Phytoestrogens and the intestinal microbiome.

The microflora of the digestive tract is composed of a unique set of bacteria, yeasts, viruses and other microorganisms, generally known as the microbiome. The microbiome exhibits considerable inter-individual variability, with up to two-thirds of the microflora differing between individuals. Because of this, the variable intestinal microflora is responsible for many differences in metabolic, hormonal and immunological processes in humans and animals. Significant differences have been observed in the metabolism of phytoestrogens, naturally occurring substances that possess estrogenic or anti-estrogenic activity. These substances occur predominately in legumes, especially in soy and many soy products. Because of their effects, phytoestrogens are used as an alternative therapy for menopausal disorders and benign prostate hyperplasia. In connection with the worldwide expansion of soy products as part of healthy lifestyles including vegetarianism and veganism, phytoestrogens have become a regular part of everyday life. The activity of phytoestrogens is strongly dependent on the microbiome. Their metabolites have stronger estrogenic activity than the natural substances themselves, and because of the variability in microbiomes, there are large differences in the effects of phytoestrogens among individuals.

The relationship between structure and in vitro antibacterial activity of selected isoflavones and their metabolites with special focus on antistaphylococcal effect of demethyltexasin.

UNLABELLED: In this study, we tested 15 naturally occurring isoflavones and their metabolites for their possible antibacterial properties against nine Gram-positive and Gram-negative bacteria. The in vitro antibacterial activity was determined using the broth microdilution method, and the results were expressed as minimum inhibitory concentrations (MICs). 6,7,4'-trihydroxyisoflavone (demethyltexasin), 7,3',4'-trihydroxyisoflavone (hydroxydaidzein), 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A), 7,8,4'-trihydroxyisoflavone (demethylretusin) and 5,7,4'-trihydroxyisoflavone (genistein) produced significant antibacterial activity (MICs ≥ 16 µg ml(-1)). The most effective compound, demethyltexasin, was subsequently tested for its growth-inhibitory effect against Staphylococcus aureus, and it exhibited significant antistaphylococcal effects against various standard strains and clinical isolates, including methicillin and tetracycline resistant ones with the MICs ranging from 16 to 128 µg ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the structure-activity relationship (SAR) analysis identified ortho-dihydroxyisoflavones as a class of antibacterially effective compounds emphasizing the hydroxyl groups at C-5, 6 and 7 positions as crucial supposition for the antibacterial action of plant isoflavones and their metabolites. Demethyltexasin, an isoflavones' metabolite present in the human body through enterohepatic recycling of soya bean isoflavones (daidzein, genistein), showed the most potent antibacterial activity, especially against various strains of Staphylococcus aureus (including MDR and MRSA). The significance of this study is a deepening of the knowledge on isoflavones' SAR and identification of the antistaphylococcal activity of demethyltexasin, which suggest that metabolites of isoflavones can be even more potent antibacterial agents than their precursors.

Rapid-resolution HPLC with spectrometric detection for the determination and identification of isoflavones in soy preparations and plant extracts.

A rapid-resolution HPLC/UV-VIS DAD separation method (which takes <1 min) for the determination and identification of genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin and biochanin A in fmol quantities in submicroliter sample volumes was optimized. A linear gradient elution (0 min 22% B, 1.0 min 80% B, 1.4 min 100% B, 1.8 min 22% B) using a mobile phase containing 0.2 % (v/v) acetic acid (solvent A) and methanol (solvent B) was applied on a Zorbax SB C18 column (1.8 microm particle size) at 80 degrees C. The method was verified using samples of bits of soy and methanolic extracts from Trifolium pratense, Iresine herbstii and Ononis spinosa plants. Pseudobaptigenin glucoside, irilone, prunetin, texasin, tlatlancuayin and other isoflavones, in addition to aglycones of isoflavones and their beta-glucosides and malonyl and acetyl derivatives, were identified by UV-VIS DAD and electrospray mass spectrometric (ESI-MS) detection in the extracts.

A novel radioimmunoassay of 7-oxo-DHEA and its physiological levels.

