Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis.

Consumption of a plant-based diet can prevent the development and progression of chronic diseases associated with extensive neovascularization, including solid malignant tumors. In previous studies, we have shown that the plant-derived isoflavonoid genistein is a potent inhibitor of cell proliferation and in vitro angiogenesis. In the present study, we report that certain structurally related flavonoids are more potent inhibitors than genistein. Indeed, 3-hydroxyflavone, 3',4'-dihydroxyflavone, 2',3'-dihydroxyflavone, fisetin, apigenin, and luteolin inhibited the proliferation of normal and tumor cells, as well as in vitro angiogenesis, at half-maximal concentrations in the low micromolar range. We have previously demonstrated that genistein concentrations in the urine of subjects consuming a plant-based diet is 30-fold higher than in subjects consuming a traditional Western diet. The wider distribution and the more abundant presence of flavonoids in the plant kingdom, together with the present results, suggest that flavonoids may contribute to the preventive effect of a plant-based diet on chronic diseases, including solid tumors.

Incorporation of esterified soybean isoflavones with antioxidant activity into low density lipoprotein.

We have recently reported that dietary intake of soybean isoflavone phytoestrogens resulted in increased oxidation resistance of isolated low density lipoprotein (LDL). In order to explore the underlying mechanisms we designed two types of in vitro experiments. First, we prepared several different isoflavone fatty acid esters to increase their lipid solubility and studied their incorporation into LDL. Second, the oxidation resistance of the isoflavone-containing LDLs was investigated with Esterbauer's 'conjugated diene' method using Cu2+ as prooxidant. Unesterified daidzein and genistein as well as genistein stearic acid esters were incorporated into LDL to a relatively small extent (0.33 molecules per LDL particle, or less) and they did not significantly influence oxidation resistance. The oleic acid esters of isoflavones were incorporated more effectively, reaching a level of 2.19 molecules per LDL particle or more, and the 4',7-O-dioleates of daidzein and genistein exhibited prolongations of lag times by 46% (P<0.05) and 202% (P<0.01), respectively. A smaller but significant increase in lag time (20.5%, P<0.01) was caused by daidzein 7-mono-oleate. In summary, esterification of soybean isoflavones daidzein and genistein with fatty acids at different hydroxyl groups provided lipophilicity needed for incorporation into LDL. Some isoflavone oleic acid esters increased oxidation resistance of LDL following their incorporation.

Antioxidant protection of lipoproteins containing estrogens: in vitro evidence for low- and high-density lipoproteins as estrogen carriers.

Some recent studies have reported that low-density lipoprotein (LDL) isolated from estrogen-treated postmenopausal women exhibited increased oxidation resistance ex vivo. However, the underlying mechanisms responsible for this effect are not clear. We explored the possibility that lipophilic derivatives of 17beta-estradiol (E(2)) could be incorporated into LDL and high-density lipoprotein (HDL) particles inhibiting lipoprotein oxidation. Introduction of small amounts of esterified E(2) into lipoproteins by means of incubation of free E(2) and E(2) 17-stearate in plasma did not result in any antioxidant effect. Using an artificial transfer system (Celite dispersion), larger amounts of E(2) esters could be incorporated into lipoproteins. Concentrations ranging between 0.27 and 1.38 molecules/LDL particle for E(2) 17-stearate and between 0.36 and 1.93 molecules/LDL particle for E(2) 17-oleate resulted in increased Cu(2+)-induced oxidation resistance of LDL as indicated by statistically significant lag time prolongations. Significant prolongations of lag times were also observed for HDL following incorporation of E(2) esters using Celite as transfer system. Our results suggest that free E(2) can be esterified and incorporated into lipoproteins during incubation in plasma. However, incorporation of supraphysiologic concentrations of E(2) esters into lipoproteins by means of the artificial transfer system was required in order to reduce their oxidation susceptibility.

Phytoestrogens: new ligands for rat and human alpha-fetoprotein.

