Analysis of supplements available to UK consumers purporting to contain selective androgen receptor modulators.

Selective androgen receptor modulators (SARMs) are compounds with specific androgenic properties investigated for the treatment of conditions such as muscle wasting diseases. The reported androgenic properties have resulted in their use by athletes, and consequently they have been on the World Anti-Doping Agency prohibited list for more than a decade. SARMs have been investigated by pharmaceutical companies as potential drug candidates, but to date no SARM has demonstrated sufficient safety and efficacy to gain clinical approval by either the European Medicines Agency or the U.S. Food and Drug Administration. Despite their lack of safety approval, SARMs are often illegally marketed as dietary supplements, available for consumers to buy online. In this study, a range of supplement products marketed as SARMs were purchased and analyzed using high resolution accurate mass - mass spectrometry to evaluate the accuracy of product claims and content labeling. This study found discrepancies ranging from a supplement in which no active ingredients were found, to supplements containing undeclared prohibited analytes. Where SARMs were detected, discrepancies were observed between the concentrations measured and those detailed on the product packaging. The outcome of this experiment highlights the high risk of such supplement products to consumers. The inaccurate product claims give rise to uncertainty over both the dose taken and the identity of any of these unapproved drugs. Even for supplements for which the product labeling is correct, the lack of complete toxicity data, especially for combinations of SARMs taken as stacks, means that the safety of these supplements is unknown.

Biomarker-estimated flavan-3-ol intake is associated with lower blood pressure in cross-sectional analysis in EPIC Norfolk.

Flavan-3-ols are a group of bioactive compounds that have been shown to improve vascular function in intervention studies. They are therefore of great interest for the development of dietary recommendation for the prevention of cardio-vascular diseases. However, there are currently no reliable data from observational studies, as the high variability in the flavan-3-ol content of food makes it difficult to estimate actual intake without nutritional biomarkers. In this study, we investigated cross-sectional associations between biomarker-estimated flavan-3-ol intake and blood pressure and other CVD risk markers, as well as longitudinal associations with CVD risk in 25,618 participants of the European Prospective Investigation into Cancer (EPIC) Norfolk cohort. High flavan-3-ol intake, achievable as part of an habitual diet, was associated with a significantly lower systolic blood pressure (- 1.9 (- 2.7; - 1.1) mmHg in men and - 2.5 (- 3.3; - 1.8) mmHg in women; lowest vs highest decile of biomarker), comparable to adherence to a Mediterranean Diet or moderate salt reduction. Subgroup analyses showed that hypertensive participants had stronger inverse association between flavan-3-ol biomarker and systolic blood pressure when compared to normotensive participants. Flavanol intake could therefore have a role in the maintenance of cardiovascular health on a population scale.

Selection of a surrogate matrix for the quantification of an endogenous analyte in dried whole blood.

Aim: A surrogate matrix is needed to quantify 25-hydroxyvitamin D3 in dried whole blood (DWB). To date, there has been limited guidance on approaches for quantification of endogenous analytes in atypical matrices, such as DWB. Methods: Different surrogate matrices were investigated in a systematic process using an LC-MS/MS assay. Performance assessment was made using quality controls of DWB with different hematocrits. Results & conclusion: Suitability of both phosphate-buffered saline containing bovine serum albumin and washed red blood cells recombined with phosphate-buffered saline containing bovine serum albumin as a surrogate matrix was demonstrated across a range of concentrations and hematocrits representative of expected endogenous analyte samples.

Evaluation of (-)-epicatechin metabolites as recovery biomarker of dietary flavan-3-ol intake.