A novel radioimmunoassay (RIA) of unconjugated 7-oxo-dehydroepiandrosterone (7-oxo-DHEA) in human serum was developed for the first time. This steroid is an intermediate in the biosynthesis of immunomodulatory 7-hydroxylated DHEA metabolites, and has been shown to possess thermogenic properties. The method employs polyclonal rabbit antiserum to (19E)-3beta-hydroxy-7,17,19-trione-19-O-(carboxymethyloxime):BSA conjugate and a homologous radioiodinated derivative with tyrosine methyl ester. The cross reactivity of the antiserum with structurally closest 7-hydroxyepimers of DHEA was lower than 1.7%, with DHEA 0.4%, with all other related steroid less than 0.4%. The method includes ether extraction of serum (0.5 ml), followed by RIA. Its detection limit was 0.06 pmol (18 pg)/tube, the average intra- and inter-assay coefficients of variation were 4.1% and 8.3%, respectively. Mean recovery of serum spiked with 7-oxo-DHEA varied between 78.8% and 112%. Its levels in three serum pools were compared with a low-resolution gas chromatography-mass spectrometry method with satisfactory results. The method has been used for determination of 7-oxo-DHEA in serum samples of 215 subjects (91 males and 124 females) without overt endocrine disorders, aged 5-71 years. The over-all mean+/-S.D. was 0.280+/-0.227, the median 0.239 nmol/l. No significant sex differences were recorded. The only group which differed significantly from all other ones were males below 10 years, significantly lower values than in other age groups were found also in the first two age groups of females.

Isoflavonoids are present in Arabidopsis thaliana despite the absence of any homologue to known isoflavonoid synthases.

Extracts from Arabidopsis thaliana leaves and inflorescence stalks and from Lepidium sativa (Brassicaceae) seedlings were analysed by HPLC-MS-SIM and by five isoflavonoid-specific ELISA methods after the HPLC fractionation of samples, in order to determine presence of isoflavonoids. Individual ELISAs were specific for daidzein, genistein, biochanin A and for their derivatives substituted either at the 4'- or at the 7- positions. Both analytical approaches revealed homologous spectra of isoflavonoids in both plant species. As the ononin specific immunoassay was not available this compound was only detected by HPLC-MS. Formononetin and prunetin represented the main aglycones followed by biochanin A, daidzein and genistein; sissotrin was the most abundant isoflavonoid glycoside followed by ononin, daidzin and genistin. The content of individual compounds ranged from a few micrograms up to 2.2 mg kg(-1) (dry weight). Expression profiles of A. thaliana genes homologous to enzymes involved in isoflavonoid synthesis and metabolism were extracted from publicly available transcriptomic datasets for various tissues. Genes likely to be involved in important steps of the isoflavonoid metabolism in A. thaliana were identified. However, in accord with the previously published data, no homologue to isoflavone synthases known from the Fabaceae plants was found. These aryl migrating enzymes belong to the CYP93C family that is absent in A. thaliana. We conclude that another gene must be responsible for biosynthesis of the isoflavone skeleton in Brassicaceae.

Neuroactive steroids, their precursors and polar conjugates during parturition and postpartum in maternal and umbilical blood: 3.3beta-hydroxy-5-ene steroids.

Five 3beta-hydroxy-5-ene steroids involved in the metabolic route from pregnenolone sulfate to dehydroepiandrosterone and its sulfate, of which three are known allosteric modulators of neurotransmitter receptors, were monitored in the serum of 20 women around parturition. In addition, their levels in maternal and umbilical serum were compared at delivery. On the basis of these data, a scheme of steroid biosynthesis in maternal organism during the critical stages around parturition is proposed. In maternal serum, all the steroids except dehydroepiandrosterone sulfate decreased during labor and even first day after delivery, although their changes were less distinct the more distant from pregnenolone sulfate (PregS) in the metabolic pathway. Calculation of product/immediate precursor ratios in maternal serum over all stages around parturition enabled identification of the respective changes in the activities of the relevant enzymes. The ratio of 17-hydroxypregnenolone/pregnenolone did not change significantly, while that of dehydroepiandrosterone/17-hydroxypregnenolone grew, indicating increased C17,20 side chain cleavage on the account of C17-hydroxylation both catalyzed by C17-hydroxylase-C17,20-lyase. As was shown by factor analysis, the changes in the maternal steroids were associated with a single common factor, which strongly correlated with all the steroids except dehydroepiandrosterone sulfate. The lack of change in the pregnenolone sulfate/pregnenolone ratio and a marked increase of the ratio dehydroepiandrosterone sulfate to unconjugated dehydroepiandrosterone indicate a different means of formation of both steroid sulfates. On the basis of these data, a scheme of steroid biosynthesis in maternal organism during the critical stages around parturition is proposed.

