Adrian A. Franke

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.

The Polymerase Chain Reaction: DNA Extraction and Amplification

The polymerase chain reaction (PCR) is a method of cloning DNA without the use of microorganisms (Saiki et al. 1985, Mullis et al. 1986). In five short years since the development of PCR, this technology has been modified for many uses (Innes et al. 1990) and has essentially revolutionized molecular biology (Guyer and Koshland 1989). PCR allows the rapid selection, isolation and amplification of DNA regions of interest from small amounts of tissue and can be used to help prepare DNA for sequencing. Because it works well for small amounts of tissue and for small pieces of DNA, PCR allows the examination of nucleotide sequences from ancient preserved specimens which have been dried, frozen, hidden in anaerobic sediments, or soaked in alcohol or formalin (Paabo 1990). The greatly increased speed of extraction, amplification, and sequencing has made nucleotide sequence data available on the large scale necessary for population biological and systematic studies. If sequencing studies turn up consistent differences in nucleotide sequence among populations or taxa, PCR can be combined with RFLP analysis to rapidly screen for these known differences. Dot blot analysis with allele specific probes or allele specific PCR primers can be used for a similar purpose (Innes et al. 1990).

Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii.

Heterocyclic amines (HAAs) and polycyclic hydrocarbons are suspected colorectal cancer (CRC) carcinogens that are found in well-done meat. They require metabolic activation by phase I enzymes, such as the smoking-inducible CYP1A isoenzymes. N-acetyltransferase 2 (NAT2) also play a role in the further activation of HAAs. We conducted a population-based case-control study in Hawaii to test the associations of preference for well-done red meat and HAA intake with colon and rectal cancers, as well as the modifying effects of NAT2 and CYP1A2. We interviewed 727 Japanese, Caucasian or Native Hawaiian cases and 727 controls matched on sex, age, and ethnicity. HAA intake was estimated based on consumption of meat and fish for each of several cooking methods and doneness levels. A subgroup of 349 cases and 467 controls was phenotyped for CYP1A2 by a caffeine test. We found that preference for well-done red meat was associated with a 8.8-fold increased risk of CRC (95% CI: 1.7-44.9) among ever-smokers with the NAT2 and CYP1A2 rapid phenotypes, compared to ever-smokers with low NAT2 and CYP1A2 activities and who preferred their red meat rare or medium. A dose-dependent association was also found between the HAA intake estimates and male rectal cancer, with a two- to three-fold increase in risk from the low (T(1)) to high (T(3)) tertile of intake for each HAA. This association was strongest for MeIQx. HAA intake was not associated with male colon cancer or colon or rectal cancer in women. These data provide support to the hypothesis that exposure to pyrolysis products through consumption of well-done meat increases the risk of CRC, particularly in individuals who smoke and are genetically susceptible (as determined by a rapid phenotype for both NAT2 and CYP1A2). An attempt to examine the risk associated with specific HAAs suggested that the main HAAs increase risk of rectal cancer in men and that they do not appreciably affect risk of rectal cancer in women or of colon cancer in either sex.

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.

Circulating Carotenoids and Risk of Breast Cancer: Pooled Analysis of Eight Prospective Studies

BACKGROUND Carotenoids, micronutrients in fruits and vegetables, may reduce breast cancer risk. Most, but not all, past studies of circulating carotenoids and breast cancer have found an inverse association with at least one carotenoid, although the specific carotenoid has varied across studies. METHODS We conducted a pooled analysis of eight cohort studies comprising more than 80% of the worlds published prospective data on plasma or serum carotenoids and breast cancer, including 3055 case subjects and 3956 matched control subjects. To account for laboratory differences and examine population differences across studies, we recalibrated participant carotenoid levels to a common standard by reassaying 20 plasma or serum samples from each cohort together at the same laboratory. Using conditional logistic regression, adjusting for several breast cancer risk factors, we calculated relative risks (RRs) and 95% confidence intervals (CIs) using quintiles defined among the control subjects from all studies. All P values are two-sided. RESULTS Statistically significant inverse associations with breast cancer were observed for α-carotene (top vs bottom quintile RR = 0.87, 95% CI = 0.71 to 1.05, P(trend) = .04), β-carotene (RR = 0.83, 95% CI = 0.70 to 0.98, P(trend) = .02), lutein+zeaxanthin (RR = 0.84, 95% CI = 0.70 to 1.01, P(trend) = .05), lycopene (RR = 0.78, 95% CI = 0.62 to 0.99, P(trend) = .02), and total carotenoids (RR = 0.81, 95% CI = 0.68 to 0.96, P(trend) = .01). β-Cryptoxanthin was not statistically significantly associated with risk. Tests for heterogeneity across studies were not statistically significant. For several carotenoids, associations appeared stronger for estrogen receptor negative (ER(-)) than for ER(+) tumors (eg, β-carotene: ER(-): top vs bottom quintile RR = 0.52, 95% CI = 0.36 to 0.77, P(trend) = .001; ER(+): RR = 0.83, 95% CI = 0.66 to 1.04, P(trend) = .06; P(heterogeneity) = .01). CONCLUSIONS This comprehensive prospective analysis suggests women with higher circulating levels of α-carotene, β-carotene, lutein+zeaxanthin, lycopene, and total carotenoids may be at reduced risk of breast cancer.

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.