The binding of the lignans, enterolactone, enterodiol, nordihydroguaiaretic acid (NDGA), and the isoflavonic phytoestrogen equol, to human and rat alpha-fetoprotein (AFP) was studied. They had differential inhibitory effects (NDGA greater than equol greater than enterolactone greater than enterodiol) on the binding of estrone and estradiol to rat AFP and the binding of unsaturated fatty acid to both rat and human AFP. Inhibition was dose-dependent. The apparent dissociation constants (Kd) for phytoestrogens binding to AFP were: Kd NDGA = 5 +/- 1.2.10(-7) M, Kd equol = 6.7 +/- 0.8.10(-6) M, Kd enterolactone = 1.7 +/- 0.4.10(-5) M and Kd enterodiol = 2.2 +/- 0.6.10(-5) M. The Kd for estrone binding to rat AFP was increased by increasing concentrations of equol, but the number of esterone binding sites remained unchanged. This, plus the results of double-reciprocal plots, suggests that they compete for the same site(s). NDGA also competitively inhibited estrone binding at low NDGA concentrations (increased Kd), but high concentrations induced conformational changes in rat AFP, as both Kd and the number of binding sites (n) were altered. Both rat and human AFPs underwent changes in electrophoretic behaviour and loss of immunoreactivity with increasing NDGA, suggesting that NDGA binding induces conformational changes in the AFPs. However, equol did not alter the electrophoretic or immunological properties of either rat or human AFP, providing further evidence for qualitative differences in the effects of these diphenols. These findings indicate that phytoestrogens could play a role in AFP-dependent normal and pathological growth and development.

Accumulation of high-density lipoprotein-derived estradiol-17beta fatty acid esters in low-density lipoprotein particles.

Estrogens are known to be powerful antioxidants in lipid-aqueous systems, as demonstrated by their inhibition of low-density lipoprotein (LDL) oxidation in vitro. Studies reporting that endogenous human estrogens could be rendered fat-soluble by esterification with fatty acids in vivo, and the subsequent detection of such esters in blood and fat tissue suggested a possible mechanism explaining how estrogens might protect LDL. Because of their lipophilicity, esterified estrogens may become incorporated in the lipoprotein structure, providing antioxidant potential for the particles. We incubated labeled 17beta-estradiol with ovarian follicular fluid and with plasma in the absence and presence of the LCAT inhibitor DTNB. This was followed by ultracentrifugal isolation of LDL and high-density lipoprotein and analysis of the radioactive label in the "ester" and "free" fractions purified from these lipoproteins. The results indicated that LCAT-mediated synthesis of esterified 17beta-estradiol occurred in high-density lipoprotein particles, and suggested a novel cholesterol ester transfer protein-mediated mechanism for their transfer to LDL particles.

Urinary isoflavonoid and lignan excretion on a Western diet: relation to soy, vegetable, and fruit intake.

Dietary isoflavone and lignan phytoestrogens are potential chemopreventive agents. This has led to a need to monitor exposure to these compounds in human populations and to determine which components of a mixed diet contribute to the exposure. Typically, urinary isoflavonoid excretion is associated with soy consumption and that of lignans is associated with whole grains. However, other plant foods are known to contain phytoestrogen precursors. The purpose of this study was to examine the association between urinary isoflavonoid and lignan excretion and intakes of vegetables and fruits (V&F). Isoflavonoids (genistein, daidzein, O-desmethylangolensin, and equol) and lignans (enterolactone, enterodiol, and matairesinol) were measured in urine collected for 3 days from 49 male and 49 female volunteers (age, 18-37 years) reporting a wide range of habitual V&F intakes. Dietary intakes were assessed using 5-day diet records and a food frequency questionnaire. V&F groupings (total V&F, total V, total F, soyfoods, and V&F grouped by botanical families) were used to assess the relationship between V&F intake and urinary isoflavonoid and lignan excretion. Pearson correlations were performed. Intake of soyfoods was correlated significantly with urinary genistein (r = 0.40; P = 0.0001), O-desmethylangolensin (r = 0.37; P = 0.0002), daidzein (r = 034; P = 0.0007), and the sum of isoflavonoids (r = 0.39; P = 0.0001). There was no association between equol excretion and soy intake or between the isoflavonoids and any other V&F groupings. In addition, isoflavonoid excretion was correlated positively with intake of high-fat and processed meats, particularly among men who did not consume soy. This suggests that, even in the United States, on a Western diet, soyfoods are the primary contributors to isoflavone intake; however, additional "hidden sources" of soy may also contribute to exposure. In contrast, a variety of fiber-containing foods contributed to lignan excretion; the sum of the urinary lignans, enterodiol, enterolactone, and matairesinol, was associated with intake of total F (r = 0.27; P = 0.008), total V&F (r = 0.25; P = 0.01), soyfoods (r = 0.28; P = 0.006), and dietary fiber (r = 0.36; P = 0.0003). Overall, urinary phytoestrogens (isoflavonoids + lignans) were significantly higher in "high" compared with "low" V&F consumers. Compared with the "low" V&F group, the "high" group consumed diets that were, on average, higher in fiber and carbohydrate and soyfoods and lower in fat; thus, the urinary phytoestrogens may also be a useful marker of healthier dietary patterns.