Time-resolved fluoroimmunoassay of plasma and urine O-desmethylangolensin.

We present a method for the determination of the phytoestrogen metabolite O-desmethylangolensin (O-DMA) in plasma (serum) and in urine. O-DMA is a metabolite of daidzein, which occurs in soybeans. It has been suggested that isoflavones may afford protection against breast and prostate cancer and therefore, also the metabolites are of interest. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After the synthesis of 4"-O-carboxymethyl-O-DMA, this compound is coupled to bovine serum albumin, and then used as antigen in immunization of rabbits. The tracers with the europium chelate are synthesized using the same 4"-O-derivative of the alpha-methyldeoxybenzoin. After enzymatic hydrolysis and ether extraction the immunoassay is carried out by time resolved fluoroimmunoassay (TR-FIA). Cross-reactivity was tested with angolensin, dihydrogenistein, dihydrodaidzein, equol, 6'-OH-angolensin, trans-4-OH-equol, 6'-OH-O-DMA, cis-4-OH-equol and 5-OH-equol. The antiserum cross-reacted only with angolensin. This cross-reactivity seems not to influence the results, which were highly specific. Plasma samples are hydrolyzed and extracted. Urine samples are analyzed directly after hydrolysis without extraction. The correlation coefficient between the plasma TR-FIA results and the GC-MS results was high; r value was 0.985. The correlation coefficient between the urine TR-FIA results and the GC-MS results was high over the entire range of concentrations (0-1500 nmol/l); r value was 0.976, but lower in the low concentration range (0-100 nmol/l), i.e. value was 0.631. The intra-assay coefficients of variation (CVs) for plasma O-DMA concentrations and for urine O-DMA concentrations at three different concentrations varied 2.8-7.7 and 3.0-6.0%, respectively and the inter-assay CVs varied 3.8-8.9 and 4.4-6.6%, respectively. The working range of the plasma and urine O-DMA assays was 0.5-512 nmol/l.

7-Hydroxydehydroepiandrosterone epimers in human serum and saliva. Comparison of gas chromatography-mass spectrometry and radioimmunoassay.

Recent reports demonstrated that 7-hydroxylated metabolites of dehydroepiandrosterone (DHEA) possess immunomodulatory and antiglucocorticoid properties. Increased 7alpha-OH-DHEA levels were found in patients with Alzheimer's disease. Hence, measurement of steroids in patients with autoimmune diseases or disturbances in the central nervous system could be of interest. A new sensitive GC-MS method for the determination of 7-hydroxydehydroepiandrosterone epimers was developed and compared with previously developed radioimmunoassays. Besides serum, these steroids were, for the first time, measured in saliva where their concentrations were about five times lower. 7alpha- and 7beta-epimer levels correlated well in both body fluids and they were larger in male.

7-Hydroxydehydroepiandrosterone--a natural antiglucocorticoid and a candidate for steroid replacement therapy?

7-Hydroxylated metabolites of dehydroepiandrosterone (DHEA) are believed to be responsible for at least some immunomodulatory and antiglucocorticoid effects of DHEA and hence are considered candidates for hormone replacement therapy. Our experiments in vitro brought the evidence that 3beta, 7beta-dihydroxy-5-androsten-3-one (7beta-OH-DHEA), but not DHEA and its 7alpha-hydroxyisomer, could counteract the immunosuppressive effect of dexamethasone on the formation of plaques in culture of murine spleen lymphocytes. In another experiment, DHEA and after a 3-weeks pause 3beta-hydroxy-5-androstene-7,17-dione (7-oxo-DHEA) were applied transdermally to 6 male volunteers on 5 consecutive days. Blood levels of DHEA, its 7-hydroxylated metabolites, and in the first case also dehydroepiandrosterone sulphate (DHEAS), were measured before, during and one day after the end of treatment. Application of DHEA increased significantly not only DHEA and DHEAS, but also its both 7-hydroxyisomers. Application of 7-oxo-DHEA also led to a significant increase of both 7-hydroxyisomers of DHEA, with 7beta-OH-DHEA being the preferred metabolite the concentration of which was increased more than three times.

