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Biomedical subjects

A A Franke

Publications and source records attributed to A A Franke.

At least 19 recordsLinked to original sources

Urinary isoflavonoid excretion and soy consumption in three generations of Japanese women in Hawaii.

OBJECTIVE: To explore soy intake and urinary isoflavonoid excretion within several generations of American-Japanese women based on the hypothesis that earlier generations excrete higher levels of urinary isoflavonoids, in particular the metabolite equol, than later generations. SUBJECTS: A convenience sample of 43 women from 19 families aged 18-78 years, all of whom reported at least 50% Japanese ancestry. INTERVENTIONS: Each woman collected overnight urine samples at baseline and after consuming one serving of soymilk, both samples were analyzed for the isoflavonoids daidzein, genistein and equol using liquid chromatography-mass spectrometry. RESULTS: Median isoflavone intakes during the last year were 7.2 mg/day for the first generation, 7.3 mg/day for the second generation and 6.3 mg/day for the third generation (P=0.36). At baseline, the median isoflavonoid excretion for the first generation was nonsignificantly higher than for later generations (190, 86 and 42 nmol/h; P=0.20) but after intervention, the median urinary isoflavonoid excretion was very similar for the three groups: 2465, 1895 and 2775 nmol/h (P=0.70). Following intervention, a nonsignificantly higher proportion of older than younger women (53 vs 32 and 33%; P=0.41) excreted the metabolite equol. The respective median equol excretion rates by generation following intervention were 39.5, 4.2 and 3.5 nmol/h (P=0.04). CONCLUSIONS: This small investigation among three generations of Japanese-Americans detected a higher equol production among older women after a soy challenge, but no difference in the excretion of total isoflavonoids after a standardized dose of soymilk was observed.

Adolescent↗

Serum prostate-specific antigen but not testosterone levels decrease in a randomized soy intervention among men.

BACKGROUND: Low prostate cancer incidence and high soy intake in Asian countries suggest a possible protective effect of soy foods against prostate cancer. The goal of this pilot study was to evaluate the feasibility of a randomized, crossover soy trial among men and to investigate the effects of daily soy intake on serum prostate-specific antigen (PSA) and testosterone levels. METHODS: We randomized 24 men to a high or a low soy diet for 3 months. After a 1-month washout period, the men crossed over to the other treatment. During the high soy diet, the men consumed two daily soy servings; during the low soy diet, they maintained their usual diet. During the entire study each man donated four blood samples and five overnight urine samples. Dietary compliance was assessed by soy calendars, 24-h dietary recalls, and urinary isoflavone excretion measured by high-pressure liquid chromatography with photodiode array detection. Blood samples were analyzed for serum testosterone and PSA by radioimmunoassay. When necessary, variables were log transformed. Two sample t-tests compared the two groups before each study period. Mixed models incorporating the repeated measurements were used to evaluate the effect of the soy diet on urinary isoflavone excretion and serum analytes. RESULTS: Twenty-three men aged 58.7+/-7.2 years completed the study. The compliance with the study regimen was high according to self-reported soy food intake and urinary isoflavone excretion. No significant between-group and within-group differences were detected. During the high soy diet, dietary isoflavone intake and urinary isoflavone excretion increased significantly as compared to the low soy diet. A 14% decline in serum PSA levels (P=0.10), but no change in testosterone (P=0.70), was observed during the high soy diet in contrast to the low soy diet. CONCLUSION: The high adherence as shown by three measures of compliance in this pilot trial demonstrated the feasibility of an intervention based on soy foods among free-living men.

Biomarkers, Tumor↗

Urinary isoflavone excretion as a compliance measure in a soy intervention among young girls: a pilot study.

