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Original Research Communication |
1 From the Department of Nutrition, the School of Public Health, the School of Medicine, the University of North Carolina, Chapel Hill; the Research Triangle Institute, Research Triangle Park, NC; and the Chemopreventive Agent Development Research Group, the Division of Cancer Prevention, the National Cancer Institute, Rockville, MD.
2 Supported by the National Institutes of Health (N01-CN-65117) and by grants from the National Institutes of Health to the University of North Carolina Clinical Nutrition Research Unit (DK56350), to the University of North Carolina General Clinical Research Center (RR00046), and to the Lineberger Cancer Research Center (CA16086). Protein Technologies International provided the formulations of unconjugated isoflavones.
3 Address reprint requests to SH Zeisel, CB #74002212, McGavran-Greenberg Building, University of North Carolina, Chapel Hill, NC 27599-7400. E-mail: steven_zeisel{at}unc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: We conducted safety studies of purified unconjugated genistein, daidzein, and glycitein, and defined pharmacokinetic parameters for their absorption and metabolism.
Design: Thirty healthy men ingested a single dose of 1 of 2 isoflavone preparations purified from soy. The delivered doses of genistein (1, 2, 4, 8, or 16 mg/kg body wt) were higher than those previously administered to humans. Formulation A was composed of 90 ± 5% genistein, 10% daidzein, and 1% glycitein. Formulation B was composed of 43% genistein, 21% daidzein, and 2% glycitein.
Results: We observed no clinically significant behavioral or physical changes after treatment. We observed elevations in lipoprotein lipase and hypophosphatemia that were possibly related to the treatment but that were associated with no clinical toxicity. Considerable quantities of isoflavones were excreted in urine as conjugates. The terminal elimination rate, elimination half-life, area under the curve, maximum plasma concentration, apparent systemic clearance, and volume of distribution were estimated for genistein and daidzein. The mean elimination half-lives with both formulations were 3.2 h for free genistein and 4.2 h for free daidzein. The mean pseudo half-lives were 9.2 h for total genistein and 8.2 h for total daidzein.
Conclusions: Dietary supplements of purified unconjugated isoflavones administered to humans in single doses exceeding normal dietary intake manyfold resulted in minimal clinical toxicity. Genistein and daidzein (free and total) were rapidly cleared from plasma and excreted in urine.
Key Words: Genistein • daidzein • soy isoflavones • cancer • toxicity • pharmacokinetics • healthy men • North Carolina
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genistein and daidzein inhibit the proliferation of different types of cancer cells (9–11), normal 3T3 cells (12), and normal colonic mucosa (13) in tissue culture. The exact mechanism for this effect is not yet elucidated, but many hypotheses have been suggested. Soy isoflavones activate estrogen receptors (2), inhibit the activity of growth-promoting steroid hormones by inhibiting the enzymes progesterone 5
-reductase and 17 ß-hydroxysteroid dehydrogenase (14), have antioxidant activities (15, 16), inhibit protein-tyrosine kinase–mediated signal transduction (17), inhibit DNA topoisomerase (ATP-hydrolyzing) (18, 19), inhibit transforming growth factor ß1-mediated signal transduction (20), and inhibit angiogenesis (11, 21). Given these disparate effects of isoflavone administration, it is possible that the administration of large doses might be associated with some toxicity.
The purposes of the present study were to conduct safety and pharmacokinetic studies of escalating doses of purified unconjugated isoflavones containing genistein, daidzein, and glycitein and to define the pharmacokinetics of genistein and daidzein absorption and metabolism in healthy men. These unconjugated isoflavones were given in higher doses than previously administered to humans. Free genistein and daidzein concentrations in humans treated with these unconjugated isoflavones are reported here for the first time.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 mg/d, or chronic disease, including a recent history of cancer. None of the subjects had a soy isoflavone intake >10 mg/d on the basis of a diet history, and subjects were instructed to consume no soy products from the time of screening until after study day 30. Before acceptance into the study, the subjects' good health was verified by a medical history and physical examination conducted by a licensed medical doctor, screening laboratory tests, chest X-ray, and electrocardiogram. Fifty subjects were screened for the study; 18 were deemed ineligible or chose not to participate and 32 were deemed eligible on the basis of the inclusion and exclusion criteria and were enrolled in the study. Two subjects were subsequently dropped from the study because of preexisting conditions that were not detected at screening. The subjects provided written, informed consent, and the study was conducted in accordance with the guidelines of the Institutional Review Board of the School of Medicine at the University of North Carolina (UNC) at Chapel Hill and the Research Triangle Institute (Research Triangle Park, NC). We operated under an Investigational New Drug application (no. 54137) for both preparations, which was obtained from the Food and Drug Administration by the National Cancer Institute (NCI).
