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American Journal of Clinical Nutrition, Vol. 87, No. 5, 1314-1323, May 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Pharmacokinetics of the soybean isoflavone daidzein in its aglycone and glucoside form: a randomized, double-blind, crossover study1,2,3

Corinna E Rüfer, Achim Bub, Jutta Möseneder, Peter Winterhalter, Melanie Stürtz and Sabine E Kulling

1 From the Department of Physiology and Biochemistry of Nutrition, Max Rubner–Institute, Karlsruhe, Germany (CER, AB, and JM); the Institute of Food Chemistry, Technical University of Braunschweig, Braunschweig, Germany (PW and MS); and the Institute of Nutritional Science, Food Chemistry, University of Potsdam, Nuthetal, Germany (SEK)

2 Supported by the Deutsche Forschungsgemeinschaft (grant Ku 1079/6-1).

3 Reprints not available. Address correspondence to SE Kulling, Food Chemistry, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. E-mail: kulling{at}uni-potsdam.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: There are conflicting results in the literature on the bioavailability of isoflavones in the aglycone and the glucoside forms.

Objective: The objective was to investigate the pharmacokinetics of the soy isoflavone daidzein (DAI) on oral administration of both the aglycone and glucoside form in a human intervention study. In addition, the pharmacokinetics of the bacterial and oxidative metabolites of DAI was assessed.

Design: Seven German men aged 22–30 y participated in a randomized, double-blind study in a crossover design. After ingestion of pure DAI or pure daidzein-7-O-β-D-glucoside (DG) (1 mg DAI aglycone equivalent/kg body weight), blood samples were drawn before isoflavone administration and 1, 2, 3, 4.5, 6, 8, 10, 12, 24, and 48 h after the dose. Urine was collected before and 0–6, 6–12, and 12–24 h after the intake of the isoflavones. The concentrations of DAI and its major bacterial and oxidative metabolites in plasma and urine were measured with isotope dilution capillary gas chromatography–mass spectrometry.

Results: The systemic bioavailability (area under the curve; AUCinf), the maximal plasma concentration (Cmax), and the cumulative recovery of DAI in urine after administration of DG were 3–6 times greater than after the ingestion of DAI. Except for equol, which was formed by only one volunteer, all other quantified metabolites exhibited 2–12 times greater AUCinf, Cmax, and urinary recoveries after consumption of DG.

Conclusion: Our results show that DG exhibits a greater bioavailability than its aglycone when ingested in an isolated form.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phytoestrogens of the isoflavone family are found most abundantly in soy and soy-derived foods. The most common representatives are the isoflavones daidzein (DAI) and genistein. In soybeans the glucoside forms predominate, and variation in food processing alters the relative content of glucosides compared with aglycones (Figure 1Go) (1). Isoflavone intake is associated with a broad variety of beneficial health effects (2–5). However, some studies have raised concerns about potential adverse effects from isoflavone intake in infants, as the result of early estrogenic exposure (6, 7), and in adults, as the consequence of an elevated breast cancer risk in woman with occult tumors (8). Thus, many studies have measured these compounds in food items and determined their concentrations in biological fluids, such as blood plasma and urine as well as feces after intake of soy products (9–12). In addition, their metabolic fate in the human body is of high interest. It was reported that, for instance, DAI is converted by the gut microflora to dihydrodaidzein (DHD), which can be further metabolized to both S-equol and O-desmethylangolensin (O-DMA) (Figure 1Go) (13–15). For the oxidative metabolism, Kulling et al (16, 17) showed that DAI and genistein undergo hydroxylation, predominantly in the C-6, -8, and -3' position, catalyzed by cytochrome P450 enzymes in vitro with the use of rat and human liver microsomes, as well as in human urine (Figure 1Go). Other reports have focused on the formation of phase II metabolism and found that the monoglucuronides are the predominant phase II metabolites in plasma and urine (18).


Figure 1
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  		FIGURE 1.. Chemical structures of isoflavone aglycones, the corresponding glucosides, and the oxidative and bacterial metabolites.

 
A substantial difference between the intake of soy food items in the Japanese, Chinese, and Indonesian populations compared with the US and European populations is that in the Asian countries people consume a traditional diet that is rich in fermented soy product, whereas in the Western countries mainly nonfermented soy products or soy supplements are consumed (19). Fermented soy products, for instance natto or tempeh, have aglycones as the major form, whereas in nonfermented soy products, eg, soy milk or soy supplements, the glucoside form predominates (1). To date, no benefit-risk assessment of soy supplements has been conducted. Consequently, knowledge about the bioavailability of different isoflavone forms is fundamental and still remains incomplete.