OBJECTIVE: To investigate the compliance of young girls with a soy intervention. DESIGN: An 8-week dietary intervention and urine sample collection. SETTING: Free-living girls. SUBJECTS: A convenience sample of 8- to 14-y-old girls (20 started and 17 finished the study) recruited through flyers distributed to staff members and previous study participants. INTERVENTION: The girls consumed one daily serving of soymilk, soy nuts, or tofu, completed 3-day food records, kept daily soy intake logs, and collected weekly urine samples. MAIN OUTCOME MEASURES: Compliance with the intervention was evaluated by daily soy intake logs, 3-day food records analyzed by the center's Food Composition and Food Groups Servings Databases, and weekly urinary isoflavone excretion using high-pressure liquid chromatography. The statistical analysis included paired t-tests, analysis of variance, and Spearman's rank-order correlation coefficients. RESULTS: Daily soy intake logs indicated a mean intake of 6.28 servings out of a maximum of 7.0 servings per week. The food records revealed a six-fold increase in isoflavone intake during the study period (P<0.01) which was confirmed by an increase in urinary isoflavone excretion of similar magnitude (23.3-142.1 nmol/mg creatinine, P=0.02). CONCLUSIONS: This study demonstrated the ability of young girls to consume one daily soy serving and the usefulness of urinary isoflavones as a primary compliance measure. The high urinary isoflavone excretion levels detected in girls as compared to adult women suggest less intestinal degradation and/or greater absorption of isoflavones in nonadult populations. This finding requires further investigations into the pharmacokinetics of isoflavones.

Adolescent↗

Association of tocopherols with circulating autoantibody levels against an oxidized DNA nucleoside in humans.

Autoantibodies against oxidized DNA bases are found in vivo and have been used as an indicator of oxidative damage, yet little is known concerning their individual variation and relation to serum micronutrients. Human plasma anti-5-hydroxymethyl-2'-deoxyuridine (HMdU) autoantibody (aAb) levels were repeatedly determined in 41 women and 11 men, and found to have small within-individual variation over time, but large between-individual differences. A positive association in both women (r = .5762, p = .0001) and men (r = .415, p = .2) between plasma total tocopherols and antibody levels was observed. Autoantibody levels were lower in postmenopausal women (8.37 +/- 1.61 vs. 17.18 +/- 2.85 in premenopausal women, p < .01), independently of plasma tocopherol. However, aAb titers in postmenopausal women were still significantly associated with plasma tocopherol levels and adjustment for menopausal status in women yielded a highly significant correlation between HMdU aAb levels and total tocopherol (r = .7342, p = .0001). Plasma malondialdehyde equivalents (MDA), a measure of lipid peroxidation, were also higher in individuals with either high plasma alpha-tocopherol or high beta+gamma-tocopherol levels. The positive association of tocopherols with markers of oxidative damage may reflect a response to the generation of endogenous oxidants associated with enhanced immune function. The decrease in aAb level in postmenopausal women may similarly reflect decreased immune function associated with decreased estrogen levels.

Adult↗

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 gamma-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 gamma-tocopherol from sesame seeds, walnuts, or soy oil. We observed that consumption of as little as 5 mg of gamma-tocopherol per day over a three-day period from sesame seeds, but not from walnuts or soy oil, significantly elevated serum gamma-tocopherol levels (19.1% increase, p = 0.03) and depressed plasma beta-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 gamma-tocopherol and alter plasma tocopherol ratios in humans and is consistent with the effects of dietary sesame seeds observed in rats leading to elevated plasma gamma-tocopherol and enhanced vitamin E bioactivity.

Adult↗

Hydroxymatairesinol, a novel enterolactone precursor with antitumor properties from coniferous tree (Picea abies).

The potential for the extraction of the plant lignan hydroxymatairesinol (HMR) in large scale from Norway spruce (Picea abies) has given us the opportunity to study the metabolism and biological actions of HMR in animals. HMR, the most abundant single component of spruce lignans, was metabolized to enterolactone (ENL) as the major metabolite in rats after oral administration. The amounts of urinary ENL increased with the dose of HMR (from 3 to 50 mg/kg), and only minor amounts of unmetabolized HMR isomers and other lignans were found in urine. HMR (15 mg/kg body wt po) given for 51 days decreased the number of growing tumors and increased the proportion of regressing and stabilized tumors in the rat dimethylbenz[a]anthracene-induced mammary tumor model. HMR (50 mg/kg body wt) did not exert estrogenic or antiestrogenic activity in the uterine growth test in immature rats. HMR also showed no antiandrogenic responses in the growth of accessory sex glands in adult male rats. Neither ENL nor enterodiol showed estrogenic or antiestrogenic activity via a classical alpha- or beta-type estrogen receptor-mediated pathway in vitro at < 1.0 microM. HMR was an effective antioxidant in vitro.