Reproducibility and validity of radioimmunoassays for urinary hormones and metabolites in pre- and postmenopausal women.

The reproducibility of RIAs of circulating sex hormones has been evaluated as part of recent epidemiological investigations, but none seem to have addressed the reproducibility or validity of RIAs for urinary hormones or their metabolites. As part of a case-control study of breast cancer in Asian-American women, 12-h overnight urine samples were obtained, and a methodological study was conducted to identify laboratories capable of assaying urinary hormones. For the reproducibility component of this study, two laboratories with extensive experience in hormone assays measured urinary estrone, estradiol, estriol, pregnanediol glucuronide, and estrone glucuronide using samples from 15 women (5 midfollicular, 5 midluteal, and 5 postmenopausal). Variance estimates from these measurements were used to calculate the laboratory variability (coefficient of variation) and to assess the magnitude of the biological variability among the women in relation to the total variability (intraclass correlation coefficient). For the validity component, urinary estrone, estradiol, and estriol levels were measured in the same samples by gas chromatography-mass spectroscopy in the laboratory of Dr. Herman Adlercreutz (University of Helsinki, Helsinki, Finland). We found that the degree of assay reproducibility differed between the laboratories, but that laboratory variability was usually low compared with the range of hormone values among women, particularly for the estrogens. Values for estrone and estradiol were well correlated among all of the laboratories. For estriol, the RIAs tended to overestimate levels compared with gas chromatography-mass spectroscopy. In one laboratory, assays for pregnanediol glucuronide and estrone glucuronide were consistently reproduced; in the other, the reproducibility of the RIA for pregnanediol glucuronide was problematic, and estrone glucuronide was not measured. Despite some limitations, urinary hormones and their metabolites can be reliably measured by current RIAs in large investigations attempting to link hormone level to disease risk and may be particularly advantageous for studies of postmenopausal women, where serum concentrations of estrone and estradiol are low and assay measurements are not as dependable.

Quantifying estrogen metabolism: an evaluation of the reproducibility and validity of enzyme immunoassays for 2-hydroxyestrone and 16alpha-hydroxyestrone in urine.

Rapid and simple enzyme immunoassays (EIAs) were recently developed to measure 2-hydroxyestrone and 16alpha-hydroxyestrone in unextracted urine. The balance between these competing estrogen metabolism pathways may serve as a biomarker of breast cancer risk. Before testing these assays in epidemiologic studies, we evaluated their reproducibility, and validity relative to gas chromatography-mass spectroscopy (GC-MS). Overnight 12-hr urine collections from five midfollicular premenopausal women, five midluteal premenopausal women, and five postmenopausal women were aliquoted and stored at -70 degrees C. Two aliquots from each woman were assayed with the EIAs in a random, blinded order, monthly over 4 months and 1 year later. Reproducibility over 4 months was good for both metabolites in premenopausal women (coefficient of variation = 8-14%) and satisfactory in postmenopausal women (approximately 19%). Reproducibility over 12 months remained good in premenopausal women, but was poor in postmenopausal women, with mean readings increasing 50 to 100%. Wide variation in estrogen metabolite levels enabled a single EIA measurement to characterize individual differences among premenopausal women in midfollicular (intraclass correlation coefficient = 98-99%) and midluteal phase (85-91%). A narrower range in metabolite levels among postmenopausal women reduced discrimination (78-82%). The correlation between EIA and GC-MS measurement was excellent for both metabolites (r>0.9), except for 2-hydroxyestrone in postmenopausal women (r=0.6). Analysis of absolute agreement suggested that both EIAs were less sensitive than GC-MS, and each detected nonspecific background. The low concentration of estrogen metabolites in urine from postmenopausal women may explain the problems with reproducibility and validity in this menstrual group. Accordingly, more sensitive EIAs have been developed and are now being evaluated.