Isoflavone preparations
The 2 isoflavone preparations were from Protein Technologies International (PTI; St Louis) via the NCI. Formulation A (PTI G-4660) contained 97% total unconjugated isoflavones (lot no. 95H7600; 90 ± 5% genistein, 10% daidzein, and 1% glycitein); formulation B (lot no. 115H7625; PTI G-2535) contained 70% total unconjugated isoflavones (43% genistein, 21% daidzein, and 2% glycitein). The isoflavone composition and concentration of each formulation were independently analyzed by 2 laboratories: Ralston Analytic Laboratories (St Louis) and Sigma Chemical Laboratories (St Louis). The preparations were stable at 40 and 70°C for 6 mo and at 25°C for 3 y (assays performed at the University Pharmaceuticals of Maryland, Inc, Baltimore).
Capsules containing the isoflavone preparation were compounded by the Investigational Pharmacy at the UNC at Chapel Hill, and study personnel were blinded to the formulation. The initial dose of genistein given to the subjects was 1 mg/kg body wt. The dose was escalated subsequently in standard increments of 100% of the previous dose. The dose schedule of genistein and daidzein, by formulation, is shown in Table 1
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The inpatient study period was defined as the 26–30 h of inpatient admission to the UNC General Clinical Research Center (GCRC) that followed a 10-h fast at home. During the inpatient period, baseline laboratory tests were conducted, the unconjugated isoflavones were administered, and blood and urine samples were collected for pharmacokinetic testing. Blood samples were drawn from the antecubital vein for measurement of isoflavones before isoflavone administration and 0.5, 1, 1.5, 2, 3, 4.5, 6, 7.5, 9, 10.5, 12, 13.5, 15, 16.5, 18, 19.5, and 24 h postdose. Urine was collected 3, 6, 12, and 24 h postdose. The subjects were discharged after the final plasma and urine samples were collected. The meals consumed throughout the inpatient study period were isoenergetic and had a standardized macronutrient composition: 55% of energy as carbohydrate, 30% as fat, and 15% as protein. All meals were consumed at specified times during the study period to standardize any effect of the time of food consumption on drug disposition. The outpatient (follow-up) study period involved visits to the UNC GCRC on days 3, 6, 14, and 30 postdose.
Toxicity analyses were conducted on study samples taken during screening, the inpatient study period, and the outpatient study period. Pharmacokinetic analyses were conducted on study samples taken during the inpatient study period. CCS Associates (Mountain View, CA), the monitoring agency designated by the NCI, conducted a complete evaluation of the study procedures and of the documentation of study events.
Clinical measurements
Laboratory tests were performed at screening and on days 1 (predose and 24 h postdose), 3, 6, 14, and 30. The variables measured were sodium, potassium, chloride, carbon dioxide, blood urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, total protein, albumin, uric acid, total bilirubin, calcium, 
-glutamyltransferase, complete blood count with differential (white blood cells, red blood cells, hemoglobin, hematocrit, platelet count, mean cell volume, mean cell hemoglobin, mean cell hemoglobin adjusted for the cell volume, red blood cell distribution width, neutrophils, lymphocytes, monocytes, eosinophils, and basophils), prothrombin time, partial thromboplastin time, fasting glucose, cholesterol, triacylglycerols, phosphorous, magnesium, amylase, lipase, and fibrinogen. The urinalysis included measurements of specific gravity, pH, protein, glucose, and blood. The laboratory tests were conducted at the McLendon Clinical Laboratory of the UNC Hospitals. This laboratory is certified according to the Clinical Laboratory Improvement Act and College of American Pathologists and maintains quality-assurance logs and standard operating procedures. Subjects also received a chest X-ray and an electrocardiogram at screening and on day 30. Chest X-rays were performed in the Radiology Department of the UNC Hospitals and were read by radiologists. Electrocardiograms were performed at the UNC GCRC and were read by the study physicians. Any abnormal results were sent to the Cardiology Department of the UNC Hospitals for further interpretation.