Human studies on the bioavailability of isoflavones in the aglycone and glucoside forms showed contradictory results, suggesting absorption of the aglycones to a lesser (20), the same (21, 22), or a greater (23, 24) degree than the corresponding glucosides. Some studies used isoflavone extracts in a pharmaceutical formulation; others used soy food items, such as soy milk. Therefore, several factors besides the chemical form of the isoflavones might have contributed to the observed pharmacokinetics of the isoflavones in these studies. Our aim was to shed light on the pharmacokinetics of the isoflavone DAI, as well as its major bacterial and oxidative metabolites, after consumption of pure DAI in both the aglycone and glucoside form in a human intervention study with a crossover design.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isoflavones
Both forms of DAI, aglycone and 7-O-β-D-glucoside (DG), were isolated and purified by high-speed countercurrent chromatography. A commercially available DAI preparation (TCI Tokyo Casei, Tokyo, Japan) was purified by high-speed countercurrent chromatography with the use of the biphasic solvent system ratio of hexane to ethyl acetate to methanol to water (6.1:3.8:5:5; vol:vol). The denser layer was used as the mobile phase. Pure DAI was obtained from soy flour according to the method described by Degenhardt and Winterhalter (25). Characterization of both compounds was performed by HPLC–diode array detector–ion mass spectroscopy and by 1H nuclear magnetic resonance spectroscopy. Purity was 99.4% for DAI and 98.8% for DG. The 3,4,8-13C-DAI and 1,2,3-13C-O-DMA were obtained from Nigel Botting, University of St Andrews (St Andrews, United Kingdom). DHD, O-DMA, equol, and 6-, 8-, and 3'-hydroxy (OH)–DAI were purchased from Plantech (Reading, United Kingdom). 2-Methoxy-7-hydroxy-isoflavone (2-OCH3-7-OH-IF) was synthesized as described by Wähälä et al (26). Enzymes and all other chemicals used were obtained from Sigma-Aldrich (Taufkirchen, Germany).

Subjects
Seven nonsmoking German men aged 22–30 y participated in this study. They were in good health as determined by a screening history and medical examination. They refrained from taking dietary supplements and medications for 3 mo and from antibiotics for 6 mo before and during the study. Body mass indexes (in kg/m2) ranged between 19 and 24. Body fat was calculated by bioelectrical impedance analysis and was determined to be 9–15%. The study protocol was approved by the Ethical Committee of Landesärztekammer Baden Württemberg, Germany. All participants had given their written consent.

Study design
Run-in-phase
The subjects refrained from soy-containing food for 2 wk. For this purpose a list of food items containing isoflavones, including traditional soy-based foods (soy beans, soy sauce, soy flour, tofu, miso, tempeh, soy desserts) and hidden sources of soy (soy flour or soy lecithin added to bakery products from specific bakeries in the neighborhood, soy lecithin added to frozen foods, soy protein isolate added to bread spreads or meat alternatives), was given to each participant, and they were instructed to avoid such products. Body weight of the volunteers was monitored 1 d before the intervention started to prepare the correct amount of isoflavones to be ingested.

Intervention period
The 7 men were randomly assigned to group A or group B (n = 3 and 4, respectively). Group A consumed a hard gelatin capsule with DAI; group B consumed a hard gelatin capsule with DG during the first period of the intervention. After the washout period (2 wk between the DAI and DG intakes), group A consumed DG during the second period and group B consumed DAI.

The dose of DAI and DG (1 mg of isoflavone calculated as aglycone equivalent/kg body weight) was given in a hard gelatin capsule with the first breakfast after a 12 h overnight fasting. This breakfast as well as the following meals consumed throughout the 2 study periods were isoenergetic and had standardized macronutrient compositions. All meals were consumed at specified times during the study period. The participants remained at the Human Nutrition Unit of the Max Rubner–Institute during the first 24 h of the study period. After the collection of the 24-h postdose blood sample, the volunteers were allowed to consume food ad libitum with the restriction to avoid soy-containing food.

Blood and urine collection
Blood samples were collected before (baseline) isoflavone administration and 1, 2, 3, 4.5, 6, 8, 10, 12, 24, and 48 h after the dose. Blood was obtained by an indwelling cannula for samples up to 12 h and thereafter by venipuncture. Plasma was collected after centrifugation at 1500 x g for 10 min at 4 °C. Aliquots were stored at –80 °C until analysis. During the intervention period, urine was collected before and 0–6, 6–12, and 12–24 h after the intake of the isoflavone preparations. Sodium-azide (0.1%) was added to prevent bacterial growths, and aliquots were stored at –80 °C.

Analytic methods
Cleanup of plasma samples
The concentrations of DAI and its bacterial metabolites, DHD, O-DMA, and equol, and the oxidative metabolites, 6-, 8-, and 3'-OH-DAI, were measured by capillary gas chromatography–mass spectrometry (GC-MS) with the use of 2 stable isotopically labeled internal standards, as well as 2-OCH3-7-OH-IF as a third internal standard. These internal standards were added to the plasma before its extraction and cleanup. Total isoflavonoids were determined after extraction and enzymatic hydrolysis of the conjugates with a mix of β-glucuronidase and sulfatase. Unconjugated isoflavonoids were measured separately without enzymatic hydrolysis.

Plasma (0.5 mL or 0.25 mL after administration of DAI and DG, respectively) was equilibrated with 125 and 250 pmol, respectively, of the internal standard 3,4,8-13C-DAI and 100 pmol of each 1,2,3-13C-O-DMA and 2-OCH3-7-OH-IF for 30 min at room temperature (RT). After dilution of the samples with 10 volumes of 0.5 mol/L triethylammonium sulfate (pH 5), they were incubated for 15 min at RT as well as at 64 °C before passage through a preconditioned RP-18 cartridge (Waters, Eschborn, Germany). The column was rinsed with 5 mL distilled water, and the isoflavonoids were then eluted with 5 mL methanol. The eluate was evaporated to dryness under a stream of nitrogen gas, reconstituted with 3 mL sodium acetate buffer (0.15 mol/L; pH 5) and hydrolyzed at 37 °C overnight with 4000 U β-glucuronidase and 150 U sulfatase (both from Helix pomatia). After hydrolysis, the samples were extracted 3 times with 3 mL ethyl acetate, and the combined organic extracts were evaporated to dryness under a stream of nitrogen. Isoflavonoids were converted to the trimethylsilyl ether derivatives for analysis by GC-MS by the addition of N,O-bis (trimethylsilyl)-trifluoracetamide and incubation for 2 h at RT.