Data from dietary intervention studies suggest that intake of (-)-epicatechin mediates beneficial vascular effects in humans. However, population-based investigations are required to evaluate associations between habitual intake and health and these studies rely on accurate estimates of intake, which nutritional biomarkers can provide. Here, we evaluate a series of structurally related (-)-epicatechin metabolites (SREM), particularly (-)-epicatechin-3'-glucuronide, (-)-epicatechin-3'-sulfate and 3'-O-methyl-(-)-epicatechin-5-sulfate (SREMB), as flavan-3-ol and (-)-epicatechin intake. SREMB in urine proved to be a specific indicator of (-)-epicatechin intake, showing also a strong correlation with the amount of (-)-epicatechin ingested (R2: 0.86 (95% CI 0.8l; 0.92). The median recovery of (-)-epicatechin as SREMB in 24 h urine was 10% (IQR 7-13%) and we found SREMB in the majority of participants of EPIC Norfolk (83% of 24,341) with a mean concentration of 2.4 ± 3.2 µmol/L. Our results show that SREMB are suitable as biomarker of (-)-epicatechin intake. According to evaluation criteria from IARC and the Institute of Medicine, the results obtained support use of SREMB as a recovery biomarker to estimate actual intake of (-)-epicatechin.

Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies.

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk.

Development and validation of a high-throughput assay for the quantification of multiple green tea-derived catechins in human plasma.

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra-batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high-throughput sample analysis studies.

Influence of delivery mode on the urinary excretion of nandrolone metabolites.

INTRODUCTION: This study examined the influence of a supplement matrix on the excretion pattern of nandrolone metabolites in response to ingestion of a trace amount of 19-norandrostenedione. METHODS: Ten male and nine female volunteers (mean ± SD: age = 26 ± 3 yr, height = 1.71 ± 0.09 m, body mass = 70.9 ± 13.2 kg) were recruited. On two occasions, subjects entered the laboratory in the morning after an overnight fast. After an initial urine collection, subjects ingested either 500 mL of plain water or a commercially available energy bar; 10 µg of 19-norandrostenedione was added to each. The volume of each urine sample passed during the next 24 h was measured, and an aliquot was retained for analysis. All samples were analyzed for the metabolites 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) by gas chromatography-mass spectrometry. RESULTS: The total volume of urine passed was higher in the water trial (2.10 ± 0.52 L) than in the bar trial (1.85 ± 0.55 L; P = 0.040). Baseline urinary 19-NA concentrations were all below the limit of quantification for the assay. Peak urinary 19-NA was lower (P = 0.002) in the water trial (4.80 ± 2.84 ng·mL(-1)) than in the bar trial (8.46 ± 4.44 ng·mL(-1)). The time elapsed between ingestion of the supplement and the peak urinary 19-NA concentration was longer (P = 0.023) on the bar trial (4.6 ± 2.4 h) than on the water trial (2.8 ± 1.9 h). There was no difference in the total recovery of 19-NA + 19-NE between the liquid and solid supplements (water 30 ± 10%; bar 28 ± 12%; P < 0.140). CONCLUSIONS: Peak 19-NA concentrations were higher, and occurred later, when the 19-norandrostenedione was added to a solid supplement. This may be due to a slower rate of absorption and/or a reduced diuresis, resulting in a longer period for the metabolites to accumulate in the urine.

Quantification of 19-nortestosterone sulphate and boldenone sulphate in urine from male horses using liquid chromatography/tandem mass spectrometry.

Following administration of the anabolic steroid 19-nortestosterone or its esters to the horse, a major urinary metabolite is 19-nortestosterone-17beta-sulphate. The detection of 19-nortestosterone in urine from untreated animals has led to it being considered a naturally occurring steroid in the male horse. Recently, we have demonstrated that the majority of the 19-nortestosterone found in extracts of 'normal' urine from male horses arises as an artefact through decarboxylation of the 19-carboxylic acid of testosterone. The aim of this investigation was to establish if direct analysis of 19-nortestosterone-17beta-sulphate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) had potential for the detection of 19-nortestosterone misuse in the male horse. The high concentrations of sulphate conjugates of the female sex hormones naturally present in male equine urine were overcome by selective hydrolysis of the aryl sulphates using glucuronidase from Helix pomatia; this was shown to have little or no activity for alkyl sulphates such as 19-nortestosterone-17beta-sulphate. The 'free' phenolic steroids were removed by solid-phase extraction (SPE) prior to LC/MS/MS analysis. The method also allowed for the quantification of the sulphate conjugate of boldenone, a further anabolic steroid endogenous in the male equine with potential for abuse in sports. The method was applied to the quantification of these analytes in a population of samples. This paper reports the results of that study along with the development and validation of the LC/MS/MS method. The results indicate that while 19-nortestosterone-17beta-sulphate is present at low levels as an endogenous substance in urine from 'normal' male horses, its use as an effective threshold substance may be viable.