4-Butyrolactone↗

Comparative effects of all-trans beta-carotene vs. 9-cis beta-carotene on carcinogen-induced neoplastic transformation and connexin 43 expression in murine 10T1/2 cells and on the differentiation of human keratinocytes.

9-cis beta-Carotene was extracted from a commercial extract of the algae Dunaliella salina (Betatene), and its actions on proliferation and gene expression were examined in murine 10T1/2 cells and human HaCaT keratinocytes. The 9-cis isomer was less active than all-trans beta-carotene in reducing proliferation and in upregulating expression of connexin 43 in 10T1/2 cells. However, it had comparable ability to suppress carcinogen-induced neoplastic transformation. When tested in HaCaT cells in organotypic culture, it was less active in inducing connexin 43 expression and suppressing expression of keratin K1. In this assay the all-trans isomer was highly active at 10(-6) M, whereas 10(-5) M 9-cis beta-carotene was required to produce a comparable effect. Only small reductions in expression of the basal keratin 5 were seen. All-trans and 9-cis retinoic acids, potential metabolites of beta-carotene isomers, were studied in the same systems. In contrast to the carotenoids, the 9-cis isomer of retinoic acid was approximately 10-fold more active in suppressing neoplastic transformation and inducing connexin 43 expression in both cell types than the all-trans isomer. The retinoic acid isomers were about equipotent in suppressing K1 expression. Cellular levels of 9-cis beta-carotene were approximately 3.5-fold lower than levels of all-trans beta-carotene, suggesting that part, but not all, of this decreased activity of the 9-cis isomer was due to decreased cell uptake. Thus 9-cis beta-carotene is consistently less active than the all-trans isomer; that 9-cis retinoic acid is, in general, much more potent than the all-trans isomer suggests little or no conversion from the carotenoid to the retinoid under these culture conditions.

Animals↗

Modulation of hepatic lipoprotein synthesis and secretion by taxifolin, a plant flavonoid.

In the present study, the effects of taxifolin, a plant flavonoid, on lipid, apolipoprotein B (apoB), and apolipoprotein A-I (apoA-I) synthesis and secretion were determined in HepG2 cells. Pretreatment of cells with (+/-)-taxifolin led to an inhibition of cholesterol synthesis in a dose- and time-dependent manner, with an 86 +/- 3% inhibition at 200 microM observed within 24 h. As to the mechanism underlying this inhibitory effect, taxifolin was shown to inhibit the activity of HMG-CoA reductase by 47 +/- 7%. In addition, cellular cholesterol esterification, and triacylglycerol and phospholipid syntheses, were also significantly suppressed in the presence of taxifolin. ApoA-I and apoB synthesis and secretion were then studied by pulse-chase experiments. ApoA-I secretion was found to increase by 36 +/- 10%. In contrast, an average reduction of 61 +/- 8% in labeled apoB in the medium was apparent with taxifolin. This effect on secretion appeared not to be exerted at the transcriptional level. Rather, the effect on apoB secretion was found to be exerted in the early stages of apoB degradation and to be sensitive to dithiothreitol (DTT) and insensitive to N-acetyl-leucyl-leucyl-norleucinal, suggesting a proteolytic pathway involving a DTT-sensitive protease. Fractionation of secreted apoB revealed a slight shift in the distribution of secreted apoB-containing lipoproteins. Cholesteryl ester, rather than triacylglycerol, was shown to be the lipid that primarily regulated apoB secretion. In summary, our data suggest that taxifolin decreases hepatic lipid synthesis with a concomitant decrease and increase in apoB and apoA-I secretion, respectively.

Anticholesteremic Agents↗

Isoflavone levels in soy foods consumed by multiethnic populations in Singapore and Hawaii.