The concentrations of individual unconjugated isoflavonoids were measured separately after solid-phase extraction and omission of the hydrolysis step with the use of 1 mL plasma. Liquid-liquid extraction and derivatization were carried out as described above.

Cleanup of urine samples
Urinary isoflavonoids were measured after the addition of the 3 internal standards before sample preparation. Total isoflavonoids (free + conjugated) were measured after extraction and enzymatic hydrolysis of the conjugates with a mix of β-glucuronidase and sulfatase (both from Helix pomatia). Unconjugated isoflavonoids (aglycone) were measured without enzymatic hydrolysis. To investigate the amount of isoflavonoid glucuronides, β-glucuronidase from bovine liver was used, which does not contain any detectable sulfatase activity. Isoflavonoid sulfates were measured by the addition of sulfatase from Patella vulgata and the β-glucuronidase inhibitor d-saccharic acid-1,4-lactone. In both cases, the absolute amounts of isoflavonoid glucuronides and sulfates were obtained by subtracting the amounts of the unconjugated isoflavonoids.

After centrifugation for 5 min at 3000 x g and RT, 5 nmol 3,4,8-13C-DAI as well as 500 pmol 1,2,3-13C-O-DMA and 2-OCH3-7-OH-IF were added to 0.5–3 mL urine. The samples were diluted with 5 mL sodium acetate buffer (0.15 mol/L; pH 5) and hydrolyzed overnight at 37 °C with 4000 U β-glucuronidase and 150 U sulfatase (both from Helix pomatia), 1000 Fishman U β-glucuronidase (from bovine liver), or 150 U sulfatase (from Patella vulgata) in the presence of 100 µmol/L d-saccharic acid-1,4-lactone, respectively. To determine the unconjugated fraction the hydrolysis step was omitted. Subsequently, isoflavonoids were isolated by liquid-liquid extraction as described above. After evaporation to dryness under a stream of nitrogen, the samples were dissolved in 3 mL sodium acetate buffer (0.15 mol/L; pH 5) and subjected to solid-phase extraction as already described for plasma samples. The dried samples were derivatized with the use of N,O-bis (trimethylsilyl)-trifluoracetamide and measured by GC-MS.

GC-MS conditions
Isoflavonoid trimethylsilyl ether derivatives were separated and quantified by GC-MS analysis. GC-MS was carried out on an Agilent Technologies system (gas chromatograph model HP6890 connected to a quadrupol mass detector model 5973; Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved on a nonpolar capillary column (MDN-5S, 30 m x 0.25 mm inside diameter, 0.25-µm film thickness; Supelco, Munich, Germany) with the use of a helium carrier gas flow of 1.2 mL/min and a linear temperature gradient (60 °C for 2 min then 30 °C/min to 250 °C, hold for 10 min, then 1 °C/min to 275 °C and hold for 5 min). The injector port temperature was set to 250 °C. The injection volume was 1 µL in the splitless mode. Mass spectra were obtained by electron impact ionization at 70 eV and an ion source temperature of 230 °C. The selected ion monitoring was applied to monitor 2 ions for each analyte, one of which was used for quantification, and the other was used as confirmation of the presence of the analyte. The following ions were monitored (first for quantification and second for confirmation): mass-to-charge ratio (m/z) 398 and 383 for DAI; m/z 401 and 386 for 3,4,8-13C-DAI; m/z 281 and 459 for O-DMA; m/z 282 and 462 for 1,2,3-13C-O-DMA; m/z 386 and 192 for equol; m/z 281 and 400 for DHD; m/z 486 and 471 for 6-, 8-, and 3'-OH-DAI; and m/z 281 and 340 for 2-OCH3-7-OH-IF. The individual isoflavonoids were quantified by comparing the peak area in the specific ion channels at the correct retention time determined from commercially available reference compounds with the peak area response for the internal standard. This area ratio was then interpolated against calibration curves in the linear range between 0 and 25 pmol constructed for known amounts of the individual isoflavonoids. Ten calibration concentrations were used for each isoflavonoid, with 4 injections being made at each concentration. The limits of detection were between 8 and 70 fmol. The recoveries ranged between 93% and 105% obtained after spiking plasma samples with reference compounds and subtracting the basal values from the blank plasma. The intra- and interassay coefficients were always <5% for all quantified isoflavonoids.

Determination of plasma pharmacokinetics and statistical analysis
Peak plasma isoflavonoid concentration (Cmax) and the time required to attain Cmax (tmax) were obtained directly by the visual inspection of each subject's plasma concentration-time profile. The area under the plasma concentration-time profile from 0 h to infinity (AUCinf) and the half-life of elimination (t1/2) were calculated by a noncompartmental approach with the use of PK SOLUTIONS 2.0 (Summit Research Services, Montrose, CO) computer software. The slope of the terminal log-linear portion of the concentration-time profile was determined by least-squares regression analysis and used as the elimination rate constant {lambda}z. The t1/2 was calculated as 0.693/{lambda}z. The AUC from time zero to the last quantifiable point (48 h) (AUC0–48) was determined with the use of the trapezoidal rule. Extrapolated AUCinf was calculated as the sum of AUC0–48 and C48/{lambda}z (27). Because plasma sampling was continued for >4 t1/2, the AUC0–48 was sufficient to cover at least 93% of the total AUC (AUCinf). Therefore, calculating AUCinf seems to be a reasonable assumption for determining the total amount of isoflavones absorbed by the body.