Grading of toxicity
The NCI's Common Toxicity Criteria for assessment of the toxicity of chemopreventive agents (22) was used to assign a severity grade to adverse events.
Isoflavone measurement
Genistein and daidzein standards were obtained from INDOFINE Chemical Company (Somerville, NJ). Dimethylsulfoxide, methanol, acetonitrile, and methyl t-butyl ether (MTBE)—all of which were ultraviolet grade—were obtained from Allied Signal, Inc (Morristown, NJ). Reagent-grade ammonium acetate, ammonium formate, formic acid, and ß-glucuronidase and aryl-sulfatase enzymes from Helix pomatia (Type H-2, catalog number G-0876) and the internal standards 2,4,4'-trihydroxybenzophenone (95%) and 4-hydroxybenzophenone (98%) were obtained from Sigma-Aldrich (St Louis). Glacial acetic acid (American Chemical Society certified) was purchased from Fisher Scientific (St Louis).
Free and total isoflavones in plasma and total isoflavones in urine were measured with use of a modified version of the method of Supko and Phillips (23). The method of Franke et al (24) was adapted for the measurement of free isoflavones in urine. In the present study, total daidzein and total genistein are each defined as the amount of nonconjugated analyte plus the amount of nonconjugated analyte that is released after treatment of the biological matrix with ß-glucuronidase and aryl-sulfatase enzymes. Because the various conjugated isoflavones are not available in pure form, unconjugated genistein and daidzein were used to prepare calibrators and controls.
The HPLC instrumentation (Waters, Milford, MA) consisted of a 600 pumping system, a 717 automatic injector, a 2487 ultraviolet detector set at 260 nm, a Nova-Pak C8 column (3.9 x 150 mm) and Nova-Pak Sentry Guard column maintained at 30°C with an Eppendorf (Westbury, NY) TC50 column heater, and a Millenium chromatography information system (Waters) capable of providing peak retention times, areas, and heights. All mobile-phase systems were prepared by volume, and all gradients were linear. Injection volumes were typically 100 µL. Concentrations of the isoflavones genistein and daidzein were calculated from the ratios of the peak heights for these analytes compared with those of the internal standards 2,4,4'-trihydroxybenzophenone (95%) and 4-hydroxybenzophenone (98%). Calibration standards and positive controls, each prepared separately in the appropriate matrix, were analyzed along with each set of samples. A linear regression analysis of each set of calibration standards in the appropriate matrix was performed with the use of 1/x weighting to obtain the calibration equation relating the ratios of the peak height to the concentration of the respective analyte for that sample set.
In HPLC system 1 the mobile phase consisted of mixtures of 0.02 mol aqueous ammonium formate/L, pH 4.0 (A), and acetonitrile (B) at a flow rate of 1 mL/min. The mobile-phase composition was held constant at 20% B for 1 min after sample introduction, increased over 5 min to 45% B, maintained at 45% B for 6 min, and increased over 2 min to 90% B, where it was maintained for an additional 2 min. In HPLC system 2 the mobile phases consisted of mixtures of 10% glacial acetic acid in deionized water (C) and acetonitrile (D). The mobile-phase composition was maintained at 10% D for 0.5 min after sample introduction, changed over 1.5 min to 20% D and maintained for 12 min, and then changed over 2 min to 80% D and maintained for 7 min. The mobile-phase flow rate was 0.8 mL/min for the first 19 min and 1.5 mL/min for the remaining 4 min.