Concentrations and glucosidic conjugation patterns of isoflavones were determined in soy foods consumed by multiethnic populations in Singapore and Hawaii. Six raw and 11 cooked food groups traditionally consumed in Singapore and 8 food groups consumed in Hawaii were analyzed by reversed-phase high-pressure liquid chromatography with diode array detection. Mean total isoflavone levels varied between 35 and 7500 ppm, with the lowest values found in soy milk and burgers and the highest levels observed in soybean and its seeds and in supplements. Total isoflavone levels and conjugation patterns varied as a function of soybean variety, storage conditions, and food processing. A large contribution to the differences in total isoflavone content between food groups was due to the water content in foods and to leaching of polar analytes into the water phase during boiling. Soy protein drinks and traditional soy foods were found to possess very similar isoflavone amounts considering usual serving sizes.

Beverages↗

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.

Adult↗

Serum levels and metabolic clearance of the isoflavones genistein and daidzein in hemodialysis patients.

Genistein and daidzein are biologically active isoflavones that are especially abundant in soybeans. After intestinal absorption, circulating genistein and daidzein are eliminated primarily by the kidneys. This study was undertaken to assess the metabolism of genistein and daidzein in patients with end-stage renal disease (ESRD) on hemodialysis therapy, and to test whether this treatment modality can replace the lack of kidney function, with respect to the elimination of the isoflavones. Twenty-three hemodialysis patients and 10 healthy subjects were studied. While consuming a self-selected low isoflavone diet, baseline blood levels were undetectable in eight of 10 healthy subjects and in 14 of 23 dialysis patients. The remaining participants had detectable levels, with the nine dialysis patients displaying much higher blood concentrations than the two healthy control subjects. After the evening intake of one dose of an isoflavone-rich soy protein isolate drink, the early morning blood levels of genistein and daidzein were higher in seven dialysis patients than in eight healthy subjects (genistein 1271+/-321 versus 425+/-104, P<0.05; daidzein 1304+/-352 versus 292+/-78, P<0.05). The blood clearance of the isoflavones was studied in two healthy subjects and in three dialysis patients. Genistein and daidzein were eliminated within 2 d in the healthy subjects, but had not returned to baseline in two of three ESRD patients, 7 d after intake. The half-life of both compounds was estimated to be 10-fold longer in the ESRD patients than in the healthy subjects. Finally, genistein and daidzein levels were measured before and after dialysis in five patients, both while on their regular diet and after one dose of a soy protein isolate drink. In both instances, the dialysis treatment did not affect the blood isoflavone levels. In conclusion, approximately one-third of hemodialysis patients eating the standard American renal diet experience high blood levels of the isoflavones genistein and daidzein, while the remaining two-thirds have undetectable levels. After ingestion of isoflavone-rich food such as soy products, all patients have detectable levels that remain very high for several days due to lack of renal excretion.

Adult↗

Exploring the feasibility and effects of a high-fruit and -vegetable diet in healthy women.

Based on reports that fruits and vegetables may protect against breast cancer, this randomized intervention study tested the feasibility of increasing fruit and vegetable intake among healthy women to 9 daily servings through individual dietary counseling and group activities. Adherence to the dietary recommendations was monitored by 24-h food recalls, log sheets, and plasma carotenoid assessments. To explore possible cancer protective mechanisms of fruits and vegetables, we investigated the treatment effect on plasma phenol levels and on thiobarbituric acid-reactive substances measured as malondialdehyde equivalents, a possible marker of oxidative damage. At baseline, women in the intervention (n = 13) and control (n = 16) group reported an average daily consumption of 3.3 and 3.2 fruit and vegetable servings, respectively. After 3 and 6 months of intervention, intake in the intervention group had increased to 8.3 and 7.4 servings, whereas the control group reported an average of 4.2 and 4.1 daily servings. An increase of plasma carotenoid levels from 1249 microg/liter at baseline to 1854 and 1827 microg/liter after 3 and 6 months confirmed compliance with the dietary recommendations in the intervention group. Plasma carotenoid levels among controls changed slightly from 1165 to 1231 and 1291 microg/liter Whereas total phenol levels did not respond according to our hypothesis, malondialdehyde levels decreased slightly in the intervention group. These results suggest that motivated women can substantially increase their fruit and vegetable intake, which leads to a notable increase in plasma carotenoid levels.

Adult↗

Urinary excretion of isoflavonoids and the risk of breast cancer.

