An overview of the health effects of isoflavones with an emphasis on prostate cancer risk and prostate-specific antigen levels

Soybean isoflavones possess hormonal and nonhormonal properties that may reduce the risk of coronary heart disease, osteoporosis, and certain cancers, and alleviate hot flashes in menopausal women. Among the various cancers whose risk may be reduced by isoflavones, there is particular interest in prostate cancer. Eleven trials have examined the effects of isoflavones on serum prostate-specific antigen (PSA) levels. The dose of isoflavones in these trials from supplements or soy protein ranged from 60 to 900 mg/day (typical Japanese intake is 30-50 mg/day), subject number/group ranged from 8 to 62, and study duration from 20 days to 1 year. Isoflavones did not affect serum PSA in healthy subjects. In contrast, in 4 of 8 trials involving prostate cancer patients, isoflavones significantly favorably affected PSA although in no studies was there an absolute decrease in PSA concentrations. The mechanism by which isoflavones affect PSA could not be determined from the existing research, although hormonal changes do not seem to be a factor. The clinical evidence is sufficiently encouraging to justify considering additional Phase II and III clinical trials investigating the efficacy of soy isoflavones in different populations of prostate cancer patients alone and in combination with other treatments.     

Analysis of isoflavones in foods and dietary supplements

Isoflavones are phytochemicals found in many plants. Because of their structural similarity to beta-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases. Daidzein, genistein, and glycitein are present as free forms or derivatives in foods containing soy or soy protein extracts. The analysis of isoflavones has become more complex, because preparations contain isoflavones from multiple sources (e.g., red clover, kudzu). Red clover contains primarily formononetin and biochanin A, while kudzu extracts, which are becoming increasingly common in dietary supplements, contain puerarin and daidzein, among other components. Isoflavones are present in foods and dietary supplements as free compounds, glucoside derivatives, 6"-O-malonyl-7-O-beta-D-glucoside derivatives, and 6"-O-acetyl-7-O-beta-D-glucoside derivatives. High-performance liquid chromatography (HPLC)/tandem mass spectrometry has been applied to the identification of isoflavone derivatives based on the fragmentation pattern of the parent ion, providing high selectivity and sensitivity in the quantitation of isoflavones in complex mixtures. HPLC with ultraviolet detection is often chosen for routine analysis, but a preliminary acid or basic hydrolysis of isoflavone derivatives is often required for the investigation of samples containing extracts from multiple sources. Several internal standards have been used in the analysis of isoflavones from a single botanical source (e.g., soy, red clover), but the identification of a general internal standard remains a challenging process.     

Simple Extraction and Rapid Quantitative Analysis of Isoflavones in Soybean Seeds

A simple extraction and rapid HPLC method, suitable to separate and quantify the isoflavonoids in soybean seeds, are proposed and discussed. The method has been applied to the separation and quantification of seed isoflavonoids in five naturally grown soy cultivars; the total amount of isoflavones ranged from 1.83 g kg^–1 to 11.88 g kg^–1 of fresh weight. The detection limits (200 g/kg^–1), evaluated for genistein and daidzein, are presented together with a list of the soy polyphenols analysed by HPLC-DAD and HPLC-MS.     

Development and validation of a liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of tamoxifen, anastrozole, and letrozole in human plasma and its application to a clinical study

