression in Breast Cell Lines

A high intake of isoflavones is associated with a reduction of breast cancer risk among Japanese women. The aim of this study was to quantify BRCA2 tumor suppressor gene expression after treatment of cells with the phytoestrogens daidzein and genistein, the main compounds of soy. The effects of 5μg/ml genistein and 20μg/ml daidzein on BRCA2 expression were studied in two human mammary tumor cell lines, MCF7 andMDA-MB-231, and one normal human breast epithelial cell line, MCF10a. BRCA2 mRNA was evaluated by quantitative real time RT-PCR and the amount of BRCA2 protein was measured by affinity chromatography. With genistein, we observed a 60% increase of BRCA2 mRNA in MDA-MB-231 and MCF10a, which are, respectively, estrogen receptors a-/β+and α-/β-, and no variation in MCF7, which is ERα+/β+. Daidzein had no effect on BRCA2 mRNA expression. The level of BRCA2 protein with both food components also remained unchanged in all three cell lines. This suggests regulation of BRCA2 between the mRNA and protein levels. Treatment with actinomycin D and cycloheximide demonstrated that the increase in BRCA2 mRNA was not blocked by cycloheximide, indicating that de novo protein synthesis was required in MDA-MB-23, although de novo protein synthesis was not required in MCF10a for the genistein. With actinomycin D, genistein had a positive action on the transcriptional level of BRCA2 mRNA in MDA-MB-231 and MCF10a.Theuse ofananti-estrogen suggested that the action of daidzein and genistein on BRCA2 expression in human breast cell lines might not be mediated through the ER.

The Clinical Importance of the Metabolite Equol—A Clue to the Effectiveness of Soy and Its Isoflavones

Equol [7-hydroxy-3-(4'-hydroxyphenyl)-chroman] is a nonsteroidal estrogen of the isoflavone class. It is exclusively a product of intestinal bacterial metabolism of dietary isoflavones and it possesses estrogenic activity, having affinity for both estrogen receptors, ERalpha and ERbeta. Equol is superior to all other isoflavones in its antioxidant activity. It is the end product of the biotransformation of the phytoestrogen daidzein, one of the two main isoflavones found in abundance in soybeans and most soy foods. Once formed, it is relatively stable; however, equol is not produced in all healthy adults in response to dietary challenge with soy or daidzein. Several recent dietary intervention studies examining the health effects of soy isoflavones allude to the potential importance of equol by establishing that maximal clinical responses to soy protein diets are observed in people who are good "equol-producers." It is now apparent that there are two distinct subpopulations of people and that "bacterio-typing" individuals for their ability to make equol may hold the clue to the effectiveness of soy protein diets in the treatment or prevention of hormone-dependent conditions. In reviewing the history of equol, its biological properties, factors influencing its formation and clinical data, we propose a new paradigm. The clinical effectiveness of soy protein in cardiovascular, bone and menopausal health may be a function of the ability to biotransform soy isoflavones to the more potent estrogenic isoflavone, equol. The failure to distinguish those subjects who are "equol-producers" from "nonequol producers" in previous clinical studies could plausibly explain the variance in reported data on the health benefits of soy.

Daidzein and genistein content of cereals.

To analyse 75 cereals and three soy flours commonly eaten in Europe for the phytoestrogens daidzein and genistein. The phytoestrogens daidzein and genistein were extracted from dried foods, and the two isoflavones quantified after hydrolytic removal of any conjugated carbohydrate. Completeness of extraction and any procedural losses of the isoflavones were accounted for using synthetic daidzin (7-O-glucosyl-4'-hydroxyisoflavone) and genistin (7-O-glucosyl-4'5-dihydroxyisoflavone) as internal standards. Foods from the Cambridge UK area were purchased, prepared for eating, which included cooking if necessary, and freeze dried. Three stock soy flours were also analysed. Eighteen of the foods assayed contained trace or no detectable daidzein or genistein. The soy flours were rich sources, containing 1639-2117 mg/kg. The concentration of the two isoflavones in the remaining foods ranged from 33 to 11,873 micro g/kg. These analyses will supply useful information to investigators determining the intake of phytoestrogens in cereal products in order to relate intakes to potential biological activities. This work was supported by the United Kingdom Medical Research Council, Ministry of Agriculture Fisheries and Food (contract FS2034) and the United States of America Army (contract DAMD 17-97-1-7028).