Dietary phytoestrogens and cancer: in vitro and in vivo studies.

Thirty postmenopausal women (11 omnivores, 10 vegetarians and 9 apparently healthy women with surgically removed breast cancer) were investigated with regard to the association of their urinary excretion of estrogens, lignans and isoflavonoids (all diphenols) with plasma sex hormone binding globulin (SHBG). A statistically significant positive correlation between urinary total diphenol excretion and plasma SHBG was found which remained statistically significant after elimination of the confounding effect of body mass determined by body mass index (BMI). Furthermore we found a statistically significant negative correlation between plasma SHBG and urinary excretion of 16 alpha-hydroxyestrone and estriol which also remained significant after eliminating the effect of BMI. Furthermore we observed that enterolactone (Enl) stimulates the synthesis of SHBG by HepG2 liver cancer cells in culture acting synergistically with estradiol and at physiological concentrations. Enl was rapidly conjugated by the liver cells, mainly to its monosulfate. Several lignans and the isoflavonoids daidzein and equol were found to compete with estradiol for binding to the rat uterine type II estrogen binding site (the s.c. bioflavonoid receptor). It is suggested that lignans and isoflavonoids may affect uptake and metabolism of sex hormones by participating in the regulation of plasma SHBG levels and in this way influence their biological activity and that they may inhibit cancer cell growth like some flavonoids by competing with estradiol for the type II estrogen binding sites.

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.

Lignan and isoflavonoid conjugates in human urine.

Lignans and isoflavonoids are two groups of diphenolic phytoestrogens of plant origin which have gained increasing interest because of their possible cancer protective properties. High excretion of these compounds occur in populations at low risk of breast, prostate and colon cancer consuming either high amounts of whole-grain (lignans and some isoflavonoids) or soy products (isoflavonoids and some lignans). We determined the pattern of conjugation of the phytoestrogens in four urine samples from vegetarian or semivegetarian women and in two samples from men. Seven compounds were investigated: enterodiol, enterolactone, matairesinol, diadzein, equol, genistein and O-desmethylangolensin. The fractions quantified are the free fraction, mono- and disulfate, as well as the mono-, di- and sulfoglucuronide fractions. For the fractionation and purification we used ion-exchange chromatography and the determination of the concentrations of each compound in all fractions was done by isotope dilution gas chromatography-mass spectrometry (GLC-MS) using deuterated internal standards of all diphenols. More than 60% of all compounds determined, occurred in the monoglucuronide fraction. Daidzein, enterodiol and equol are excreted to a relatively high extent as sulfoglucuronides and genistein as diglucuronide. We conclude that the general pattern of lignan and isoflavonoid conjugates in urine is similar to that of endogenous estrogens.

Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens.

Isoflavonoid phytoestrogens and lignans in plants are known to be constituents of animal and human food and recently they have been found in human urine and other biological materials. These compounds have received increasing attention because of their interesting biological properties and possible role in human cancer and other diseases. The present study demonstrates that the main mammalian lignan enterolactone (trans-2,3-bis[(3-hydroxyphenyl)methyl]-butyrolactone) and some other diphenols are moderate or weak inhibitors of human estrogen synthetase (aromatase) and that this lignan binds to or near the substrate region of the active site of the P-450 enzyme. The inhibition is competitive with respect to testosterone and androstenedione, and the lignan affinity is 1/75-1/300 that of these natural substrates. It is suggested that the high concentration of lignans in vegetarians, by inhibiting aromatase in peripheral and/or cancer cells and lowering estrogen levels, may play a protective role as antipromotional compounds during growth of estrogen-dependent cancers.

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.

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.

Familial correlations, segregation analysis, and nongenetic correlates of soy isoflavone-metabolizing phenotypes.