Phytanic acid: measurement of plasma concentrations by gas-liquid chromatography-mass spectrometry analysis and associations with diet and other plasma fatty acids.

Epidemiological data suggest that a diet rich in animal foods may be associated with an increased risk of several cancers, including cancers of the prostate, colorectum and breast, but the possible mechanism is unclear. It is hypothesised that phytanic acid, a C20 branched-chain fatty acid found predominantly in foods from ruminant animals, may be involved in early cancer development because it has been shown to up regulate activity of alpha-methylacyl-coenzyme A racemase, an enzyme commonly found to be over-expressed in tumour cells compared with normal tissue. However, little is known about the distribution of plasma phytanic acid concentrations or its dietary determinants in the general population. The primary aim of the present cross-sectional study was to determine circulating phytanic acid concentrations among ninety-six meat-eating, lacto-ovo-vegetarian and vegan women, aged 20-69 years, recruited into the Oxford component of the European Prospective Investigation into Cancer and Nutrition (EPIC-Oxford). Meat-eaters had, on average, a 6.7-fold higher geometric mean plasma phytanic acid concentration than the vegans (5.77 v. 0.86 micromol/l; P < 0.0001) and a 47 % higher mean concentration than the vegetarians (5.77 v. 3.93 micromol/l; P = 0.016). The strongest determinant of plasma phytanic acid concentration appeared to be dairy fat intake (r 0.68; P < 0.0001); phytanic acid levels were not associated with age or other lifestyle factors. These data show that a diet high in fat from dairy products is associated with increased plasma phytanic acid concentration, which may play a role in cancer development.

The application of ultra-performance liquid chromatography/tandem mass spectrometry to the detection and quantitation of apolipoproteins in human serum.

The detection and quantitation of apolipoproteins, important markers for coronary heart disease, in serum by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM) is reported. A tryptic digest of depleted human serum was analysed by nanoflow LC/MS/MS at a flow rate of 300 nL/min and several apolipoproteins (Apo), including Apo A1, A2, A4, C1, C2, C3, D, F and M, were successfully identified. The analysis of the same depleted serum digest by ultra-performance (UP)LC/MS/MS operating at 700 microL/min resulted in comparable sensitivity and selectivity to the nanoflow method, but with a dramatic ( approximately 20-fold) reduction in run time. The potential of UPLC/MS/MS for the rapid quantitation of proteins in biological matrices by representative tryptic peptides was further investigated using Apo A1 and its corresponding stable isotopically labelled tryptic AQUA peptide (DYVSQFEGSALGK). A set of serum-based Apo A1 calibrators from a clinical analyser kit were digested without depletion following the addition of the AQUA peptide and analysed using UPLC/MS/MS. A linear calibration curve was generated from peak area ratios to the labelled peptide with a coefficient of correlation of 0.9989. Standard curves were also generated for other apolipoproteins together with Apo B100, Apo E, lecithin cholesterol acyltransferase and albumin, which were also detected in the standards. The concentration of Apo A1 in five fresh undepleted human serum samples and a quality control (QC) sample were determined using both the UPLC/MS/MS method and a clinical analyser. Results were comparable and the quantitative study, involving 80 injections which took hours rather than days to complete, demonstrates the high-throughput potential of UPLC/MS/MS to quantify multiple serum proteins without the need for antibodies, and thus provide an alternative to the use of clinical analysers for serum protein biomarkers.

Variations in plasma phytoestrogen concentrations in European adults.