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).

Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay.

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestrogens as tracers, was developed for the quantitative determination of enterolactone, genistein and daidzein in human urine. The aim was to create a method for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases. After the synthesis of the 5'-carboxymethoxy derivative of enterolactone and 4'-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen were used in preparing the europium tracers. After the enzymatic hydrolysis, the TR-FIA was carried out using the Victor 1420 multilabel counter. The method has sufficient sensitivity to measure the phytoestrogens at concentrations even below 5 nmol/l. The intra- and inter-assay coefficients of variation, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively. We measured urinary enterolactone, genistein and daidzein in 215 samples from Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, <0.1 and 0.6 and below 0.6 micromol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than those measured by gas chromatography-mass spectrometry (GC-MS). However, the assay results by the present method showed strong correlation with those obtained by GC-MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens.

[Selected phytoestrogens with potential beneficial effects on risk of certain environmental diseases]. / Vybrané fytoestrogeny jako mozné príznivé faktory z hlediska rizika nekterých civilizacních onemocnení.

Evidence has been accumulated that some phytoestrogens act as protective factors against development of cancer and also cardiovascular diseases. These are phytoestrogens of isoflavone and lignane series, found especially in soy. Beneficial effect of these compounds may be explained by a complexity of their actions at various levels: they interact with estrogen receptors, some of them are inhibitors of the key enzymes responsible in the final effect for cell growth and proliferation, and, due to their chemical nature they are scavengers of free radicals. In the presented work the authors, in collaboration with Finnish partners from the University of Helsinki, developed original immunoassays for determination of main phytoestrogens of isoflavone series--daidzein, genistein, formononoetin, biochanin A, their metabolite equal and lignane enterolactone. The methods are sensitive enough for follow-up of actual levels of phytoestrogens in serum. By using these methods, the levels of phytoestrogens in Czech population have been established. The group of patients suffering from osteoporosis has been investigated, too. Significantly lower levels of isoflavonoids in comparison with sex- and age-matched healthy subject have been found in the patients. The methods have also enabled us to follow up the dynamics of these compounds in the organism, as well as to determine their content in food and its sources. The original detection and quantification of four above mentioned isoflavonoids in beer is an example.

Elimination of cross-reactivity by addition of an excess of cross-reactant for radioimmunoassay of 17alpha-hydroxypregnenolone.

Polyclonal antiserum against 3beta,17alpha-dihydroxypregn-5-en-20-one-19-O-(carboxymethyl )-oxime bovine serum albumin (17alpha-hydroxypregnenolone-19-CMO:BSA), was raised in rabbits. Its main structural determinants were the substituents on D-ring as demonstrated by its 107% cross-reaction with 17alpha-hydroxyprogesterone. This unspecificity was almost completely eliminated by addition of the excess of the cross-reactant directly to the analytical system. The contribution of the cross-reactant from the sample in such a system became negligible due to saturation of the populations of polyclonal antibodies recognizing the analyte as well as the cross-reactant. The possible interference of 17alpha-hydroxypregnenolone-3-sulfate was avoided by inserting ether extraction. The analytical system appeared to be stable to differences in cross-reactant concentrations even in samples from patients with pathologically elevated serum levels of 17alpha-hydroxyprogesterone. The radioimmunoassay was compared with the system using the unspecific antiserum alone, but after separation of the cross-reactants by HPLC. As demonstrated by parallel measurement of 125 samples of human plasma from both sexes and various ages either before and/or after adrenocorticotropin stimulation and 17 samples with elevated basal of human plasma 17alpha-hydroxyprogesterone levels, an excellent correlation was achieved between both methods. The method, based on a simple addition of the cross-reactant, avoids the time-consuming chromatographic separation and, in comparison with the other approaches for improving the specificity of polyclonal antisera, is efficient and rapid. Mathematical analysis of the relations in equilibrium demonstrates that such a simple approach is an efficient way for improvement of immunoassay specificity using some polyclonal antisera.