All statistical calculations were performed with the use of STATVIEW program version 5.0 (SAS Institute, Cary NC). Results are reported as means ± SDs. The differences between the mean values of Cmax, tmax, t1/2, AUCinf, and urinary excretion, as well as of the different enzymatic hydrolyzed fractions (phase II conjugates, free, and total) for DAI and its metabolites after administration of the 2 isoflavone preparations, were statistically analyzed with the use of the paired Student's t test. Changes between the baseline (0 h) and the following time points among the 2 treatment groups were tested for significance by repeated-measures analysis of variance. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma pharmacokinetics of daidzein
The group mean plasma total DAI appearance and disappearance curves (after hydrolysis of the phase II conjugates to the aglycone) for the 7 men after consuming a single bolus dose of 1 mg/kg body weight of DAI and DG (calculated as aglycone equivalent), respectively, is shown in Figure 2Go. Before consuming the isoflavone preparations, plasma DAI concentrations were <20 nmol/L, indicating that the volunteers had refrained from eating soy products. In 5 of the 7 volunteers after ingestion of DAI and in 4 of 7 volunteers after consumption of DG, the plasma profiles were characterized by a rapid increase in the DAI concentration during the first 3 h followed by a slight decline and a second rise that attained Cmax at 8–10 h. In case of the remaining 2 and 3 subjects, respectively, the curves displayed a rapid increase to Cmax at 8–10 h. For both compounds, tmax was not significantly different. The systemic bioavailability as determined by comparing the plasma concentration-time curve (AUCinf) was found to be 4.5 times and the Cmax 6 times greater after administration of DG than after DAI. After consumption of DG, t1/2 was significantly longer. The results are summarized in Table 1Go. Unconjugated DAI concentrations were relatively low on administration of the 2 isoflavone preparations. In the first hour, the proportions of free DAI increased and accounted for 12.9% of total DAI after the administration of DAI and 11.0% after the ingestion of DG (Figure 3Go). After 4.5 h, steady state was established and the unconjugated DAI fraction was averaged, 3.4% and 3.1% after the administration of DAI and DG, respectively.


Figure 2
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  		FIGURE 2.. Mean (± SD) plasma appearance and disappearance curves for total daidzein (DAI) (after hydrolysis of the phase II conjugates to the aglycone) in 7 men after consumption of a single bolus dose of 1 mg/kg body weight DAI (solid line) and daidzein-7-O-β-D-glucoside (DG; short dashed line) (calculated as aglycone equivalent). Plasma concentrations differ significantly over time between the 2 treatment groups (P < 0.0001, repeated-measures ANOVA).

 

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  		TABLE 1. Plasma pharmacokinetic variables for daidzein in 7 men after consumption of a single bolus dose of 1 mg/kg body weight of daidzein-glucoside (calculated as aglycone equivalent)1

 

Figure 3
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  		FIGURE 3.. Mean (± SD) plasma appearance and disappearance curves for unconjugated daidzein (DAI) after consumption of DAI (solid line) and daidzein-7-O-β-D-glucoside (DG; short dashed line) (n = 7). Plasma concentrations do not differ significantly over time between the 2 treatment groups (repeated-measures ANOVA).

 
Plasma pharmacokinetics of the bacterial and oxidative metabolites
The bacterial metabolites DHD and O-DMA as well as the oxidative metabolites 6-, 8-, and 3'-OH-DAI were detected in the plasma of all 7 subjects. Only 1 of the 7 subjects was an equol producer as classified by Setchell et al (28).

The total mean plasma appearance and disappearance curves of the bacterial metabolites DHD, O-DMA, and equol, as well as of the oxidative metabolites 6-, 8-, and 3'-OH-DAI, are depicted in Figure 4Go and Figure 5Go . As for DAI, plasma isoflavonoid concentrations were low before the consumption of the isoflavone preparations (<10 nmol/L). In the case of DHD, O-DMA, and equol, there was a time lag of {approx}6–8 h before they appeared in substantial amounts in the plasma. For the oxidative metabolites, there was a delay of 2 h in their plasma appearance after ingestion of the isoflavone preparations. Plasma concentrations were always higher after the consumption of DG with the exception of equol. The pharmacokinetic variables as well as the unconjugated fractions of the quantified bacterial metabolites (Table 2Go) and the variables of the oxidative metabolites (Table 3Go) are summarized. It was not possible to determine the unconjugated fractions of the oxidative metabolites in plasma because the free plasma concentrations were below the limit of detection.


Figure 4
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  		FIGURE 4.. Plasma appearance and disappearance curves for total dihydrodaidzein (DHD), O-desmethylangolensin (O-DMA), and equol after consumption of daidzein (DAI; solid line) and daidzein-7-O-β-D-glucoside (DG; short dashed line). For DHD and O-DMA, the mean (±SD) profiles are depicted. Plasma DHD and O-DMA concentrations differ significantly over time between the 2 treatment groups (n = 7) (repeated-measures ANOVA, P < 0.01 for DHD and P < 0.0001 for O-DMA). Equol plasma concentration profiles of the only equol producer are shown (no statistical analysis was performed).

 

Figure 5
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  		FIGURE 5.. Mean (±SD) plasma appearance and disappearance curves for total 6-, 8-, and 3'-OH-DAI after consumption of daidzein (DAI; solid line) and daidzein-7-O-β-D-glucoside (DG; short dashed line) (n = 7). Only plasma 6-OH-DAI concentrations differ significantly over time between the 2 treatment groups (P < 0.05, repeated-measures ANOVA).