The extraction and chromatographic procedures enabled the measurement of free (nonconjugated) genistein and daidzein and total genistein and daidzein in plasma and urine (Figure 1). The performance of the analytic methods for these analytes was validated over the expected concentration ranges in each biological matrix (Table 2). Linearity was confirmed for each assay (r2 0.985). Genistein and daidzein were stable in both plasma and urine for 90 d when stored at about -20°C. Freeze-thaw stability (ie, 2 freezing and thawing cycles) was also shown.
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Urinary free isoflavones
After centrifugation (1200 x g, 10 min, 25°C) at low speed to precipitate particulate material in the urine, 10-mL aliquots of urine were mixed with 2.5 mL of 0.2 mol aqueous ammonium acetate buffer/L (pH 4.0; buffer 1) and 87 nmol of the internal standard 2,4,4'-trihydroxybenzophenone in a 20-mL silylated vial. The buffered urine samples were then added to C18 solid-phase extraction columns (Bond-Elut; Varian Inc, Harbor City, CA) prewet with 3 mL methanol followed by 3 mL buffer 1. Each solid-phase extraction column was then washed with 2 mL buffer 1, followed by 4 mL methanol:buffer 1 (1:4, by vol). Tests during method development showed that no measurable analytes were eluted from the solid-phase extraction column by these wash steps. Genistein, daidzein, and the internal standard were eluted from the column with 4 mL methanol:buffer 1 (3:2, by vol). The eluent was passed through a 0.45-µm filter and transferred to a silylated autoinjector vial for analysis with HPLC system 2.
Plasma total isoflavones
Plasma (250 µL), in 0.75 mL buffer 1, was treated overnight (15–18 h) at 
37°C in a 10-mL disposable centrifuge tube with 0.5 mL enzyme solution freshly prepared in buffer 1 and containing 3000 U ß-glucuronidase, 115 U aryl-sulfatase, and 7.5 mg ascorbic acid. After the addition of 50 nmol of the internal standard 4-hydroxybenzophenone, the hydrolyzed conjugates were extracted with 4 mL MTBE with use of an end-over-end mixer. The MTBE layer was transferred to a 10-mL silylated glass culture tube and concentrated. The residue was reconstituted in 0.25–2.0 mL methanol:0.05 mol aqueous ammonium acetate/L (3:7, by vol; pH 4.7) by vortex mixing to facilitate complete reconstitution and analyzed with HPLC system 1.
Urinary total isoflavones
Aliquots (250 µL) of the subjects' urine were treated with ß-glucuronidase and steryl-sulfatase as described above for plasma. After the hydrolysis procedure, 87 nmol of the internal standard 2,4,4'-trihydroxybenzophenone was added, and the hydrolyzed conjugates were extracted with 4 mL MTBE with use of an end-over-end mixer. The MTBE layer was transferred to a 10-mL silylated glass culture tube and concentrated. The residue was reconstituted in 0.25–2.0 mL methanol:0.05 mol ammonium acetate/L (3:7, by vol; pH 4.7) and analyzed with HPLC system 2.
Pharmacokinetic calculations
The dispositions of free and total genistein and daidzein were assessed by using standard model-independent pharmacokinetic analyses with the use of the nonlinear least-squares program WINNONLIN (version 1.5; SCI, Morrisville, NC). The terminal elimination rate constant (kel) and elimination half-life (t1/2) were estimated by linear least-squares regression of the log-transformed concentrations versus time in the terminal log-linear phase of the disposition profile. Apparent systemic clearance (Clp) and apparent volume of distribution (Vd) were estimated for genistein and daidzein. Because the doses were administered orally, Vd and Clp are actually Vd/F and Clp/F, where F is bioavailability.
The area under the curve (AUC) for plasma concentration versus time, from the time of dosing (time 0) to the time the last plasma sample, was obtained by trapezoidal integration. In cases when the analyte concentrations in plasma were less than the limit of quantification (LOQ) of the assay, the lower and upper limits for the AUC and maximum plasma concentration (Cmax) were determined by setting these concentrations equal to 0 and then equal to the LOQ. To calculate mean concentrations in plasma, values lower than the LOQ were assigned a value of 0.5 LOQ. At a given dose (mg/kg) of genistein, the relative bioavailability of the 2 formulations was estimated as the ratio of the AUC for plasma concentration versus time for the 2 formulations and as the ratio of the amount excreted in urine over 24 h (25). For daidzein, dose groups receiving similar doses of daidzein were compared.
Statistics
Descriptive statistics for continuous variables are reported as means ± SDs and ranges and as counts and percentages for categorical variables.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thirty subjects completed the study: 76% whites, 17% blacks, and 7% Asians. Their mean age was 48.1 y and their mean body mass index was 25.7. Blacks represent 23.6%, Asians represent 1.6%, and American Indian and other races represent 0.8% of the general population in the Research Triangle Park area of North Carolina.
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Seven adverse events with a grade 2 were judged to be possibly related to isoflavone administration because there was no other obvious explanation. None of these events were associated with any clinical toxicity. A summary of the adverse events judged to be possibly related to isoflavone administration is presented in Table 4 by dose and formulation. The adverse events included 2 cases of elevated lipase, 1 case of elevated amylase, 1 case of leukopenia, and 4 episodes of hypophosphatemia. The cases of elevated lipase and amylase occurred with formulation A. There were no physical symptoms of pancreatitis reported or detected during the physical examination. Hypophosphatemia occurred with 8 mg/kg genistein as formulation A in 1 case, with 4 mg/kg genistein as formulation B in 2 cases, and with 8 mg/kg genistein as formulation B in 1 case. In all cases, hypophosphatemia occurred within 24 h of isoflavone administration and all of the subjects who experienced hypophosphatemia had calcium values within normal limits. The episode of leukopenia occurred 24 h after a dose of 16 mg/kg genistein as formulation B.
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2 were judged unrelated to isoflavone administration. Five cases of elevated blood lipase occurred 3, 6, and 5 d after 1, 2, and 16 mg/kg genistein as formulation A, respectively, and 24 h after 4 and 8 mg/kg genistein as formulation B; these cases were considered to be unrelated because each of the subjects had a history of significant alcohol consumption. One episode of hyperkalemia was judged to be possibly due to hemolysis in the blood sample; all other potassium measurements in this subject were normal. One subject had leukopenia 6 d postdose, and one subject had a single elevated blood bilirubin concentration 30 d postdose that was unexplained (the subject refused to come in for a repeat measurement). An additional subject had an elevated bilirubin concentration; however, this subject had Gilbert syndrome, which is known to be associated with benign elevations in bilirubin. There were 14 adverse events with a grade of 1 that were judged to be unrelated to isoflavone administration. These adverse events included tingling of the skin, dry mouth, premature ventricular contractions, wheezing, headache, anxiety, vivid dreams, nausea, back pain, and lost desire for alcohol. No estrogenic or antiestrogenic symptoms (eg, gynecomastia and weight loss) were observed after administration of the isoflavone preparation.
Plasma and urinary isoflavones
Plasma free genistein was measurable in all subjects administered formulation B at doses 2 mg/kg and in all subjects administered formulation A at doses 8 mg/kg. Plasma free daidzein was measurable in all subjects only at doses 0.98 mg/kg (2 mg genistein/kg). Plasma total genistein was measurable in all subjects at all doses. Plasma total daidzein was measurable in all subjects that received daidzein doses 0.49 mg/kg. No genistein or daidzein were measured in plasma samples drawn predose. Reported average data and pharmacokinetic parameters are from subjects whose measurable analyte concentrations were available at 6 time points for 2 of the 3 subjects. In all other subjects, measurable analyte concentrations were available for 
3 time points for 2 of the 3 subjects. Genistein and daidzein concentrations from 0 to 24 h in the subjects who received 8 mg genistein/kg are shown in Figures 2 and 3, respectively.
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). Considerably smaller proportions of the genistein dose were excreted in urine as genistein glucuronides and sulfates. Total genistein in urine accounted for a maximum of 18% of formulation B and of 7.7% of formulation A. Some decrement in the percentage dose excreted as total analyte in urine at 24 h was seen at the higher doses of each formulation and analyte. Only for genistein in formulation A at the highest dose did the rate of urinary excretion (nmol/h) not increase as the dose increased (data not shown).
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), which suggests that free genistein and free daidzein are more likely to enter and perhaps be sequestered in the tissue compartment than are their respective conjugates. Also, free isoflavones are cleared from the plasma much more rapidly than are conjugated isoflavones. High clearance of free isoflavones may be due to metabolism (eg, conjugation), sequestration of the free isoflavones in tissues, or excretion.
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). The average t1/2 of free genistein was 3.2 h and that of free daidzein was 4.2 h. The t1/2 of total genistein and of total daidzein were actually pseudo values because each t1/2 is the combination of the metabolism and excretion rates of multiple metabolites. The average pseudo t1/2 of total genistein was 9.2 h and of total daidzein was 8.2 h. The observed Cmax and the times at which Cmax was observed (tmax) are shown in Table 5. For free isoflavones, tmax values ranged from 2.5 to 6.5 h (
: 3.5 h); for total isoflavones, tmax values ranged from 3.7 to 8.0 h (: 5.7 h).
The plasma AUCs versus dose for free and total isoflavones are shown in Figures 5 and 6, respectively. At equivalent doses of isoflavones, AUCs for free daidzein were higher than those for free genistein. Formulation B resulted in higher AUCs for all analytes than did formulation A. Mean Cmax values and mean AUCs for formulation B increased linearly with dose (r2 > 0.92) for both free and total isoflavones, although there was considerable scatter within most dose groups. For formulation A, linearity of mean Cmax values and mean AUC values with dose were established only for total genistein (r2 > 0.99). A comparison of the relative bioavailability of genistein and daidzein from formulations A and B is summarized in Table 6.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Toxicity
Genistein and daidzein may perturb phosphorus metabolism. Although minor hypophosphatemia was observed in 5 of 30 subjects within 24 h postdose, the serum calcium concentrations in these subjects did not change significantly. This finding suggests that the etiology was not related to changes in parathyroid hormone. Renal cellular phosphate transport is modulated by a tyrosine kinase that is inhibited by genistein (29). It is possible that genistein inhibits phosphate reabsorption and increases phosphorus excretion by the kidney. Also, tyrosine kinases play a key role in the regulation of bone metabolism (30). Perhaps genistein changed phosphorus deposition in bone. There is no clinical toxicity associated with this degree of hypophosphatemia, although if these changes were sustained over longer periods, low phosphorus could inhibit normal bone formation.
We observed sporadic elevations in blood lipase activity after administration of both isoflavone preparations. The assay for lipase used in the clinical laboratories at the UNC Hospitals measures pancreatic lipase and lipases secreted from the gastric and intestinal mucosa as well as lipoprotein lipase. In the one subject with elevations in amylase and lipase at the same time, we could not exclude the possibility of pancreatitis. However, lipase elevations alone in the remaining 29 subjects were not considered to indicate pancreatic damage. Genistein or daidzein might alter the regulation of activity or the distribution of lipoprotein lipase. A protein kinase that is inhibited by genistein mediates the modulation of lipoprotein lipase activity by glucose (31). Also, genistein inhibits the cyclic AMP–mediated release of lipoprotein lipase activity from fat pads (32).
We observed no estrogenic or antiestrogenic symptoms or signs in men after administration of either isoflavone formulation. Only 2 published controlled studies investigated the hormonal effects of soy intake in men. In one of these studies, consumption by men of 60 g textured vegetable protein/d for 1 mo had no significant effect on luteinizing hormone or follicle-stimulating hormone (33). In the second study, consumption by men of 355 g (12 oz) soy milk with each meal for 1 mo (providing a total of 100 mg genistein and genistin and a total of 100 mg daidzein and daidzin daily) had no significant effect on serum estradiol or testosterone but resulted in decreases in dehydroepiandrosterone sulfate and in 3-,17ß-androstanediol glucuronide (a metabolite of dihydrotestosterone) of 14% and 13%, respectively (34).
Genistein, but not daidzein, at high concentrations in vitro induces chromatid breaks, gaps, and interchanges in human lymphocytes (35) and production of micronuclei in mouse splenocytes (36). However, micronucleated splenocyte frequency did not increase in mice gavaged with 20 mg genistein·kg-1·d-1 for 5 d (36). Strick et al (37) recently reported that isoflavones cause site-specific DNA cleavage in the MLL breakpoint cluster region in vivo. Chromosomal translocations involving the MLL gene occur in 80% of infant leukemia (37). Our single-dose study was not designed to assess the genotoxicity potential of isoflavones, but studies currently underway in our laboratory using a multiple-dosing design will examine gene-damage parameters.
Pharmacokinetics
Previous studies have looked at the pharmacokinetics of conjugated isoflavones in humans after the consumption of known amounts of soy foods (34, 38–40). However, none described the pharmacokinetics of free genistein or daidzein, although the terms genistein and daidzein are used by some authors to mean total genistein and total daidzein. We now show that measurable quantities of free genistein and free daidzein are present in plasma with t1/2 values of 3.2 h (free genistein) and 4.2 h (free daidzein) after doses of the purified unconjugated isoflavones (formulation B) that are 10 times the normal daily intake of the Japanese (41) (Table 5). Total genistein and daidzein had longer t1/2 values (9.2 and 8.2 h, respectively). King and Bursill (40) reported average tmax values of 8.0 and 7.4 h, respectively, for total genistein and total daidzein when a soy-flour formulation was fed to 6 subjects. (Pseudo t1/2 values for total genistein and total daidzein in that study were estimated to be 5.7 and 4.7 h, respectively.) Pseudo t1/2 values for total genistein and total daidzein were reported by Lu and Anderson (39) to be 3.8 and 2.9 h, respectively, on the basis of urinary excretion rates after the first daily dose of soy milk (80–210 mg each of genistein and daidzein, primarily as glucosides) to 6 men in a month-long study. The results of these studies and ours indicate that genistein and daidzein are both rapidly eliminated after a single dose. On the basis of urinary excretion, Lu and Anderson (39) reported that the pseudo t1/2 values of both genistein and daidzein increase after chronic dosing in men but not in women.
King and Bursill (40) reported that after a single dose of soy flour in men, 22% of the genistein and 62% of the daidzein were excreted in urine as conjugates. After an initial daily dose of soy milk, 15% of the genistein and 47% of the daidzein were excreted in urine as conjugates (34, 39). We observed similar percentages of the isoflavones in formulation B excreted in urine (Figure 4). Providing that the isoflavone glucosides in soy milk and soy flour are converted to aglycones before absorption, these data suggest that the bioavailability of each isoflavone is similar from the 3 matrices: soy milk, soy flour, and formulation B. The lower urinary excretion of the isoflavones in formulation A than in formulation B in the present study is consistent with the lower bioavailabilities of the isoflavones from this formulation determined from plasma AUC data. However, some caution must be taken with this interpretation on the basis of previous findings in women (38, 42). It is possible that significant amounts of the isoflavones are never absorbed or are excreted in the feces, or they may be metabolized to other compounds that were not measured.
Summary
In summary, well-defined mixtures of purified unconjugated isoflavones of genistein, daidzein, and glycitein extracted from soy were administered to humans for the first time. Minimal clinical toxicity was observed, even at single doses that exceeded normal dietary intakes manyfold. We found that free and total genistein and daidzein are rapidly cleared from plasma and that 2 or 3 doses daily should not be expected to result in progressive accumulation of these isoflavones. A large portion of genistein and daidzein is excreted in urine. Soon, we plan to conduct studies similar to this in women and studies of chronic doses of isoflavones in both men and women.
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ACKNOWLEDGMENTS |
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REFERENCES |
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) and formulation B (
); free genistein: formulation A (
) and formulation B (
). n = 3 per data point.