Particular intestinal bacteria metabolize the soy isoflavone daidzein to equol and O-desmethylangolensin (O-DMA), metabolites that can be identified in urine. Individuals that harbor bacteria capable of producing equol or O-DMA are known as equol producers (approximately 30%-50% of the population) and O-DMA producers (approximately 80%-90% of the population), respectively. The equol-producer phenotype has been associated with sex hormone-related outcomes in several studies. However, the bacteria responsible for these phenotypes have not yet been identified and factors that influence the manifestation of these phenotypes are not well understood. To evaluate familial clustering of and nongenetic factors associated with these phenotypes, 410 individuals from 112 families participated in phenotyping (3-day soy challenge and Day 4 spot urine collection). In segregation analyses of the equol-producer phenotype, the Mendelian dominant model provided the most parsimonious fit to the data, suggesting that the pattern of inheritance of the equol-producer phenotype is consistent with an autosomal dominant trait. This phenotype was positively associated with education (p trend = 0.01), but not with sex, smoking, or several dietary factors. Results of the segregation analyses of the O-DMA-producer phenotype were inconclusive; no other models provided a more parsimonious fit to the data than the general model. This phenotype was inversely associated with age in a nonlinear model (p = 0.01), positively associated with age- and sex-adjusted height (odds ratio [OR] 10-cm increase = 0.38, 95% confidence interval [CI] = 0.15, 0.95) and body mass index (kg/m(2)) (OR = 0.91, 95% CI = 0.85, 0.96), but not with sex, education, smoking, or several dietary factors. These results suggest the equol-producer phenotype may be under some degree of genetic control and that there are likely other environmental factors not evaluated in the present analysis that contribute to both of these phenotypes. These results provide a foundation for further work to refine our understanding of heritable and environmental determinants of daidzein-metabolizing phenotypes.

Detection and identification of the plant lignans lariciresinol, isolariciresinol and secoisolariciresinol in human urine.

The mammalian lignans enterolactone and enterodiol are regular constituents of human urine and are excreted daily in mumol amounts. They are produced by metabolic action of intestinal bacteria from natural plant lignan precursors which are constituents of various food plants. However, natural plant lignans seem to occur very seldom in detectable amounts in human urine. The present investigation shows that only in 5% of the 150 diphenolic fractions extracted from the urine of women plant lignans other than the previously identified matairesinol could be found. The lignans lariciresinol, isolariciresinol and secoisolariciresinol were identified for the first time by comparison of their GC characteristics and mass spectra with the corresponding results of authentic synthesized reference compounds. Secoisolariciresinol is one natural precursor of the mammalian lignan enterodiol. Of the two other plant lignans, no animal or human metabolic products are known. The occurrence of chemically unchanged plant lignans in some cases in human urine could be a result of an insufficient metabolic capacity of intestinal bacteria, resulting in a decreased detoxification of phenolic plant products.

Isotope dilution gas chromatographic-mass spectrometric method for the determination of lignans and isoflavonoids in human urine, including identification of genistein.

We describe an isotope dilution gas chromatographic-mass spectrometric method for the quantitative determination of the lignans enterolactone, enterodiol and matairesinol and the isoflavonoids daidzein, equol, O-desmethylangolensin and genistein in urine. Furthermore we present the gas chromatographic/mass spectrometer identification of genistein. Urine samples were extracted on Sep-Pak cartridges, conjugated fractions were isolated by chromatography on the acetate form of DEAE-Sephadex and deuterated internal standards of all seven compounds were added to the samples before hydrolysis. The hydrolysate was extracted on a Sep-Pak cartridge and following chromatography on the acetate form of QAE-Sephadex two fractions were obtained: Fraction 1 contained equol, enterolactone, enterodiol, matairesinol and all estrogens and fraction 2 contained O-desmethylangolensin, daidzein and genistein. The latter was ready for gas chromatography/mass spectrometry, but the first one was further purified to eliminate the estrogens by chromatography on the carbonate form of QAE-Sephadex. Following silylation, the samples were analyzed by combined capillary column gas chromatography/mass spectrometry in the selective ion monitoring mode. The within-assay imprecision varied from 0.8-15.2% (mean 8.7%) and the between-assay imprecision from 4.1-13.9% (mean 9.3%), depending on compound and concentration level. The mean recovery of authentic standards added to urine extracts before hydrolysis varied from 96.6 to 105.5%. Values obtained from 10 Finnish omnivorous men are presented. Individual values for matairesinol (excretion range 3.3-59.9 nmol/24 h) and genistein (range 21.8-1180 nmol/24 h) in human urine have never been published before.