Determination of the isoflavonoids genistein and daidzein in biological samples by gas chromatography-mass spectrometry.

The marked differences in the incidences of both breast and prostate cancer between the East and the West have been attributed to habitual diet. Traditionally, Japanese and Far Eastern people in general consume large quantities of soya and soya-derived foodstuffs. Diphenolic soya phytoestrogens have weak oestrogenic and anti-oestrogenic properties and have been implicated in preventing or limiting the early processes associated with breast and prostate carcinogenesis. We have developed a gas chromatography-mass spectrometry procedure that is suitable for measurement of the phytoestrogens daidzein and genistein in serum, urine and tissue samples. In serum samples of Japanese subjects mean (standard deviation) concentrations of daidzein [men, 281 (375.5) nmol/L; women, 246 (369.4) nmol/L] and genistein (men, 493 (604.4) nmol/L; women, 502 (717.6) nmol/L] were approximately 15 times higher than the mean levels achieved in British men [daidzein, 18.2 (20.4) nmol/L; genistein, 34.1 (27.2) nmol/L] and women [daidzein, 13.5 (11.6) nmol/L; genistein, 30.1 (31.2) nmol/L]. In pharmacokinetic studies of British subjects, maximum levels of daidzein and genistein were achieved within 6-8 h after the consumption of a cereal bar containing 20 mg of soya isoflavonoids; these levels were very similar to the mean levels achieved in normal Japanese subjects. Unlike serum, the mean daidzein concentration in urine from British subjects was higher than the mean genistein concentration (1.66 and 0.72 micromol per 24 h, respectively); following soy supplementation, urinary isoflavonoid levels were increased at least 10-fold. Serum daidzein and genistein concentrations are lower in British subjects than in Japanese subjects; this may be due to dietary differences.

Detection of phytoestrogens in samples of second trimester human amniotic fluid

There is widespread concern that fetal exposure to hormonally active chemicals may adversely affect development of the reproductive tract. Therefore, the present study was performed to develop the necessary analytical methods and test the hypothesis that dietary phytoestrogens can be quantified in second trimester human amniotic fluid. Amniotic fluid samples ( n=59) from women ( n=53) undergoing routine amniocentesis between 15 and 23 weeks of gestation were analyzed by gas chromatography/mass spectrometric (GC/MS). Analytes included the phytoestrogens daidzein, genistein, formononetin, biochanin A, and coumestrol. Dietary phytoestrogens were quantified in 96.2% of second trimester amniotic fluid samples tested. The mean (±standard deviation (S.D.)) concentration of daidzein and genistein in amniotic fluid was 1.44±1.34 and 1.69±1.48 ng/ml with maximum levels of 5.52 and 6.54 ng/ml, respectively. Second trimester amniotic fluid contains quantifiable levels of dietary phytoestrogens and thus is a marker of mid pregnancy fetal exposure.

Transplacental transfer of the phytoestrogen daidzein in DA/Han rats.

Disposition and transplacental transfer of the phytoestrogen daidzein was studied in pregnant DA/Han rats on day 18 of gestation. Daidzein concentrations were determined by HPLC in maternal blood, maternal organs (liver, kidney, uterus), placenta and fetuses (liver and residual tissues) at specific times (5, 10, 20, 40 and 120 min) after intravenous administration of 10 mg/kg body weight. Early after injection, the majority of circulating daidzein was still in the aglycone form; at later time points the majority consisted of conjugates. The initially high isoflavone concentration in maternal plasma (about 25 microg/ml at 5 min) decreased rapidly within the first hour, and after 2 h total daidzein was below 1 microg/ml. Despite its efficient conjugation, daidzein was rapidly distributed in the organism: peak concentrations were attained 10 min after intravenous administration in all tissues analysed, with mean values of about 31 microg/g in maternal liver, 13 microg/g in kidneys and 5 microg/g in the uterus. Placenta contained about one-tenth the hepatic daidzein concentration, and fetal liver about 1/30 the peak concentration of maternal liver (i.e. 1.3 microg/g, which is one-third the placental concentration). Daidzein levels in tissues then declined in parallel with those in maternal blood. The data show that daidzein is transferred across the placenta of DA/Han rats to fetuses. This is indicative of a rapid transfer from the mother to the fetus, but also that efficient hepatic extraction of daidzein from the maternal blood occurs. Since dietary phytoestrogens account for a significant proportion of human exposure to potential endocrine modulators, and since the placenta does not represent a barrier to daidzein or related estrogenic isoflavones, the consequences of these exposures early in life should be examined and monitored carefully.