Isoflavonoids are a group of biologically active phytochemicals that humans are exposed to mainly through soy food intake. Because of the similar chemical structure of these compounds and estradiol, it has been hypothesized that isoflavonoids may be related to the risk of breast cancer. Overnight urine samples from 60 incident breast cancer cases and their individually matched controls were assayed for urinary excretion rates of five major isoflavonoids (daidzein, genistein, glycitein, equol, and O-desmethylangolensin) and total phenols. These subjects were from a large population-based case-control study conducted in Shanghai, and urine samples from breast cancer cases were collected before any cancer therapy to minimize the potential influence of the disease and its sequelae on study results. Urinary excretion of total phenols and all individual isoflavonoids, particularly glycitein, was substantially lower in breast cancer cases than controls. For total isoflavonoids, the mean excretion was 13.95 nmol/mg creatinine (SD, 20.76 nmol/mg creatinine) for cases and 19.52 nmol/mg creatinine (SD, 25.36 nmol/mg creatinine) for controls (P for difference = 0.04). The case-control difference was more evident when median levels of these compounds were compared, with the median excretion of all major isoflavonoids being 50-65% lower in cases than in controls. Individuals in the highest tertile of daidzein, glycitein, and total isoflavonoids had about half the cancer risk of those in the lowest tertile. The adjusted odds ratio for breast cancer was 0.14 (95% confidence interval, 0.02-0.88) for women whose urinary excretion of both phenol and total isoflavonoids was in the upper 50% compared with those in the lower 50%. The results from this study support the hypothesis that a high intake of soy foods may reduce the risk of breast cancer.

Adult↗

Inhibition of neoplastic transformation and bioavailability of dietary flavonoid agents.

Evaluation of unknown biological effects of chemicals including food plant products requires the assessment of bioactivity and bioavailability. Epidemiologic studies show consistently a cancer protective effect of fruit and vegetable consumption, but there is little understanding of which phytochemicals account for this observation. Commonly studied antioxidant micronutrients are less consistently correlated with cancer protection relative to the food groups themselves, suggesting that other phytochemicals or a combination of food products play key roles in preventing cancer. We investigated the effects of the predominant dietary flavonoids and isoflavonoids at inhibiting neoplastic transformation induced by 3-methylcholanthrene in C3H 10T1/2 murine fibroblasts. We found that most phenolic agents tested were equal to or superior to known chemopreventive agents such as carotenoids or vitamins in effectiveness. Hesperetin, hesperidin and catechin were the most potent agents among the flavonoids tested, inhibiting transformation completely when applied at 1.0 microM after exposure to the carcinogen. Structure-activity comparison revealed that among the compounds tested, flavonoids with a vicinal diphenol structure in ring 'B' and a saturated 'C' ring exhibited the strongest effects. Most agents tested showed dose-dependent patterns. Interestingly, the soy isoflavonoids were weakly active except when applied in combination, suggesting a synergistic effect. In addition, HPLC techniques were developed for determining the bioavailability of isoflavonoids in human biological fluids including urine, plasma and breast milk. We observed a relatively fast absorption, distribution and elimination of isoflavonoids including a biphasic pattern probably due to enterohepatic circulation. Total peak isoflavone levels in urine, plasma and in breast milk were found to be 60 microM, 2 microM and 0.2 microM, respectively and were reached 8-12 hours after consumption of soy foods. Levels detected in human body fluids were found to be highly active at inhibiting neoplastic transformation, especially considering synergistic effects observed for combinations of daidzein and genistein, the predominant isoflavonoids occurring in soy foods.

Animals↗

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.

Adult↗

HPLC analysis of isoflavonoids and other phenolic agents from foods and from human fluids.

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), glycitein (3), and genistein (4), including their malonyl (a) and acetyl (b) esters, and their mammalian metabolites equol (6) and O-desmethylangolensin (7), as well as formononetin (2), biochanin-A (5), and coumestrol (8) 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-microl injections were found to be 1.09, 0.53, 3.28, and 1.00 pmoles for daidzein, genistein, equol, and O-desmethylangolensin (DMA), respectively, by monitoring at the individual compound's 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.

Chromatography, High Pressure Liquid↗