Identification of lignans and phytoestrogens in urine of chimpanzees.

It was recently observed that the urinary excretion of animal lignans is low in postmenopausal breast cancer patients compared to normal omnivorous and vegetarian women. In addition, the mean excretion of the isoflavonic phytoestrogen equol tended to be lower. Because nonhuman primates appear to be remarkably resistant to the carcinogenic effect of estrogens, we investigated the possible occurrence of lignans and phytoestrogens in the urine of chimpanzees on their regular diet. Five major diphenols were isolated and identified by capillary gas chromatography and mass spectrometry by comparison with synthesized authentic reference compounds. Three of these compounds, the phytoestrogen equol and its precursor daidzein, the lignan enterolactone, were according to preliminary assays excreted in very large amounts. In addition, the lignan enterodiol and the daidzein metabolite O-desmethylangolensin were identified. It is concluded that the chimpanzee excretes both isoflavonic phytoestrogens and lignans in urine, apparently in high concentrations. It is suggested that these compounds may play a role in the maintenance of the resistance against carcinogenic effects of estrogens, which nonhuman primates possess, because both equol and enterolactone have been shown to have antiestrogenic properties in animals. However, much further work is necessary before the possible biological role of these compounds may be established.

Antiproliferative efficacy of lipophilic soy isoflavone phytoestrogens delivered by low density lipoprotein particles into cultured U937 cells.

Some fat-soluble bioactive substances incorporated into low density lipoprotein (LDL) may be delivered into cells via LDL receptor pathway influencing cellular functions. In this study, we synthesized a number of fat-soluble isoflavone esters and investigated their incorporation into LDL as well as their delivery into U937 cells. Using an artificial transfer system (Celite dispersion), genistein and daidzein oleates and daidzein dilinoleate were efficiently incorporated into LDL with concentrations ranging between 2.7 to 16.9 isoflavone molecules/LDL particle, while much smaller amounts of unesterified isoflavones and genistein stearates were transferred into LDL. LDL containing 7-oleates or 4',7-dioleates of genistein and daidzein significantly reduced U937 cell proliferation by 36-43%. The strongest inhibitory effect was shown by daidzein 4',7-dilinoleate with 93% reduction of cell proliferation. Neither of the 4'-oleates of genistein and daidzein contained in LDLs exhibited antiproliferative effects although they were incorporated into LDL. In summary, we demonstrated that isoflavones made fat-soluble by esterification can be incorporated into LDL in vitro and delivered into cultured U937 cells via the LDL-receptor pathway, reducing the cell proliferation.

Effect of diet on lignans and isoflavonoid phytoestrogens in chimpanzees.

Diphenolic compounds belonging to the classes of lignans and isoflavonoids have been identified in urine of man and animals, including the chimpanzee. Some of these compounds, formed by intestinal bacteria from plant lignans and phytoestrogens, have been shown in animal studies to exhibit biological activities that suggest they could function as cancer-protective compounds. The effect of diet on urinary excretion of these compounds in the adult male chimpanzee has been studied. It was found that the chimpanzees consuming their regular food excreted large amounts of the isoflavonoid phytoestrogens, equol (mean +/- SE) (127.5 +/- 34.0 nmol/mg cr.) and daidzein (20.7 +/- 9.0 nmol/mg cr.) and the lignan, enterolactone (14.1 + 3.5 nmol/mg cr.). Small amounts of the lignan, enterodiol, (0.4 +/- 0.2 nmol/mg cr.) were also excreted. On all other four test diets (high protein, high carbohydrate, high vegetable, and high fat), the excretion was less, particularly on a high fat diet where the excretion of all diphenolic compounds was reduced by more than 90% to a level observed in omnivorous human subjects or women with breast cancer. These results suggest that diet profoundly influences the excretion of both animal lignans and phytoestrogens in urine. Because non-human primates are particularly resistant to mammary and genital carcinoma on estrogen treatment, the present data suggest that the very high levels of phytoestrogens and lignans as found during exposure to the regular diet may partially account for why these primates are so resistant to hormonal manipulations to induce cancer.