Age relationships and sex differences in serum levels of pregnenolone and 17-hydroxypregnenolone in healthy subjects.

17-Hydroxypregnenolone (3beta,17alpha-dihydroxypregn-5-en-20-one) and pregnenolone (3beta-hydroxypregn-5-en-20-one) were determined by radioimmunoassay following HPLC separation in serum of healthy subjects of both sexes from 2 to 66 years old (29 girls, 85 women, 30 boys, 89 men). The effects of age and sex on the levels of both steroids were investigated and the upper limits of normal in age groups were determined. The 17-hydroxypregnenolone levels as a function of age were characterized by a statistically significant maximum at the age of 18 and 20 years followed by a local minimum at the age of 39 and 37 years and by a statistically insignificant local maximum at the age of 55 and 49 years in men and women, respectively. Pregnenolone age-dependence was similar and the statistically significant maximum was reached at the age of 17 and 16 years, the local minimum occurred at the age of 37 and 38 years and the second, statistically insignificant, local maximum at the age of 48 and 47 years in men and women, respectively. Both 17-hydroxypregnenolone and pregnenolone in both sexes exhibited similarly shaped peaks with age. Both peaks of the polynomial fit in 17-hydroxypregnenolone were more pronounced in men than in women (13.0 and 9.20 nmol/l in the first peak; 7.72 and 4.78 in the second peak respectively). The situation with pregnenolone was the opposite. Both peaks of the polynomial fit in pregnenolone were lower in men than in women (2.29 and 3.21 nmol/l in the first peak; 0.92 and 1.78 in the second peak, respectively). The higher serum levels of pregnenolone at puberty and during fertile age and their wider variance in comparison with men could, be explained by the different gonadal steroidogenesis depending on the menstrual cycle, where the pregnenolone serves as a substrate for progesterone formation. The age dependencies of 17-hydroxypregnenolone and pregnenolone in women resembled that of unconjugated dehydroepiandrosterone. These results indicate that the increased metabolic activity in gonads in adolescence concerns not only dehydroepiandrosterone as the product of the 5-ene metabolic pathway but also its precursors.

Immunoassay of 7-hydroxysteroids: 2. Radioimmunoassay of 7alpha-hydroxy-dehydroepiandrosterone.

High sensitivity radioimmunoassay of 3beta, 7alpha-dihydroxy-5-androsten-17-one (7alpha-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [(125)I]iodotyrosine methyl ester as a tracer. Sensitivity of the assay amounted to 3.12 fmol (0.95 pg)/tube, precision as a mean intra- and interassay coefficient of variation was 7.1 and 10.6%, respectively, and the average recovery of the analyte added to steroid-free serum was 110%. Out of the steroids occurring in human serum which may interfere with the assay, the only important cross-reactants were dehydroepiandrosterone and 3beta, 7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) with cross-reactivities of 1.95 and 1.16%, respectively. The levels of free (unconjugated) 7alpha-OH-DHEA have been determined in 29 sera from healthy volunteers (23 females and 6 males), and from 48 patients (43 females and 5 males) in which dehydroepiandrosterone and its sulfate (DHEA/S) had been measured for various endocrinopathies. The levels in healthy subjects ranged from 0.21 to 6.57 (mean 2.33+/-1.50) nM, while those of the patients from 0 to 5. 99 (mean 1.46+/-1.52) nM. The levels of 7alpha-OH-DHEA in patients significantly correlated with those of DHEA and its sulfate.

Immunoassay of phytoestrogens in human plasma.

Highly sensitive plasma immunoassay methods based on time-resolved fluorometry were developed for plasma enterolactone, genistein, and daidzein. For daidzein and genistein three types of methods and for enterolactone two different methods were developed and validated and the results compared with our gas chromatographic-mass spectrometric reference method. All three compounds may be determined in duplicate in a 2-300 mu plasma sample, even in subjects with low phytoestrogen values.