Dietary phytoestrogens may play a role in chronic disease occurrence. The aim of our study was to assess the variability of plasma concentrations in European populations. We included 15 geographical regions in 9 European countries (Denmark, France, Germany, Greece, Italy, Spain, Sweden, The Netherlands, and UK) and a 16th region, Oxford, UK, where participants were recruited from among vegans and vegetarians. All subjects were participants of the European Prospective Investigation into Cancer and Nutrition (EPIC). Plasma concentrations of 3 isoflavones (daidzein, genistein, and glycitein), 2 metabolites of daidzein [O-desmethylangolensin (O-DMA) and equol] and 2 mammalian lignans (enterodiol and enterolactone) were measured in 1414 participants. We computed geometric means for each region and used multivariate regression analysis to assess the influence of region, adjusted for gender, age, BMI, alcohol intake, smoking status, and laboratory batch. Many subjects had concentrations below the detection limit [0.1 microg/L (0.4 nmol/L)] for glycitein (80%), O-DMA (73%) and equol (62%). Excluding subjects from Oxford, UK, the highest concentrations of isoflavones were in subjects from the Netherlands and Cambridge, UK [2-6 microg/L (7-24 nmol/L); P < 0.05], whereas concentrations for lignans were highest in Denmark [8 microg/L (27 nmol/L); P < 0.05]. Isoflavones varied 8- to 13-fold, whereas lignans varied 4-fold. In the vegetarian/vegan cohort of Oxford, concentrations of isoflavones were 5-50 times higher than in nonvegetarian regions. Region was the most important determinant of plasma concentrations for all 7 phytoestrogens. Despite the fact that plasma concentrations of phytoestrogens in Europe were low compared with Asian populations, they varied substantially among subjects from the 16 different regions.

High throughput quantification of phytoestrogens in human urine and serum using liquid chromatography/tandem mass spectrometry (LC-MS/MS).

Phytoestrogens are currently the subject of intense study owing to their potential protective effects against a number of complex diseases. However, in order to investigate the interactions between phytoestrogens and disease state effectively, it is necessary to have analytical methods which are sensitive, reproducible, and require low sample volumes. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (equol and O-desmethylangolensin), three lignans (secoisolariciresinol, enterodiol, and enterolactone), and one flavanone (naringenin) in human urine and serum. A high throughput of samples has been achieved via the use of 96-well plate sample extraction and liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis incorporating column switching, thus making the assay suitable for use on large sample numbers, such as those found in epidemiological studies. The robustness of the assay was proven via the comparison of data generated on two different LC-MS/MS systems, with and without column switching.

Plasma phytoestrogens and subsequent breast cancer risk.

PURPOSE: Phytoestrogens are plant compounds that are structurally and functionally similar to mammalian estrogens. By competing for estrogen receptors, phytoestrogens possibly inhibit binding of the more potent endogenous estrogens and decrease their potential effects on breast cancer risk. We investigated the association between plasma phytoestrogen levels and breast cancer risk in a prospective manner. PATIENTS AND METHODS: We performed a nested case-control study within the Prospect cohort, one of the two Dutch cohorts participating in the European Prospective Investigation into Cancer and Nutrition. A total of 383 women (87 pre- or perimenopausal women [mean age, 52 years] and 296 postmenopausal women [mean age, 59 years]) who developed breast cancer were selected as case subjects and were matched to 383 controls, on date of blood sampling. Plasma levels of isoflavones (daidzein, genistein, glycitein, O-desmethylangolensin, and equol) and lignans (enterodiol and enterolactone) were measured. The isotope dilution liquid chromatography/tandem mass-spectrometry method incorporating triply 13C-labeled standards was used for all analyses. Breast cancer odds ratios were calculated for tertiles of phytoestrogen plasma levels using conditional logistic regression analysis. RESULTS: For genistein, the risk estimate for the highest versus the lowest tertile was 0.68 (95% CI, 0.47 to 0.98). Similar protective effects, although not statistically significant, were seen for the other isoflavones. Lignan levels did not appear to be related to breast cancer risk. Results were the same in pre- or perimenopausal women, and in postmenopausal women. CONCLUSION: High genistein circulation levels are associated with reduced breast cancer risk in the Dutch population. No effects of lignans on breast cancer risk were observed.

Phytoestrogen exposure, polymorphisms in COMT, CYP19, ESR1, and SHBG genes, and their associations with prostate cancer risk.

Prospective phytoestrogen exposure was assessed using both biomarkers and estimates of intake in 89 British men recruited into the Norfolk arm of the European Prospective Investigation into Cancer and Nutrition study, men who subsequently developed prostate cancer. Results were compared with those from 178 healthy men matched by age and date of recruitment. Levels of seven phytoestrogens (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) were measured in spot urine and serum samples. Five single-nucleotide polymorphisms in COMT, CYP19, ESR1, and SHBG genes were genotyped. Urinary levels of all phytoestrogens correlated strongly with serum levels. Correlation coefficients ranged from 0.63 (glycitein) to 0.88 (daidzein) (P < 0.001). Urinary and serum levels correlated significantly with isoflavone intake assessed from food diaries (R = 0.15-0.20; P < 0.05) but not with that from a food-frequency questionnaire. Odds ratios for phytoestrogen exposure, as assessed using the four methods, were not significantly associated with prostate cancer risk (P = 0.15-0.94). Men with the CC genotype for the ESRI PvuII polymorphism had significantly higher risk for prostate cancer compared with men with the TT genotype [adjusted odds ratio = 4.65 (1.60-13.49); P = 0.005]. Our results utilizing a combined prospective exposure provide no evidence that phytoestrogens alter prostate cancer risk in British men, whereas the C allele for the PvuII polymorphism may be associated with increased risk.

Purification of the crude solution from Helix pomatia for use as beta-glucuronidase and aryl sulfatase in phytoestrogen assays.

Phytoestrogens occur in a variety of foods and are thought to offer a protective effect against a number of complex diseases. Due to the diversity of phytoestrogen conjugates formed in the human body, most assays include an enzymatic hydrolysis step prior to analysis. beta-Glucuronidase from Helix pomatia, which also contains sulfatase activity, is popular for this task but contains appreciable levels of some phytoestrogens and related compounds, which could affect accurate quantification at low concentrations. Use of solid phase extraction on a polymeric resin has been found to remove the majority of these compounds from the enzyme, without affecting the enzyme activity for almost all of the analytes tested.

Polymorphisms in the CYP19 gene may affect the positive correlations between serum and urine phytoestrogen metabolites and plasma androgen concentrations in men.

Phytoestrogens have been hypothesized to protect against prostate cancer via modulation of circulating androgen concentrations. We conducted a cross-sectional study of 267 men in the Norfolk arm of the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort with 2 aims: first, to investigate the association between phytoestrogen exposure (measured from diet, urine, and serum) and plasma concentrations of sex hormone-binding globulin (SHBG), androstanediol glucuronide, testosterone and Free Androgen Index (FAI); and second, whether the association may be modified by polymorphisms in CYP19 and SHBG genes. Dietary daidzein and genistein intakes were obtained from food diaries and computed using an in-house food composition database. Urinary and serum concentrations of 3 isoflavones (daidzein, genistein, glycitein), 2 daidzein metabolites O-desmethylangolensin (O-DMA) and 2 lignan metabolites (enterodiol and enterolactone) were measured using mass spectrometry. There was no association between dietary, urinary, and serum phytoestrogens and plasma SHBG concentrations. Enterolactone was positively associated with plasma androstanediol glucuronide concentrations (urinary enterolactone: r = 0.127, P = 0.043; serum enterolactone: r = 0.172, P = 0.006) and FAI (urinary enterolactone: r = 0.115, P = 0.067; serum enterolactone: r = 0.158, P = 0.011). Both urinary and serum equol were associated with plasma testosterone (urinary equol: r = 0.332, P = 0.013; serum equol: r = 0.318, P = 0.018) and FAI (urinary equol: r = 0.297, P = 0.027; serum equol: r = 0.380, P = 0.004) among men with the TT genotype but not the CC or CT genotypes (r = -0.029 to -0.134, P = 0.091-0.717) for the CYP19 3'untranslated region (UTR) T-C polymorphism. Urinary and serum enterolactone showed similar genotype-dependent associations with testosterone but not with FAI. In this first study on phytoestrogen-gene associations in men, we conclude that enterolactone and equol are positively associated with plasma androgen concentrations, and interactions with CYP19 gene may be involved.

Optimization of conditions for the enzymatic hydrolysis of phytoestrogen conjugates in urine and plasma.

Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens.

Phytoestrogen exposure correlation with plasma estradiol in postmenopausal women in European Prospective Investigation of Cancer and Nutrition-Norfolk may involve diet-gene interactions.

Cross-sectional studies investigating the relationship between phytoestrogens in diet, urine, or blood with plasma estradiol and sex hormone binding globulin (SHBG) have been inconclusive. We investigated the relationship among phytoestrogen exposure, polymorphisms in the ESR1, COMT, CYP19, and SHBG genes, and plasma estradiol and SHBG levels in 125 free-living postmenopausal women taking part in a cohort study (European Prospective Investigation of Cancer and Nutrition-Norfolk) using three different markers: dietary, urinary, and serum phytoestrogens. Phytoestrogen levels (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) in spot urine and serum were analyzed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry, respectively. Plasma estradiol and SHBG were measured by immunoassays. Adjusting for age and body mass index, urinary daidzein, genistein, glycitein, and serum daidzein and glycitein were negatively correlated with plasma estradiol (R = -0.199 to -0.277, P <0.03), with particularly strong associations found in the 18 women with CC genotype for ESR1 PvuII polymorphism (R = -0.597 to -0.834, P < 0.03). The negative correlations observed between isoflavones and estradiol in women as a whole became no longer significant when we excluded women with ESR1 PvuII CC genotype, indicating that the correlations observed were due mainly to this group of women. There was no relationship between dietary isoflavones and plasma estradiol and no association was found between any of the dietary, urinary, and serum phytoestrogen and plasma SHBG or between these factors and polymorphisms in CYP19, SHBG, and COMT. We conclude that higher isoflavone exposure is associated with lower plasma estradiol in postmenopausal women and that this preliminary study is suggestive of the involvement of diet-gene interactions.

Phytoestrogen concentrations in serum and spot urine as biomarkers for dietary phytoestrogen intake and their relation to breast cancer risk in European prospective investigation of cancer and nutrition-norfolk.

Subjects of this study consisted of 333 women (aged 45-75 years) drawn from a large United Kingdom prospective study of diet and cancer, the European Prospective Investigation of Cancer and Nutrition-Norfolk study. Using newly developed gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry methods incorporating triply (13)C-labeled standards, seven phytoestrogens (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) were measured in 114 spot urines and 97 available serum samples from women who later developed breast cancer. Results were compared with those from 219 urines and 187 serum samples from healthy controls matched by age and date of recruitment. Dietary levels were low, but even so, mean serum levels of phytoestrogens were up to 600 times greater than postmenopausal estradiol levels. Phytoestrogen concentrations in spot urine (adjusted for urinary creatinine) correlated strongly with that in serum, with Pearson correlation coefficients > 0.8. There were significant relationships (P < 0.02) between both urinary and serum concentrations of isoflavones across increasing tertiles of dietary intakes. Urinary enterodiol and enterolactone and serum enterolactone were significantly correlated with dietary fiber intake (r = 0.13-0.29). Exposure to all isoflavones was associated with increased breast cancer risk, significantly so for equol and daidzein. For a doubling of levels, odds ratios increased by 20-45% [log(2) odds ratio = 1.34 (1.06-1.70; P = 0.013) for urine equol, 1.46 (1.05-2.02; P = 0.024) for serum equol, and 1.22 (1.01-1.48; P = 0.044) for serum daidzein]. These estimates of risk are similar to those established for estrogens and androgens in postmenopausal breast cancer but need confirmation in larger studies.

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.