There is substantial evidence that circulating estrogens promote the proliferation of breast cancer. Consequently, adjuvant hormonal treatment strategies targeting estrogen action have been established. Such hormonal therapies include selective estrogen receptor modulators, such as tamoxifen, which interfere at the estrogen receptors directly, or non-steroidal aromatase inhibitors, such as anastrozole and letrozole, which inhibit estrogen synthesis through blocking the aromatase, a key enzyme of estrogen production. Despite considerable therapeutic success, in several cases, the use of these drugs is limited by side effects that have been described to significantly impair the adherence of patients to endocrine treatment. However, objective data concerning patient adherence and its clinical relevance are limited. One promising approach to check patient-reported adherence is drug monitoring in human plasma. Therefore, a liquid chromatography–tandem mass spectrometry method to determine the plasma concentrations of tamoxifen, anastrozole, and letrozole has been developed and fully validated according to guidelines for clinical and forensic toxicology. The validation criteria evaluated were selectivity, linearity, accuracy and precision, limit of quantification, recovery and matrix effects, sample stability, and carryover. The six-point calibration curves showed linearity over the range of concentrations from 25 to 500 ng/ml for tamoxifen, 5 to 200 ng/ml for anastrozole, and 10 to 300 ng/ml for letrozole. The intra- and inter-day precision and accuracies were always better than 15%. The validated procedure was successfully applied to a clinical study (Patient-Reported Outcomes in Breast Cancer Patients undergoing Endocrine Therapy, PRO-BETh). A major aim of PRO-BETh study is the comprehensive evaluation of adherence to treatment in pre- and post-menopausal women with breast cancer. Plasma samples of 310 breast cancer patients undergoing anti-estrogen therapy were analyzed. Eight samples did not contain a quantifiable amount of drug, strongly indicating non-adherence of the corresponding patients to adjuvant breast cancer treatment. Furthermore, plasma concentrations at the lower end of the observed plasma level distribution might represent a hint but not a confirmation for non-adherence in terms of non-daily and irregular intake of the prescribed drug.     

Quantitative determination of acetyl glucoside isoflavones and their metabolites in human urine using combined liquid chromatography-mass spectrometry

To investigate whether the bioavailability of isoflavones could be an alternative to fermented soy foods, the conjugated forms of soy nutritional supplement (containing 98% acetyl glucoside isoflavones) were consumed by eight human volunteers (three were Asian people and five were British). Their daily urine samples were collected before and after a 5-week consumption of supplementation period. Conjugated isoflavones of genistein, daidzein and glycitein were hydrolyzed by enzyme, extracted with methyl tert-butyl ether and analysed using liquid chromatography coupled with electrospray tandem mass spectrometry. Daidzein, genistein, glycitein, dihydrogenistein, dihydrodaidzein and O-desmethylangolensin were identified and quantified simultaneously with high recoveries. The levels of free isoflavones and total isoflavones were compared, and isoflavone glucuronides were identified much higher than the corresponding sulfates or aglycone isoflavones. This method provided the measurement of isoflavones with high sensitivity and specificity and simplified the sample pre-treatment procedure. The limitation of detections of dihydrodaidzein, 3'-hydroxydaidzein, glycitein, daidzein, genistein, dihydrogenistein and O-desmethylangolensin were 37, 23.5, 12.2, 15.4, 14.8, 2.20 and 0.31 pmol, respectively. Only 0.5 ml of urine sample was needed.     

Computational design and experimental discovery of an antiestrogenic peptide derived from alpha-fetoprotein

Breast cancer is the most common cancer among women, and tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment. Many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Consequently, there is an ongoing need for breast cancer drugs that have different molecular targets. Previous work has shown that 8-mer and cyclic 9-mer peptides inhibit breast cancer in mouse and rat models, interacting with an unsolved receptor, while peptides smaller than eight amino acids did not. We show that the use of replica exchange molecular dynamics predicts the structure and dynamics of active peptides, leading to the discovery of smaller peptides with full biological activity. Simulations identified smaller peptide analogues with the same conserved reverse turn demonstrated in the larger peptides. These analogues were synthesized and shown to inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition.     