Radioimmunoassay of free genistein in human serum.

Two radioimmunoassay (RIA) systems for genistein have been established, based on polyclonal antibodies against genistein-4'-O-(carboxymethyl)ether-bovine serum albumin and genistein-7-O-(carboxymethyl)ether-bovine serum albumin conjugates. The sensitivities of assays were 4.44 and 10.4 fmol (1.2 and 2.8 pg)/tube, respectively, the intraassay coefficients of variation ranged from 3.54 to 9.30%, the interassay C.V. varied from 6.72 to 19.7%, depending on the type of method and on genistein concentration. The cross-reactivities with other chemically related compounds (with exception of genistein derivatives at the position used for construction of the immunogen) were 5.5 and 6.1% for daidzein and 3.9 and 0.04% for formononetin in RIAs using reagents prepared through positions 4'- and 7- of genistein, respectively. The method was used for measurement of genistein levels in 26 omnivore subjects and in three volunteers after consumption of a meal prepared from 125 g of cooked whole soybeans. The values obtained in ether extracts from human sera were almost identical for both RIA systems, indicating that both RIAs measure the same entity.

Identification of isoflavonoids in beer.

The isoflavonoids, genistein (4',5,7-trihydroxyisoflavone), biochanin A (5,7-dihydroxy-4'-methoxyisoflavone), daidzein (4',7-dihydroxyisoflavone), and formononetin (7-hydroxy-4'-methoxyisoflavone) are supposed to be health-promoting dietary factors of plant origin. They are particularly abundant in seeds and other parts of many plant species belonging to Leguminosae. The most popular source of isoflavonoids in human diet is soy. Here, evidence is presented that isoflavonoids are regularly found in beer. Diethyl ether extracts of beer were fractionated on thin-layer chromatography-silica, (straight phase) and rechromatographed using a reversed phase high-performance liquid chromatography octadecylsilica column. The fractions were analyzed by two recently developed radioimmunoassays, the first of them being specific for diadzein/formononetin and the second one specific for genistein/biochanin A. The immunoreactivity was found only in fractions with the mobility corresponding to the positions of standards on control chromatograms. Additionally, 26 samples of bottled beer were analyzed for isoflavonoid content using the combination of reversed phase high-performance liquid chromatography and radioimmunoassay. The sum of the four isoflavonoids ranged from 1.26 to 29 nmol/L in individual beers. Formononetin was the major isoflavonoid (0.19-14.99 nmol/L), whereas the concentration of daidzein was several times lower (0.08-2.5 nmol/L). Genistein and biochanin A concentrations were comparable, ranging from 0.169-6.74 nmol/L and from 0.820-4.84 nmol/L for genistein and biochanin A, respectively. It is concluded that beer contains significant amounts of biologically active isoflavonoid phytoestrogens.

Immunoassay of 7-hydroxysteroids: 1. Radioimmunoassay of 7beta-hydroxy dehydroepiandrosterone.

High sensitivity radioimmunoassay of 3beta,7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against its 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [125I]iodotyrosine methyl ester as a tracer. Alternatively, [3H]tracer has been prepared, which was recognized by the antiserum as well, but the assay sensitivity was lower. The identity of measured immunoreactive material was confirmed by high performance liquid chromatography which separated 7beta-OH-DHEA from its 7alpha-isomer. Using radioiodinated tracer, the sensitivity of the method was 3.48 fmol (1.06 pg) per tube, the mean recovery of standard added to steroid-free serum was 98.5%. Free (unconjugated and not-esterified) 7beta-OH-DHEA amounted in average 5.8% of the total 7beta-OH-DHEA present in human serum. It was measured in 42 normal subjects (28 females and 14 males) and in 92 randomly selected patients with various endocrinopathies. The mean values +/- SD in normals were 2.05 +/- 1.02 nmol l(-1), the broad range of values from undetectable levels to 10.3 nmol l(-1) was found in the patients. Serum levels of free 7beta-OH-DHEA in the patients significantly correlated with DHEA and its sulfate.