 

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  		TABLE 2. Plasma pharmacokinetic variables for dihydrodaidzein, O-desmethylangolensin, and equol and the percentage of the unconjugated fraction of the bacterial metabolites in plasma after ingestion of a single oral dose of daidzein or daidzein-glucoside1

 

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  		TABLE 3. Plasma pharmacokinetic variables for 6-, 8-, and 3'-hydroxy-daidzein after ingestion of a single oral dose of daidzein or daidzein-glucoside1

 
Urinary excretion of daidzein
The mean urinary excretion of total daidzein according to fraction (0, 0–6, 6–12, and 12–24 h) for the 7 participants is shown in Figure 6Go. The urinary profiles were similar for all 7 participants. Most of the excreted DAI was recovered between 6 and 12 h. The cumulative recovery over 24 h was {approx}3 times greater after DG than after DAI consumption. The cumulative recovery averaged 11.6% after the ingestion of DAI and 38.9% after the administration of DG. The results are shown in Table 4Go. The mean fractions of DAI glucuronide, sulfate, and aglycone during the 24-h collection period are summarized in Table 5Go. Addition of the sulfate and glucuronide fractions (88.5% and 88.9%, respectively, after consumption of DAI and DG; Table 5Go) did not equal the total aglycone values as determined with the combined β-glucuronidase and sulfatase treatment (100%). This indicates that conjugates other than those recognized by β-glucuronidase and sulfatase alone must have been present in urine, presumably mixed conjugates.


Figure 6
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  		FIGURE 6.. Mean (±SD) urinary excretion of total daidzein (DAI), DAI glucuronide, sulfate, and aglycone after consumption of DAI and daidzein-7-O-β-D-glucoside (DG), respectively (n = 7). The values differ significantly for all fractions between the 2 isoflavone preparations (paired Student's t test, P < 0.05 for 0–6 h as well as 6–12 h and P < 0.01 for 12–24 h for total DAI, DAI glucuronide, sulfate, and aglycone).

 

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  		TABLE 4. Urinary total daidzein and urinary total isoflavonoids (including daidzein and its bacterial and oxidative metabolites) in 7 men after consumption of a single bolus dose of daidzein and daidzein-glucoside expressed as the amount recovered and as fractional absorption after hydrolysis of the phase II conjugates to the aglycone1

 

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  		TABLE 5. Mean fractions of daidzein glucuronide, sulfate, and aglycone during the 24-h collection period after hydrolysis with β-glucuronidase and sulfatase (total, set 100%), with β-glucuronidase (minus free = glucuronides), with sulfatase (minus free = sulfates), and without hydrolysis (aglycone)1

 
Urinary excretion of the bacterial and oxidative metabolites
The mean excreted amounts during the 24-h collection period and the mean fractions of the glucuronides, sulfates, and aglycones for the bacterial and oxidative metabolites, respectively, for the 7 participants are summarized in Table 6Go and Table 7Go . The urinary profiles were similar for the volunteers. Most of the excreted bacterial metabolites was recovered between 12 and 24 h and of the oxidative metabolite between 6 and 12 h (data not shown). Again, addition of the sulfate and glucuronide fractions did not equal the total aglycone values, especially in the case of the oxidative metabolites, indicating mixed conjugates. The cumulative recovery over 24 h, including all quantified metabolites, averaged 16.6% after the consumption of DAI and 53.3% after the administration of DG (Table 4Go).


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  		TABLE 6. Mean fractions of the glucuronides, sulfates, and aglycones of dihydrodaidzein, O-desmethylangolensin, and equol during the 24-h collection period with β-glucuronidase and sulfatase (total, set 100%), with β-glucuronidase (minus free = glucuronides), with sulfatase (minus free = sulfates), and without hydrolysis (aglycone) and the total amount excreted for the quantified metabolites1

 

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  		TABLE 7. Mean fractions of the glucuronides, sulfates, and aglycones of the oxidative metabolites during the 24-h collection period after hydrolysis with β-glucuronidase and sulfatase (total, set 100%), with β-glucuronidase (minus free = glucuronides), with sulfatase (minus free = sulfates), and without hydrolysis (aglycone) and the total amount excreted for the quantified metabolites1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Contradictory results about the bioavailability of isoflavone aglycones and glucosides have been published and have lead to confusion. Our study in which pure isoflavones were used clearly shows that DG exhibits a greater bioavailability than its aglycone as determined by AUCinf that gives the total amount of isoflavone absorbed by the body. The results are in accordance with those of Setchell et al (20). However, Setchell et al (20) used a dose-normalized AUC to compare the bioavailability, and their study was not conducted in a crossover design. Because there is still no consensus whether the bioavailability correlates linearly with increasing doses (29–32), studies administering different doses are difficult to interpret. Other published human studies investigating the bioavailability of aglycones compared with their glucosides used isoflavone supplements, soy extracts, or soy food (21–24) and found a greater or the same bioavailability of the aglycones. In those studies several additional factors such as the kind of pharmaceutical formulation, the interaction between the various isoflavone compounds, or the nutrient composition of the food matrix might have a substantial effect on the pharmacokinetics of the isoflavone glucosides and aglycones. For example, Richelle et al (21) used a commercial isoflavone preparation named Fujiflavone P10 (Fujicco, Japan), which is described as a patented isoflavone-cyclodextrin-clathrate formulation leading to high water-soluble soy isoflavones (US patent 5847108). It is well known that cyclodextrins enhance the physicochemical properties of drugs through complexation and in consequence their bioavailability. This is already shown in the case of the isoflavones (US patent 5847108) (33, 34).

Our study shows that the greater bioavailability of DG is not caused by differences in the metabolism and excretion, because AUCinf, Cmax, and the urinary recoveries of DAI and the formed metabolites increased by about the same factor after consumption of DG compared with DAI. However, several factors might enhance the absorption of DG: active transport of the glucoside, protection of bacterial degradation by the sugar moiety, reduced interaction with food matrix components, or higher solubility in the aqueous surface of the intestine. A greater bioavailability of the glycosidic form is reported for other flavonoids, eg, quercetin glucosides (35–37), and active transport by the human intestinal sodium-coupled glucose transporter 1 (SGLT1) is thought to contribute to their greater bioavailability (38–40). However, to date there is little evidence that isoflavone glucosides interact with SGLT1 (41, 42). Moreover, a recent publication completely denied the transport of flavonoids by SGLT1 (43).

Because no isoflavone glucosides can be found in human plasma, hydrolysis of the sugar moiety has to occur either by microbial or intestinal β-glucosidases before transport to the liver (44). Because tmax is similar after consumption of DAI and DG, hydrolysis of the O-glucosidic bond seems not to be the rate-determining step for absorption. One reported explanation for the greater bioavailability of DG is that the glucoside moiety acts as a protecting group, preventing degradation of the isoflavone skeleton by the intestinal microflora (20). Evidence supporting this theory is that the overall recovery for DAI is much less than that for DG. On the other hand, the pattern of metabolites is the same in both cases, and the degree of metabolism is not different. However, the degradation of the isoflavone skeleton and the formation of unknown metabolites cannot be excluded.

One other point is the extended binding of the aglycone to dietary ingredients, eg, proteins of the consumed breakfast. Its greater reactivity, and lipophilicity might play a role. This has been shown for quercetin and its 3-O-rhamnosylglucoside rutin in vitro (45), and a similar behavior is expected for the isoflavones (46).

However, the most important point might be the different physicochemical properties of DAI and DG as illustrated by their octanol-water partition coefficient values (log P). Log P was determined to be 2.5 and 0.3 for DAI and DG, respectively, indicating a much higher lipophilicity of DAI (47). It is well known that the aqueous boundary layer as well as the mucus layer of the intestine acts as a barrier to the diffusion, absorption, or both of lipophilic molecules (48, 49). Therefore, it makes sense that the higher water solubility of DG accounts for its greater bioavailability.

The shapes of the plasma appearance and disappearance curves of total DAI after ingestion of DAI and DG showed an early peak before Cmax in 5 and 4, respectively, of the 7 subjects. This biphasic pattern has also been reported by other researchers (12, 20–22, 50). One explanation for this behavior is that the compounds undergo an enterohepatic circulation. Another one is that the uptake occurs in the small as well as in the large intestine without contribution of an enterohepatic recycling (51).

We analyzed all known bacterial and oxidative metabolites, namely equol, DHD, O-DMA, and 6-, 8-, and 3'-OH-DAI, in human plasma and urine. The pharmacokinetics of the bacterial metabolites was addressed in a few studies only (12, 20–22, 51). In agreement with those studies, we show that, for DHD, O-DMA, and equol, there is a time lag in their appearance in plasma of {approx}6–8 h. Furthermore, most of the excreted bacterial metabolites were recovered between 12 and 24 h. These results can be explained by the colonic origin of the bacterial metabolites. To date, no human intervention study had investigated the pharmacokinetics of the oxidative metabolites. We observed a delay of 2 h in their plasma appearance. This can be attributed to their immediate formation in the enterocyte after absorption of DAI or in the hepatocyte after transport directly over the portal vein to the liver. Note that the high urinary fraction of sulfated oxidative metabolites (>20%) (Table 7Go) is of the same magnitude as the sulfated fraction of endogenous estrogens in human urine (52). These high values of estrogen-sulfates are also found in tissues and serve for the in situ estrogen production by conversion to the active aglycones (53).

The excreted amount of oxidative metabolites in the 24-h urine makes up to only 1% of the total excreted amount of isoflavonoids, that of the bacterial metabolites up to 25%. Interindividual variations of urinary excretion of DAI were low (2.4-fold after ingestion of DAI and 1.6-fold after that of DG). In contrast, interindividual variations for the bacterial and oxidative metabolites were high. For DHD it accounted for 220- and 320-fold and for O-DMA 65- and 12-fold after consumption of DAI and DG, respectively. For the total oxidative metabolites, they made up to 35-fold after consumption of both preparations. Responsible for these high variations in the case of the bacterial metabolites are the interindividual intestinal microflora. For the oxidative metabolites, one might speculate that differences in the expression of biliary and intestinal cytochrome P450 enzymes, transport proteins, or polymorphisms might play a role. The high variations in the excretion of bacterial metabolites and the low in that of DAI have already been observed in other studies (54). Interindividual variations in the excretion of the oxidative metabolites have so far not been assessed. The high interindividual variations are responsible for the missing significant differences in the pharmacokinetic variables (AUCinf, Cmax) and the urinary excretions of bacterial and oxidative metabolites after consumption of DAI and DG.

In summary, this study gives a comprehensive overview about the pharmacokinetics, including absorption mechanisms, bioavailability, metabolism, and elimination, of DAI and its bacterial and oxidative metabolites after consumption of the pure, nonformulated aglycone and glucoside.


   ACKNOWLEDGMENTS
 
We thank E Hoch and U Stadler-Prayle for technical assistance and the volunteers for taking part in this study.

The author's responsibilities were as follows—SEK: developed the initial idea and designed the study; CER: collected and analyzed the data and performed the statistical analysis; SEK, CER, and AB: drafted the manuscript; AB and JM: recruited and checked the volunteers, planned and scheduled meals, oversaw the kitchen personnel, and handled, collected, and stored all specimens; MS and PW: provided pure DAI and DG. All coauthors participated in critically revising the manuscript. None of the authors had a personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Coward LN, Barnes C, Setchell KDR, Barnes S. Genistein, daidzein, and their beta-glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets. J Agric Food Chem 1993;41:1961–7.
  2. Adlercreutz H, Goldin BR, Gorbach SL, et al. Soybean phytoestrogen intake and cancer risk. J Nutr 1995;125(suppl):757S–70S.[Abstract/Free Full Text]
  3. Thompson LU. Lignans and isoflavones. In: Eisenbrand G, Dayan AD, Elias PS, Grunow W, Schlatter J, eds. Carcinogenic and anticarcinogenic factors in food. Weinheim, Germany: Wiley-VCH, 2000:348–58.
  4. Cassidy A, Hooper L. Phytoestrogens and cardiovascular disease. J Br Menopause Soc 2006;12:49–56.[Medline]
  5. Cassidy A, Albertazzi P, Lise Nielsen I, et al. Critical review of health effects of soyabean phyto-oestrogens in post-menopausal women. Proc Nutr Soc 2006;65:76–92.[Medline]
  6. Irvine C, Fitzpatrick M, Robertson I, Woodhams D. The potential adverse effects of soybean phytoestrogens in infant feeding. N Z Med J 1995;108:208–9.[Medline]
  7. Sheehan DM. Herbal medicines, phytoestrogens and toxicity: risk:benefit considerations. Proc Soc Exp Biol Med 1998;217:379–85.[Medline]
  8. Bouker KB, Hilakivi-Clarke L. Genistein: does it prevent or promote breast cancer? Environ Health Perspect 2000;108:701–9.[Medline]
  9. Horn-Ross PL, Barnes S, Lee M, et al. Assessing phytoestrogen exposure in epidemiologic studies: development of a database (United States). Cancer Causes Control 2000;11:289–98.[Medline]
  10. Kiely M, Faughnan M, Wahala K, Brants H, Mulligan A. Phyto-oestrogen levels in foods: the design and construction of the VENUS database. Br J Nutr 2003;89(suppl):S19–23.[Medline]
  11. Xu X, Wang H-J, Murphy PA, Cook L, Hendrich S. Daidzein is a more bioavailable soymilk isoflavone than genistein in adult women. J Nutr 1994;124:825–32.[Abstract/Free Full Text]
  12. Watanabe S, Yamaguchei M, Sobue T, et al. Pharmacokinetics of soybean isoflavones in plasma, urine and feces of men after ingestion of 60 g baked soybean powder (kinako). J Nutr 1998;128:1710–5.[Abstract/Free Full Text]
  13. Chang YC, Nair MG. Metabolism of daidzein and genistein by intestinal bacteria. J Nat Prod 1995;58:1892–6.[Medline]
  14. Joannou GE, Kelly GE, Reeder AY, Waring M, Nelson C. A urinary profile study of dietary phytoestrogens. The identification and mode of metabolism of new isoflavonoids. J Steroid Biochem Mol Biol 1995;54:167–84.[Medline]
  15. Heinonen SM, Hiokkala A, Wahala K, Adlercreutz H. Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton. J Steroid Biochem Mol Biol 2003;87:285–99.[Medline]
  16. Kulling SE, Honig DM, Simat TJ, Metzler M. Oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein. J Agric Food Chem 2000;48:4963–72.[Medline]
  17. Kulling SE, Honig D, Metzler M. Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo. J Agric Food Chem 2001;49:3024–33.[Medline]
  18. Adlercreutz H, van der Wildt J, Kinzel J, et al. Lignan and isoflavonoid conjugates in human urine. J Steroid Biochem Mol Biol 1995;52:97–103.[Medline]
  19. Setchell KD. Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. Am J Clin Nutr 1998;68(suppl):1333S–46S.[Abstract]
  20. Setchell KDR, Brown NM, Desai P, et al. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr 2001;131(suppl):1362S–75S.[Abstract/Free Full Text]
  21. Richelle M, Primore-Merten S, Bodenstab S, Enslen M, Offord EA. Hydrolysis of isoflavone glycosides to aglycones by beta-glycosidase does not alter plasma and urine isoflavone pharmacokinetics in postmenopausal women. J Nutr 2002;132:2587–92.[Abstract/Free Full Text]
  22. Zubik L, Meydani M. Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women. Am J Clin Nutr 2003;77:1459–65.[Abstract/Free Full Text]
  23. Izumi T, Piskula MK, Osawa S, Obata A, et al. Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J Nutr 2000;130:1695–9.[Abstract/Free Full Text]
  24. Kano M, Takayanagi T, Harada T, Sawada Y, et al. Bioavailability of isoflavones after ingestion of soy beverages in healthy adults. J Nutr 2006;136:2291–6.[Abstract/Free Full Text]
  25. Degenhardt A, Winterhalter P. Isolation and purification of isoflavones from soy flour by high-speed countercurrent chromatography. Eur Food Res Technol 2001;213:277–80.
  26. Wähälä K, Hase T, Adlercreutz H. Synthesis and labeling of isoflavone phytoestrogens, including daidzein and genistein. Proc Soc Exp Biol Med 1995;208:27–32.[Medline]
  27. Gibaldi M, Perrier D. Pharmacokinetics. 2nd ed. New York, NY: Marcel-Dekker, 1982:348–58.
  28. Setchell KD, Brown NM, Lydeking-Olsen E. The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones. J Nutr 2002;132:3577–84.[Abstract/Free Full Text]
  29. Busby MG, Jeffcoat AR, Bloedon LT, et al. Clinical characteristics and pharmacokinetics of purified soy isoflavones: single-dose administration to healthy men. Am J Clin Nutr 2002;75:126–36.[Abstract/Free Full Text]
  30. Bloedon LT, Jeffcoat AR, Lopaczynski W, et al. Safety and pharmacokinetics of purified soy isoflavones: single-dose administration to postmenopausal women. Am J Clin Nutr 2002;76:1126–37.[Abstract/Free Full Text]
  31. Setchell KDR, Brown NM, Desai P, et al. Bioavailability, disposition, and dose-response effects of soy isoflavones when consumed by healthy women at physiologically typical dietary intakes. J Nutr 2003;133:1027–35.[Abstract/Free Full Text]
  32. Setchell KDR, Faughnan MS, Avades T, et al. Comparing the pharmacokinetics of daidzein and genistein with the use of [13C]labeled tracers in premenopausal women. Am J Clin Nutr 2003;77:411–9.[Abstract/Free Full Text]
  33. Challa R, Ahuja A, Ali J, Khar RK. Cyclodextrins in drug delivery: an updated review. AAPS Pharm Sci Tech 2005;6:E329–57.
  34. Stancanelli R, Mazzaglia A, Tommasini S, et al. The enhancement of isoflavone water solubility by complexation with modified cyclodextrins: a spectroscopic investigation with implications in the pharmaceutical analysis. J Pharm Biomed Anal 2007;44:980–4.[Medline]
  35. Hollman PC, de Vries JH, van Leeuwen SD, Mengelers MJ, Katan MB. Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr 1995;62:1276–82.[Abstract/Free Full Text]
  36. Morand C, Manach C, Crespy V, Remesy C. Quercetin 3-O-beta-glucoside is better absorbed than other quercetin forms and is not present in rat plasma. Free Radic Res 2000;33:667–76.[Medline]
  37. Lesser S, Cermak R, Wolffram S. Bioavailability of quercetin in pigs is influenced by the dietary fat content. J Nutr 2004;134:1508–11.[Abstract/Free Full Text]
  38. Gee JM, DuPont MS, Rhodes MJ, Johnson IT. Quercetin glucosides interact with the intestinal glucose transport pathway. Free Radic Biol Med 1998;25:19–25.[Medline]
  39. Walgren RA, Lin JT, Kinne RK, Walle T. Cellular uptake of dietary flavonoid quercetin 4'-beta-glucoside by sodium-dependent glucose transporter SGLT1. J Pharmacol Exp Ther 2000;294:837–43.[Abstract/Free Full Text]
  40. Mullen W, Boitier A, Stewart AJ, Crozier A. Flavonoid metabolites in human plasma and urine after the consumption of red onions: analysis by liquid chromatography with photodiode array and full scan tandem mass spectrometric detection. J Chromatogr A 2004;1058:163–8.[Medline]
  41. Oitate M, Nakaki R, Koyabu N, et al. Trans-cellular transport of genistein, a soybean-derived isoflavone, across human colon carcinoma cell line (Caco-2). Biopharm Drug Dispos 2001;22:23–9.[Medline]
  42. Murota K, Shimizu S, Miyamoto S, et al. Unique uptake and transport of isoflavone aglycones by human intestinal Caco-2 cells: comparison of isoflavonoids and flavonoids. J Nutr 2002;132:1956–61.[Abstract/Free Full Text]
  43. Kottra G, Daniel H. Flavonoid glycosides are not transported by the human Na+/glucose transporter when expressed in Xenopus laevis oocytes, but effectively inhibit electrogenic glucose uptake. J Pharmacol Exp Ther 2007;322:829–35.[Abstract/Free Full Text]
  44. Setchell KDR, Brown NM, Zimmer-Nechemias L, et al. Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability. Am J Clin Nutr 2002;76:447–53.[Abstract/Free Full Text]
  45. Rawel HM, Rohn S, Kroll J. Influence of a sugar moiety (rhamnosylglucoside) at 3-O position on the reactivity of quercetin with whey proteins. Int J Biol Macromol 2003;32:109–20.[Medline]
  46. Rawel HM, Ranthers H, Rohn S, Kroll J. Assessment of the reactivity of selected isoflavones against proteins in comparison to quercetin. J Agric Food Chem 2004;52:5263–71.[Medline]
  47. Rothwell JA, Day AJ, Morgan MRA. Experimental determination of octanol-water partition coefficients of quercetin and related flavonoids. J Agric Food Chem 2005;53:4355–60.[Medline]
  48. Larhed AW, Artursson P, Grasjö J, Björk E. Diffusion of drugs in native and purified gastrointestinal mucus. J Pharm Sci 1997;86:660–5.[Medline]
  49. Larhed AW, Artursson P, Björk E. The influence of intestinal mucus components on the diffusion of drugs. Pharm Res 1998;15:66–71.[Medline]
  50. Cassidy A, Brown JE, Hawdon A, et al. Factors affecting the bioavailability of soy isoflavones in humans after ingestion of physiologically relevant levels from different soy foods. J Nutr 2006;136:45–50.[Abstract/Free Full Text]
  51. Franke AA, Custer LJ, Hundahl SA. Determinants for urinary and plasma isoflavones in humans after soy intake. Nutr Cancer 2004;50:141–54.[Medline]
  52. Hamalainen E, Fotsis T, Adlercreutz H. A gas chromatographic method for the determination of neutral steroid profiles in urine, including studies on the effect of oxytetracycline on these profiles in men. Clin Chim Acta 1991;199:205–20.[Medline]
  53. Pasqualini JR, Gelly C, