Laurie J. Custer

RAPID HPLC ANALYSIS OF DIETARY PHYTOESTROGENS FROM LEGUMES AND FROM HUMAN URINE

Abstract Due to growing evidence suggesting that phytoestrogens might protect against various cancers, particularly against breast and prostate cancer, it is important to measure the exposure of populations to these compounds by determining levels in food and in human tissue or body fluids to assess the possible cancer protective properties of these agents. Therefore, we developed a simple and fast procedure to extract and simultaneously hydrolyze phytoestrogens and their conjugates from food items, and present a fast and selective high-performance liquid chromatography (HPLC) method for precise determinations of the most common dietary phytoestrogens genistein, biochanin-A, daidzeln, formononetin, and coumestrol using flavone as internal standard. For the first time HPLC was applied to measure these phytoestrogens and their most abundant metabolites equol and O-desmethyl-angolensin from human urine. The proposed methodology has been evaluated for losses due to thermal degradation during extraction and hydrolysis and due to sample handling during the entire work-up including solid phase extraction, and values are given for inter- and intra-assay variability. We present isoflavonoid levels of most common peas and beans used in “western” and “eastern” diets and compare isoflavonoid and coumestrol levels of raw, canned, and cooked foods which can be used in future epidemiological studies. We also determined human urinary levels with our methodology comparing values before and after soybean intake.

HPLC ANALYSIS OF ISOFLAVONOIDS AND OTHER PHENOLIC AGENTS FROM FOODS AND FROM HUMAN FLUIDS

Abstract A fast, precise and selective diode array HPLC method is presented for the extraction and analysis of soy isoflavonoids from foods and from human urine, plasma, and breast milk in support of mechanistic and epidemiologic studies assessing the potential cancer protective role of soya or isoflavones. Solid phase or solvent extraction was chosen for isolation, and enzymatic or acid hydrolysis procedures were used for aglycone production depending on the matrix to be analyzed. C-18 reversed-phase HPLC was applied to selectively separate and quantitate daidzein (1), 2 glycitein (3), 2 and genistein (4), 2 including their malonyl (a) 2 and acetyl (b) 2 esters, and their mammalian metabolites equol (6) 2 and Odesmethylangolensin (7), 2 as well as formononetin (2), 2 biochanin-A (5), 2 and coumestrol (8) 2 using a gradient elution system. UV absorbance scans and authentic standards were applied for identification purposes, additional to fluorometric monitoring, electrochemical detection, and GC/MS analysis after trimethyl silylation. Detection limits of 20-μl injections were found to be 1.09, 0.53, 3.28, and 1.00 pmoles for daidzein, genistein, equol, and Odesmethylangolensin (DMA), respectively, by monitoring at the individual compounds absorption maximum. The proposed method was applied to monitor isoflavone levels in soy foods and in human plasma, urine and breast milk after challenge with roasted soybeans. Implications of the presented results on the potential activity of isoflavones to prevent cancer by exposing newborn infants to these agents are discussed.

Usual dietary consumption of soy foods and its correlation with the excretion rate of isoflavonoids in overnight urine samples among Chinese women in shanghai

Soy foods and certain soy constituents, particularly isoflavones, have been suggested to have potential cancer-inhibitory effects in laboratory and epidemiological studies. Chinese women in Shanghai consume high levels of soy foods and have low incidence rates of breast and other hormone-related cancers. To assess the usual dietary consumption of soy foods and evaluate the correlation of soy food consumption with the urinary excretion of isoflavonoids in overnight urine samples in this population, we analyzed data from 60 healthy women included in an ongoing population-based case-control study of breast cancer in Shanghai. Usual consumption of soy foods in the previous five-year period was assessed using a food-frequency questionnaire, and urinary excretion of daidzein, genistein, glycitein, equol, and O-desmethylangolensin was measured from overnight urine samples collected at the time of dietary assessment. Virtually all women (96.7%) in Shanghai consumed soy foods at least once a week. The median intake of soy food was 100.6 g/day, with 25th and 75th percentiles of 36.8 and 238.2 g, respectively. The median intake of isoflavones was 39.26 mg/day, and there was a nearly fourfold difference between the 25th and 75th percentiles of this measurement. With the increasing intake of soy foods, urinary excretion rates of total isoflavonoids and all individual major isoflavonoids were increased in a dose-response manner (trend test p < or = 0.05). At individual levels the urinary excretion rate of total isoflavonoids was correlated closely with dietary soy food intake, with a correlation coefficient of around 0.5 (p < 0.001). These results indicate that the urinary excretion rate of total isoflavonoids measured from overnight urine samples may reflect reasonably well the usual intake of soy foods in a population with a high level of soy food consumption.

Liquid chromatographic-photodiode array mass spectrometric analysis of dietary phytoestrogens from human urine and blood.

Dietary phytoestrogens have been implicated in the prevention of chronic diseases. However, it is uncertain whether the phytoestrogens or the foods associated with phytoestrogens account for the observed effects. We report here a new liquid chromatography photodiode array mass spectrometry (LC-PDA-MS) assay for the determination of nanomolar amounts of the most prominent dietary phytoestrogens (genistein, dihydrogenistein, daidzein, dihydrodaidzein, glycitein, O-desmethylangolensin, hesperetin, naringenin, quercetin, enterodiol, enterolactone) in human plasma or serum and urine. This assay was found to be suitable for the assessment of quercetin exposure in an onion intervention study by measuring urinary quercetin levels. Other successful applications of this assay in clinical and epidemiologic studies validated the developed method and confirmed previous results on the negative association between urinary isoflavone excretion and breast cancer risk.

Effects of Dietary Sesame Seeds on Plasma Tocopherol Levels

The tocopherols, the major vitamers of vitamin E, are believed to play a role in the prevention of human aging-related diseases such as cancer and heart disease, yet little is known concerning determinants of their plasma concentrations. Evidence from animal studies suggests that the dietary source of γ-tocopherol can significantly affect plasma levels of this tocopherol as well as its functional vitamin E activity. To determine whether plasma levels of tocopherols in humans are similarly altered, a study was undertaken in which subjects (n = 9) were fed muffins containing equivalent amounts of γ-tocopherol from sesame seeds, walnuts, or soy oil. We observed that consumption of as little as 5 mg of γ-tocopherol per day over a three-day period from sesame seeds, but not from walnuts or soy oil, significantly elevated serum γ-tocopherol levels (19.1% increase, p = 0.03) and depressed plasma β-tocopherol (34% decrease, p = 0.01). No significant changes in baseline or postintervention plasma levels of cholesterol, triglycerides, or carotenoids were seen for any of the intervention groups. All subjects consuming sesame seed-containing muffins had detectable levels of the sesame lignan sesamolin in their plasma. Consumption of moderate amounts of sesame seeds appears to significantly increase plasma γ-tocopherol and alter plasma tocopherol ratios in humans and is consistent with the effects of dietary sesame seeds observed in rats Lawrence Erlbaum Associatesding to elevated plasma γ-tocopherol and enhanced vitamin E bioactivity.

Isoflavones in human breast milk and other biological fluids.

We established a method for using HPLC and diode-array ultraviolet scanning to quantitate soy isoflavonoids in foods and in human plasma, urine, and breast milk. The analytes occurring as glycoside conjugates were hydrolyzed enzymatically before HPLC analysis if extracted from biological matrices or were subjected to direct HPLC analysis after extraction from foods. We monitored the isoflavones daidzein, genistein, glycitein, formononetin, and biochanin-A and their mammalian metabolites equol and O-desmethylangolensin in human plasma, urine, and breast milk. Analytes were identified by absorbance patterns, fluorometric and electrochemical detection. and comparison with internal and external standards. In addition, we identified analytes by using gas chromatography-mass spectrometry after trimethylsilylation. The HPLC method was also used to measure concentrations of isoflavones and their glucoside conjugates in various soy-based infant formulas. Total isoflavone concentrations varied between 155 and 281 mg/kg. After one woman received a moderate challenge with 20 g roasted soybeans (equivalent to 37 mg isoflavones), we detected mean total isoflavone concentrations of approximately 2.0 micromol/L in plasma, 0.2 micromol/L in breast milk, and 3.0 micromol/h in urine. According to our measurements, with adjustment for body weight, isoflavonoid exposure is 4-6 times higher in infants fed soy-based formula than in adults eating a diet rich in soyfoods (approximately 30 g/d). Implications of the presented results for the potential cancer-preventing activity of isoflavones by exposing newborn infants to these phytochemicals are discussed.

High-performance liquid chromatographic assay of isoflavonoids and coumestrol from human urine.

A rapid, sensitive and precise diode-array reversed-phase HPLC method was developed for human urine analysis of the most common dietary isoflavones daidzein, genistein, formononetin and biochanin-A, their mammalian metabolites equol and O-desmethylangolensin, and of coumestrol, another commonly occurring phytoestrogen. Analytes were isolated and concentrated by solid-phase extraction and separated by HPLC followed by identification through retention times and UV scans, and in the case of coumestrol additionally by fluorometric response. This method was applied to monitor changes in urinary excretion of these analytes after challenge with soybeans and was evaluated for precision and recovery of analytes.

Lifestyle and nutritional correlates of cytochrome CYP1A2 activity : inverse associations with plasma lutein and alpha-tocopherol

Cytochrome CYP1A2, a liver enzyme responsible for the metabolic activation of a number of putative human carcinogens, exhibits wide inter-individual differences in activity. In order to characterize sources of variability in CYP1A2 activity, we phenotyped (with the caffeine test) 90 subjects of various ethnic backgrounds in Hawaii. Forty-three subjects were patients with in-situ colorectal cancer treated by polypectomy and 47 were healthy population controls. Subjects were also administered a detailed lifestyle questionnaire, including a quantitative food frequency questionnaire, and were assessed for plasma levels of carotenoids, tocopherols, retinol, ascorbic acid, cholesterol and triglycerides. In a stepwise multiple regression, 27% of the overall variation in CYP1A2 activity was explained by seven variables. Plasma lutein explained the largest portion of the variance (7%) and was negatively associated with CYP1A2 activity (p < 0.01), as were use of menopausal replacement estrogens (p = 0.04), plasma alpha-tocopherol (p = 0.05) and alcohol consumption (p = < 0.01). Acetaminophen use (p = 0.05), coffee consumption (p = 0.05) and plasma lycopene (p = 0.06) were positively associated with CYP1A2 activity. After adjustment for these variables, no association was found between CYP1A2 activity and sex, race, age, education, smoking, physical activity, weight, vitamin E supplements, the other plasma micronutrients measured, and dietary intakes of red meat, processed meat and cruciferous vegetables. Results were similar for colorectal cancer cases and controls. Almost two-thirds (73%) of the variability in CYP1A2 activity remained unexplained. This study confirms an enhancing effect of acetaminophen and coffee on CYP1A2 activity and suggests and inhibitory effect of estrogens, alcohol and food sources of lutein and alpha-tocopherol on this enzyme.

Isoflavones in breastfed infants after mothers consume soy.

BACKGROUND The bioavailability of isoflavones in children after soy exposure is uncertain. OBJECTIVE We aimed to compare isoflavone patterns in infants exposed to isoflavone-containing breast milk (BF), in tofu-fed (TF) infants, and in mothers consuming a soy beverage. DESIGN Eighteen nursing mothers who were not feeding soy foods to their infants consumed one daily serving of a soy protein beverage for 2-4 d and collected their own milk and urine and infant urine. Plasma was collected from infants if venous blood draws were ordered by pediatricians. Blood and urine were collected from additional children after they consumed tofu. Isoflavones were measured by liquid chromatography-mass spectrometry. RESULTS In 7 subjects, isoflavone values increased significantly from baseline after mothers ate soy: in maternal urine (x +/- SEM) from 18.4 +/- 13.0 to 135.1 +/- 26.0 nmol/mg creatinine, in breast milk from 5.1 +/- 2.2 to 70.7 +/- 19.2 nmol/L, and in infant urine from 29.8 +/- 11.6 to 111.6 +/- 18.9 nmol/mg creatinine. The mean isoflavone concentration in plasma obtained from 11 BF infants was 19.7 +/- 13.2 nmol/L. TF infants had much higher mean isoflavone values (urine, 229 +/- 129 nmol/mg creatinine; plasma, 1049 +/- 403 nmol/L). Statistically significant correlations were observed between the types of fluids investigated within mothers, between mothers and infants, and within infants. Urinary isoflavone excretion per hour adjusted for dose per body weight was 81% lower for BF infants and 24% higher for TF infants than for their mothers after eating soy. CONCLUSIONS More isoflavones appear in children than in adults after adjustment for isoflavone intake. Systemic isoflavone exposure in infants can be determined by urinary analysis.

Phytoestrogenic isoflavonoids in epidemiologic and clinical research.

Isoflavones (IFLs) are natural products to which humans have been traditionally exposed predominantly through soy foods; more recently humans are also exposed to them through soy protein addition to processed foods or through supplements. They are structurally similar to steroidal estrogens and can exert estrogenic or antiestrogenic effects depending on their concentrations and on the tissue considered. These properties qualify IFLs to be classified as phytoestrogens and are believed to account for many of the biological effects observed for soy and/or IFL exposure including benefits for bone and heart health or prevention of menopausal symptoms and certain types of cancer. In order to evaluate the function of IFLs, alone or when exposure happens through soy intake, pharmacokinetics and bioavailability are critical issues to be considered in epidemiologic and clinical research. For this purpose precise, accurate, robust, fast, and affordable techniques for IFL analyses are required.