On-line sample preparation using restricted-access media in the analysis of the soy isoflavones, genistein and daidzein, in rat serum using liquid chromatography electrospray mass spectrometry

Soy isoflavones are the subject of many investigations in experimental animals and humans regarding possible modulation of endocrine activity and chemoprevention of carcinogenesis. Genistein and daidzein, the principal biologically active isoflavones in soy, were measured using on-line solid-phase extraction (SPE) and liquid chromatography electrospray mass spectrometry (LC/ES-MS) detection in serum of rats consuming a common open-formula (NIH 31) chow that contained approximately 30 microg each of genistein and daidzein per gram of feed and a specially designed 'soy-free' chow that contained approximately 60-fold lower isoflavones. The use of a restricted-access/reverse phase trap cartridge and automated column switching permitted rapid and robust analytical performance with many injections of plasma onto a reverse phase LC column. Enzymatic deconjugation and a single centrifugation step were the only sample preparation steps required. The limit of detection for the isoflavones, based on the MS responses observed in serum from male and female rats consuming the soy-free chow, was 0.020 microM. The method, which uses deuterated isoflavones as internal standards, was determined to be accurate using spiked control serum (102-110% of added amounts) and precise using spiked control serum and incurred serum (<6% relative standard deviation). The average genistein and daidzein levels were determined in female (0.62 and 0.25 microM, respectively) and male rats (0.35 and 0.20 microM, respectively) consuming the standard diet. The sex difference observed for serum genistein concentrations was statistically significant (p < 0.0001). These results underscore the potential impact of standard open-formula diets on the results from rodent bioassays of biological activity.     

Analysis of isoflavones in soy drink by capillary zone electrophoresis coupled with electrospray ionization mass spectrometry

Capillary zone electrophoresis coupled with electrospray ionization mass spectrometry (CZE–ESI-MS) has been applied for the first time for the separation and quantification of isoflavones in soy products. The proposed method was successfully applied to the determination of seven isoflavones, including aglycones and glucosides, in soy drink. The target compounds were the glucosides daidzin and genistin, and the aglycones daidzein, genistein, formononetin, biochanin A and glycitein. During CE separation in positive mode, the analytes were present as anions, and MS detection was carried out in ESI positive-ion mode. To prevent the frequent drops in current and to improve the resolution in the separation of analytes in anionic form, a programmed nebulizing gas pressure (PNP) was applied along the analysis.     

Bioanalytical methods for determination of tamoxifen and its phase I metabolites: a review

The selective estrogen receptor modulator tamoxifen is used in the treatment of early and advanced breast cancer and in selected cases for breast cancer prevention in high-risk subjects. The cytochrome P450 enzyme system and flavin-containing monooxygenase are responsible for the extensive metabolism of tamoxifen into several phase I metabolites that vary in toxicity and potencies towards estrogen receptor (ER) alpha and ER beta.An extensive overview of publications on the determination of tamoxifen and its phase I metabolites in biological samples is presented. In these publications techniques were used such as capillary electrophoresis, liquid, gas and thin layer chromatography coupled with various detection techniques (mass spectrometry, ultraviolet or fluorescence detection, liquid scintillation counting and nuclear magnetic resonance spectroscopy). A trend is seen towards the use of liquid chromatography coupled to mass spectrometry (LC-MS). State-of-the-art LC-MS equipment allowed for identification of unknown metabolites and quantification of known metabolites reaching lower limit of quantification levels in the sub pg mL⁻¹ range. Although tamoxifen is also metabolized into phase II metabolites, the number of publications reporting on phase II metabolism of tamoxifen is scarce. Therefore the focus of this review is on phase I metabolites of tamoxifen.We conclude that in the past decades tamoxifen metabolism has been studied extensively and numerous metabolites have been identified. Assays have been developed for both the identification and quantification of tamoxifen and its metabolites in an array of biological samples. This review can be used as a resource for method transfer and development of analytical methods used to support pharmacokinetic and pharmacodynamic studies of tamoxifen and its phase I metabolites.     

A review of the animal models used to investigate the health benefits of soy isoflavones

This review considers the recent literature in which animal models were used to investigate the purported health benefits of soy isoflavones. The main conclusions are that our animal models demonstrate minimal effects in breast, prostate, and colon cancer prevention, and that, while some cancers may respond to isoflavones, it would appear that isoflavones do not prevent further development once cancer has become established. Regarding cardiovascular health, the lipid-lowering effects of isoflavones have been established, but their efficacy may be less than original research purported. However, it may be considered a bonus of habitual soy consumption that blood cholesterol levels would be reduced somewhat. With respect to osteoporosis and menopausal symptoms, animal models do not show any consistent benefit of isoflavones in preventing osteoporosis, and calcium fortification or the use of prescribed medications are likely much better approaches to combat bone loss. However, our animal models of osteoporosis and menopausal symptoms may not be entirely representative of the human situation. Perhaps the benefit of isoflavones in cognitive skills and in delaying Alzheimer's disease is an area where they can be of some advantage. However, this field is very recent and requires much more research in both humans and animal models before any definitive benefit can be propounded. On the other hand, isoflavones in moderation are probably not dangerous, as few studies have indicated adverse effects. However, large doses have been shown to increase apoptosis and cell degeneration, and in some cancer regimes, once the cancer has progressed beyond the hormone-dependent stage, high doses of isoflavones may be contraindicated. The prospect of mega-dosing from isoflavone supplements opens a new chapter in the risk assessment of isoflavone consumption.     

Synthesis of [3,4,8-C3]daidzein

The biological effects of the soy isoflavones have attracted considerable interest in recent years leading to numerous studies on dietary intake and epidemiology. Such studies require accurate and reproducible analytical methods. Herein we report the first synthesis of a multiply ¹³C-labelled daidzein derivative, [3,4,8-¹³C₃]daidzein, which has been employed as an internal standard in LC-MS and GC-MS analysis. The synthesis includes an improved three-step method for the synthesis of [2-¹³C]resorcinol as one building block.     

Photochemical Internalization of Tamoxifens Transported by a “Trojan‐Horse” Nanoconjugate into Breast‐Cancer Cell Lines

Photochemical internalization (PCI) has shown great promise as a therapeutic alternative for targeted drug delivery by light‐harnessed activation. However, it has only been applicable to therapeutic macromolecules or medium‐sized molecules. Herein we describe the use of an amphiphilic, water‐soluble porphyrin–β‐cyclodextrin conjugate (mTHPP‐βCD) as a “Trojan horse” to facilitate the endocytosis of CD‐guest tamoxifens into breast‐cancer cells. Upon irradiation, the porphyrin core of mTHPP‐βCD expedited endosomal membrane rupture and tamoxifen release into the cytosol, as documented by confocal microscopy. The sustained complexation of mTHPP‐βCD with tamoxifen was corroborated by 2D NMR spectroscopy and FRET studies. Following the application of PCI protocols with 4‐hydroxytamoxifen (4‐OHT), estrogen‐receptor β‐positive (Erβ+, but not ERβ?) cell groups exhibited extensive cytotoxicity and/or growth suspension even at 72 h after irradiation.     

Determination of isoflavones in dietary supplements containing soy, Red Clover and kudzu: extraction followed by basic or acid hydrolysis

Isoflavones are phytochemicals found in many plants. Because of their structural similarity to beta-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases. Dietary supplement preparations contain extracts from soy, Red Clover and kudzu. Soy products contain primarily genistein, daidzein, and glycitein, while Red Clover products contain primarily formononetin and biochanin A. Kudzu extracts contain puerarin and daidzein among other components. Previous methods of analysis focused on the determination of isoflavones from a single botanical source, while dietary supplements are often a blend of extracts from different plants. We developed a method for the analysis of isoflavones in dietary supplements regardless of their botanical composition, using HPLC-PDA because of its applicability to routine analysis. Isoflavones are found as free compounds, glucoside derivatives, 6'-O-malonyl-beta-d-glucoside and 6'-O-acetyl-beta-d-glucoside derivatives. In this study, the samples were extracted at room temperature with 50:50 (v/v) MeCN/water, and then analyzed before and after hydrolyzing the isoflavones by acid or basic digestion. 2'-Methoxy-flavone and 6-methoxy-flavone were used as internal standards and were added together to every sample. Daidzein, glycitein, genistein, puerarin, calycosin, pratensein, pseudobaptigenin, formononetin, biochanin A and prunetin were among the isoflavones determined.     

Potential Effects of Soy Isoflavones on the Prevention of Metabolic Syndrome

Isoflavones are polyphenols primarily contained in soybean. As phytoestrogens, isoflavones exert beneficial effects on various chronic diseases. Metabolic syndrome increases the risk of death due to arteriosclerosis in individuals with various pathological conditions, including obesity, hypertension, hyperglycemia, and dyslipidemia. Although the health benefits of soybean-derived isoflavones are widely known, their beneficial effects on the pathogenesis of metabolic syndrome are incompletely understood. This review aims to describe the association between soybean-derived isoflavone intake and the risk of metabolic syndrome development. We reviewed studies on soy isoflavones, particularly daidzein and genistein, and metabolic syndrome, using PubMed, ScienceDirect, and Web of Science. We describe the pathological characteristics of metabolic syndrome, including those contributing to multiple pathological conditions. Furthermore, we summarize the effects of soybean-derived daidzein and genistein on metabolic syndrome reported in human epidemiological studies and experiments using in vitro and in vivo models. In particular, we emphasize the role of soy isoflavones in metabolic syndrome-induced cardiovascular diseases. In conclusion, this review focuses on the potential of soy isoflavones to prevent metabolic syndrome by influencing the onset of hypertension, hyperglycemia, dyslipidemia, and arteriosclerosis and discusses the anti-inflammatory effects of isoflavones.     

Quantification of isoflavones and lignans in serum using isotope dilution liquid chromatography/tandem mass spectrometry

Phytoestrogens (isoflavones and lignans) are receiving increasing attention due to a potential protective effect against a number of complex diseases. However, in order to investigate these associations, it is necessary to accurately quantify the levels of phytoestrogens in foods and biological fluids. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (O-desmethylangolensin and equol), and two lignans (enterodiol and enterolactone) in human serum using electrospray ionisation liquid chromatography/mass spectrometry (LC/MS) with selective reaction monitoring. A simple, highly automated sample preparation procedure requires only 200 microL of sample and utilises one solid-phase extraction stage. Limits of detection are in the region of 10 pg/mL for all analytes except equol, which had a limit of detection of approximately 100 pg/mL. The method developed is suitable for measuring the concentrations of phytoestrogens in blood samples collected from large epidemiological studies. The results of the analysis of serum samples from 300 men and women living in the UK are reported.     

Determination of total soy isoflavones in dietary supplements, supplement ingredients, and soy foods by high-performance liquid chromatography with ultraviolet detection: collaborative study

An interlaboratory study was conducted to evaluate a method for determining total soy isoflavones in dietary supplements, dietary supplement ingredients, and soy foods. Isoflavones were extracted using aqueous acetonitrile containing a small amount of dimethylsulfoxide (DMSO) and all 12 of the naturally occuring isoflavones in soy were determined by high-performance liquid chromatography (HPLC) with UV detection using apigenin as an internal standard. Fifteen samples (6 pairs of blind duplicates plus 3 additional samples) of soy isoflavone ingredients, soy isoflavone dietary supplements, soy flour, and soy protein products were successfully analyzed by 13 collaborating laboratories in 6 countries. For repeatability, the relative standard deviations (RSDr) ranged from 1.07 for samples containing over 400 mglg total isoflavones to 3.31 for samples containing 0.87 mg/g total isoflavones, and for reproducibility the RSDR values ranged from 2.29 for samples containing over 400 mg/g total isoflavones to 9.36 for samples containing 0.87 mg/g total isoflavones. HorRat values ranged from 1.00 to 1.62 for all samples containing at least 0.8 mg/g total isoflavones. One sample, containing very low total isoflavones (< 0.05 mg/g), gave RSDR values of 175 and a HorRat value of 17.6. This sample was deemed to be below the usable range of the method. The method provides accurate and precise results for analysis of soy isoflavones in dietary supplements and soy foods.     

Characterization and quantification of soy isoflavone metabolites in serum of renal transplanted patients by high-performance liquid chromatography/electrospray ionization mass spectrometry

During a dietary intervention study on 16 renal transplanted patients, in which 25 g/day of animal proteins were replaced with 25 g of soy proteins, the metabolic profile of soy isoflavones in serum was characterized. This paper describes a reliable and fast liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method, in negative ion mode, allowing the characterization and simultaneous quantification of several soy isoflavone metabolites. Six metabolites were identified and quantified: daidzein ([M-H](-) at m/z 252.8), dihydrodaidzein (DHD, [M-H](-) at m/z 254.8), equol ([M-H](-) at m/z 240.9), O-desmethylangolensin (O-DMA, [M-H](-) at m/z 256.8), genistein ([M-H](-) at m/z 268.8), and dihydrogenistein (DHG, ([M-H(+)](-) at m/z 270.8). Quantification was assessed using two deuterated internal standards, D(3)-daidzein and D(4)-genistein. This method permitted a limit of quantification (LOQ, S/N = 10) and a limit of detection (LOD, S/N = 3) of 0.05 microM and 0.005 microM for all analytes, except for genistein, where the LOQ and LOD were 0.005 microM and 0.001 microM, respectively. The linearity ranges were from 0.005 to 1.5 microM for genistein, from 0.05 to 1.5 microM for DHG, and from 0.05 to 0.7 microM for the other metabolites. The relative standard deviations (RSDs) were between 0.19% and 13.9% at the LOQ concentration for all metabolites, and between 0.6% and 4.8% at the maximum concentration. On the basis of the results obtained in the dietary intervention study, it was possible to split the patients into five groups characterized by different metabolic pathways.     

An accurate and reproducible method for the quantitative analysis of isoflavones and their metabolites in rat plasma using liquid chromatography/mass spectrometry combined with photodiode array detection

To study the safety and potential health benefits of soy isoflavones, a rapid and simple method based on liquid chromatography combined with mass spectrometry (LC/MS) and photodiode array detector (PDA) was developed for the determination of isoflavones in rat plasma. The analytes included daidzein, genistein, glycitein, equol, 4-ethyl phenol, and biochanin A over a concentration range of 1.0-4320.0 nM using 75 microL of rat plasma. Rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia containing glucuronidase and sulfatase to convert the isoflavone beta-glycosides daidzin, genistin, and glycitin to their active aglycone forms. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used to extract aglycones and the internal standards (phenolphthalein beta-D glucuronide, 4-methylumbelliferyl sulfate, and apigenin) from digested plasma samples. The extract was evaporated to dryness under a nitrogen stream, reconstituted with 0.1% formic acid in water-acetonitrile (85 + 15), and injected into a Zorbax SB-CN reversed-phase column (4.6 x 75 mm, 3.5 microm particle size). The Micromass ZQ detector was operated in the positive ion selected-ion monitoring mode. The flow rate for LC was 1.0 mL/min, with a split where 25% of the effluent was introduced into the electrospray ionization probe of the MS instrument and 75% into the PDA. The chromatographic run time was 16.0 min, with delay of 10 min/injection. The interday precision and accuracy of the standard samples were <2.6% relative standard deviation and <10% relative error, respectively. Recovery of the reported isoflavones with this method varied from 86 to 100%.     

HPLC/DAD/MS and antioxidant activity of isoflavone-based food supplements

Isoflavones are polyphenolic compounds found mainly in legumes the benefits of which have been widely studied and attributed in particular to their phytoestrogenic activity. The aim of this study was to evaluate the quali-quantitative composition of food supplements based on soy isoflavones (Glycine max L.) and red clover (Trifolium pratense). Six commercial food supplements (five soy-based and one red clover-based) were analyzed by HPLC/DAD/MS. Genistein, daidzein, glycitein, biochanin A and formononetin derivatives (glycosides and acylglycosides) were identified in the analyzed samples. Also the antiradical activities (towards the DPPH* radical) and Fe2+ chelating abilities were compared.