Evaluation of triterpene glycoside estrogenic activity using LC/MS and immunoaffinity extraction.

We present a study on the mass spectral as well as the binding properties of three triterpene glycosides (cimicifugoside, cimiracemoside F, 27-deoxyactein) contained in black cohosh to the ligand binding domain of estrogen receptor beta (ER-beta). Using affinity ultrafiltration and LC/ MS detection, initial experiments using estradiol and the phytoestrogens daidzein and genistein (compounds known to bind ER-beta) were performed to serve as positive controls. The same affinity techniques and LC/MS procedures were then employed to show that neither the triterpene glycosides nor their enzymatically prepared aglycons bound significantly to ER-beta, except for 27-deoxyactein aglycon, which showed weak binding affinity (4%). Additionally, metabolites of the aglycons were prepared by incubation with female human liver microsomes and subjected to binding experiments with ER-beta. No significant binding of the metabolites to the receptor was observed. Further studies are needed to fully characterize whether these triterpene glycosides as well as other components of black cohosh in this plant extract bind to the estrogen receptor alpha (ER-alpha).

Assaying the estrogenicity of phytoestrogens in cells of different estrogen sensitive tissues

There is currently much concern that a wide range of both synthetic and naturally occuring enviromental chemicals may act as endocrine disruptors (ED), and may adversely affect humans and wildlife. We examined the estrogenic effects of the phytoestrogens daidzein (DAI), equol (EQU) and O-desmethylangolensin (O-DMA), two metabolites of DAI, in three different assays. Binding affinity to the estrogen receptor α was 1000–10,000-fold lower compared with the endogenous estrogen estradiol. In the receptor positive cell line MCF-7 the phytoestrogens induced the expression of a reporter gene. The E-SCREEN is based on the estrogen-receptor binding induced proliferation of the human breast cancer cell line MCF-7. We also adapted the E-SCREEN for the estrogen-receptor positive human ovarian cancer cell line BG-1. The tested phytoestrogens induced cell proliferation in both cell lines, but not in the receptor negative human breast cancer cell line MDA-MB-231. The phytoestrogen-induced cell proliferation could be blocked by addition of the receptor antagonist 4-hydroxytamoxifen (OHT). Combination treatments with the endogenous estrogen estradiol showed competitive effects in MCF-7 cells. These studies demonstrated that the tested phytoestrogens exerted estrogenic responses in cells derived from two different tissues, breast and ovary. Furthermore, we demonstrated that BG-1 cells are a suitable additional cell system to investigate estrogenicity of test compounds.

Toxicokinetics of the phytoestrogen daidzein in female DA/Han rats.

Female DA/Han rats were given the phytoestrogen daidzein, either intravenously (10 mg/kg b.w.) or orally by gavage (10 or 100 mg/kg b.w.). The plasma concentration-time curve determined after i.v. administration of daidzein was fitted to a triexponential model, resulting in a final half-life (gamma-phase) of approximately 4 h. The oral bioavailability of 10 mg daidzein/kg was 9.7%, while that of 100 mg/kg was 2.2%; the higher dose (100 mg/kg) was apparently absorbed to a four- to fivefold lower extent than the smaller dose. The plasma concentration time curves after oral administration of daidzein to female DA/Han rats revealed pronounced interindividual differences and multiple peaks, pointing to extensive enterohepatic circulation and/or protracted absorption from the gastrointestinal tract. As shown in a separate experiment with bile duct-cannulated rats, daidzein (i.p. 10 mg/kg b.w.) is efficiently excreted with bile: glucuronide/sulfate metabolites amounting to approximately 30% of the dose in 8 h. Conjugates were also the main circulating metabolites upon i.v. or gavage administration of daidzein, indicating efficient phase II metabolism in female DA/Han rats. Since only few data have been published on tissue levels of isoflavones, their concentrations were measured in various organs and compared to plasma levels determined at the time the animals were killed, with one exception 32 or 48 h after rats had received a single dose of daidzein (i.v. or per os). As expected, the daidzein concentrations depended upon dose and administration route. Despite notable differences in the absolute amounts of total daidzein (free plus hydrolyzed conjugates), the levels were usually three- to fivefold higher in liver and kidney than in plasma; in most samples of uteri, the concentrations were similar, or up to twofold higher, than the respective plasma levels. These data point to an uptake and storage of isoflavones and metabolites in tissues. Experimental toxicokinetics appear to be a relevant subject that should be integrated into assessments of toxicological data for endocrine modulators.

Time-resolved fluoroimmunoassay of plasma daidzein and genistein

We present a method for the determination of the phytoestrogens daidzein and genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has been suggested that they may afford protection against prostate and breast cancer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4′- O-carboxymethyl-daidzein and 4′- O-carboxymethyl-genistein the compounds are coupled to bovine serum albumin (BSA), then used as antigens to immunize rabbits. The tracers with the europium chelate are synthesized using the same 4′- O-derivative of the isoflavones. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). The antisera cross-reacted to some extent with some isoflavonoids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values were about 0.95–0.99 depending on concentration. The intra-assay coefficients of variation (CV%) for daidzein and genistein at three different concentrations vary 3.2–4.5 and 3.2–4.1, respectively. The inter-assay CVs vary 5.0–6.3 and 4.5–5.3, respectively. The working ranges of the daidzein and genistein assays are 1.0–216 and 1.7–370 nmol/l, respectively. The plasma values ( n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8 ± 6.8 and for genistein, 3.2 ± 7.6 nmol/l; median value for daidzein 1.5 and for genistein 1.4 nmol/l).

Development of ELISAs for the measurement of the dietary phytoestrogens daidzein and equol in human plasma

Microtitration plate ELISAs were developed for the isoflavone daidzein, a phytoestrogen found mainly in soya and the mammalian metabolite of daidzein, equol. The detection limits in assay buffer were 21 pg per well for daidzein and 70 pg per well for equol. The assays were both highly specific. Human plasma was extracted with an organic solvent and standard curves for daidzein and equol were obtained in the presence of the extract. Applications of the assays are discussed.

Synthesis of novel mammalian metabolites of the isoflavonoid phytoestrogens daidzein and genistein.

The synthesis of novel mammalian metabolites of dietary isoflavones, dihydrodaidzein (4',7-dihydroxyisoflavanone) 7, dihydrogenistein (4',5,7-trihydroxyisoflavanone) 9, 6'-hydroxy-O-demethylangolensin [1-(2,4,6-trihydroxyphenyl)-2-(4-hydroxyphenyl)propan-1-one] 10, and cis- and trans-4',7-dihydroxyisoflavan-4-ols 11, 12 is described, and their characteristics by physical and chemical constants given for the first time.

A microbore high performance liquid chromatography/electrospray ionization mass spectrometry method for the determination of the phytoestrogens genistein and daidzein in comminuted baby foods and soya flour.

A microbore high performance liquid chromatographic/electrospray/mass spectrometric (HPLC/ESI-MS) method has been developed for the determination of the phytoestrogens daidzein and genistein in soya flours and baby foods. The samples were hydrolysed and extracted with acetonitrile-water prior to analysis. LC was performed on a microbore Primesphere 5C8 column using a water/acetonitrile/acetic acid mobile phase at a flow rate of 60 microliters/min. Atmospheric pressure ionization in the form of pneumatically assisted electrospray mass spectrometry (ESI-MS) was used as the method of detection. The limit of detection was 0.2 mg/ kg for daidzein and 0.7 mg/kg for genistein in the flour and food samples. The method proved both robust and reliable when operated over a long time period (10 days, 463 injections) generating precision data with a coefficient of variation of 4-15%.

DNA integrity in human sperm.

Since the 1970s there have been conflicting reports of decreasing sperm counts in man and increasing testicular cancer. There is a hypothetical link between apparent adverse trends in several measures of human reproductive health and exposure to endocrine disrupters. Rodent bioassays are not suited for the large-scale screening of such chemicals because of their costs, complexity, and ethical concerns. Various in vitro assays have been used to examine the effects of these chemicals, but none has directly used semen as one of the target tissues in man. The present study has examined in the alkaline Comet assay in human sperm the effect of two estrogens--beta-estradiol and the phytoestrogen daidzein--and 1,2-epoxybutene, a metabolite of 1,3-butadiene, and compared them with the effects of the known reprotoxin, dibromochloropropane, in two fertile and two infertile frozen sperm samples and two fresh fertile samples. While differences were detected in the frozen fertile and infertile samples with flow cytometry, in the Comet assay both frozen and fresh samples exposed to the chemicals in vitro from fertile and infertile men produced similar altered responses by comparison with untreated samples. The integrity of DNA is necessary not only for the noncancerous state, but also for the accurate transmission of genetic material to the next generation. Thus this assay may be useful for examination of chemicals in fresh and frozen sperm samples.

Regulation of sex hormone-binding globulin production by isoflavonoids and patterns of isoflavonoid conjugation in HepG2 cell cultures

The effect of the isoflavonoid phytoestrogens daidzein, equol, and genistein on sex hormone-binding globulin (SHBG) levels, SHBG mRNA transcript levels, and SHBG gene methylation was studied in HepG2 cell cultures by fluoroimmunometric SHBG assay and Northern and Southern hybridizations, respectively. The effect of 17β-estradiol on these parameters was studied as a control. The metabolism of isoflavonoids in HepG2 cells was determined by isotope dilution gas chromatography-mass spectrometry, after ion-exchange chromatography. Daidzein and equol increased SHBG levels in parallel intracellularly and extracellularly, whereas genistein increased SHBG levels only within the cells, resembling thus the effect of 17β-estradiol. The difference may originate from the fact that genistein has more hydroxyl groups than daidzein and equol. The regulation of SHBG production by phytoestrogens appears to occur at the post-transcriptional level. Firstly, daidzein, equol, or genistein did not have a clear effect on the steady-state SHBG mRNA levels. Secondly, no effect on SHBG gene methylation was observed by genistein. The findings applied also to 17β-estradiol. However, as the SHBG gene was more methylated in SHBG-negative MCF-7 cells than in SHBG-positive HepG2 cells, DNA methylation may play a role in the tissue-specific activation of this gene. The metabolism of isoflavonoids in HepG2 cells yielded mainly unconjugated and sulfated compounds. Similar metabolism in hepatocytes in vivo might retain their biological activity in tissues responsive to estrogens.

Interaction of phytoestrogens and other environmental estrogens with prostaglandin synthase in vitro

The phytoestrogens daidzein, genistein, equol and coumestrol were found to stimulate microsomal prostaglandin H synthase (PHS) in vitro in a concentration-dependent manner when PHS-activity was measured by arachidonic acid-dependent oxygen uptake. These compounds were co-oxidized by PHS and the conversion of parent compounds was measured by HPLC analysis. The stimulation of PHS-cyclooxygenase by these compounds was partially reversed at high concentrations probably due to their antioxidant properties causing inhibition. In contrast, the monomethyl ethers of daidzein and genistein, formononetin and biochanin A, had little or weakly inhibitory effect on PHS, and appear to be no or poor co-substrates for PHS. Compared to the equine estrogen equilin, its metabolite d-equilenin was poorly metabolized by PHS and inhibited rather than stimulated PHS-cyclooxygenase activity in vitro. The resorcylic acid lactones zearalenone and zeranol, on the other hand, were surprisingly good inhibitors of PHS-cyclooxygenase. Furthermore, zeranol inhibited both the arachidonic acid and the hydrogenperoxide-dependent oxidation of DES in contrast to indomethacin which inhibited only cyclooxygenase-dependent cooxidation of DES. The results of this in vitro study are discussed in the context of data on synthetic and steroidal estrogens and support the idea that PHS-activity may be modulated by interaction with certain estrogenic compounds.

Identification of phytoestrogens in the urine of male dogs

It is becoming increasingly apparent that dietary factors may play a role in the etiology of hormone dependent neoplasias. It has been hypothesized that estrogens play some role in the etiology of benign prostatic hyperplasia (BPH) in the canine. The presence of estrogen receptor binding activity in a fraction of canine urine purified by high performance liquid chromatography (HPLC) that did not correspond to estriol, estradiol, estrone or any of their primary metabolites was observed in the present study. We used thermospray-mass spectrometry and GC-MS to identify the phytoestrogens daidzein, equol, formononetin and genistein in HPLC purified fractions of urine obtained from male beagles. Using the same techniques we also confirmed the presence of daidzein and genistein in the commercial diet fed to these same dogs. Using the immature rat uterine cytosol estrogen receptor assay, relative binding affinities of 0.08, 1.1, < 0.01 and 3.9% were obtained for daidzein, equol, formononetin and genistein, respectively when compared to estradiol (100%). In conclusion, phytoestrogens are present in urine of male beagles. Moreover, the commercial diet fed to these dogs contains isoflavones which can be converted to equol by intestinal microflora. These results suggest the need for investigations of phytoestrogens (e.g. equol) excreted into the urine daily and its relationship to the incidence and severity of BPH in the dog.

High-performance liquid chromatographic analysis of phytoestrogens in soy protein preparations with ultraviolet, electrochemical and thermospray mass spectrometric detection

The phytoestrogens daidzein, genistein, coumestrol, formononetin, and Biochanin A are separated on a C 18 reversed-phase column (Hypersil ODS) with methanol-0.1 M ammonium acetate buffer, pH 4.6 (60:40, v/v) as eluent. The retention and resolution are affected by buffer concentrations, pH type, and proportion of organic solvent in the mobile phase. Detection in the (low pg range) is achieved with an electrochemical detector, and the compounds are positively identified by high-performance liquid chromatography-thermospray mass spectrometry. Daidzein and genistein were found in high concentrations in all soy protein preparations analyzed.

ChemInform Abstract: Synthesis of the FHI‐Labelled Urinary Lignans, Enterolactone and Enterodiol, and the Phytoestrogen Daidzein and Its Metabolites Equol and O‐Demethylangolensin.

Chemischer InformationsdienstVolume 17, Issue 19 Natural Products ChemInform Abstract: Synthesis of the FHI-Labelled Urinary Lignans, Enterolactone and Enterodiol, and the Phytoestrogen Daidzein and Its Metabolites Equol and O-Demethylangolensin. K. + WAEHAELAE, K. + WAEHAELAESearch for more papers by this authorT. + MAEKELAE, T. + MAEKELAESearch for more papers by this authorR. + BAECKSTROEM, R. + BAECKSTROEMSearch for more papers by this authorG. BRUNOW, G. BRUNOWSearch for more papers by this authorT. HASE, T. HASESearch for more papers by this author K. + WAEHAELAE, K. + WAEHAELAESearch for more papers by this authorT. + MAEKELAE, T. + MAEKELAESearch for more papers by this authorR. + BAECKSTROEM, R. + BAECKSTROEMSearch for more papers by this authorG. BRUNOW, G. BRUNOWSearch for more papers by this authorT. HASE, T. HASESearch for more papers by this author First published: May 13, 1986 https://doi.org/10.1002/chin.198619312Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume17, Issue19May 13, 1986 RelatedInformation

Synthesis of the [2H]-labelled urinary lignans, enterolactone and enterodiol, and the phytoestrogen daidzein and its metabolites equol and O-demethyl-angolensin

Methods are described for the synthesis of [2H6]enterolactone, {[2H6]-trans-2,3-bis(3-hydroxybenzyl)-γ-butyrolactone}, [2H6]enterodiol, {[2H6]-2,3-bis(3-hydroxybenzyl)butane-1,4-diol}, [2H4]daidzein, {[2H4]-7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one}, [2H4]equol, {[2H4]-3(4-hydroxyphenyl)-2H-1-benzopyran-7-ol}, and [2H5]-O-demethylangolensin,{[2H6]-1-(2′,4′-dihydroxy-2-(p-hydroxyphenyl)propiophenone} by hydrogen–deuterium exchange at aromatic rings using PBr3 or NaOD in deuterium oxide or labelled trifluoroacetic acid. The structures, isotopic purities and positions of uptake of deuterium were determined by n.m.r. and mass spectrometry (m.s.)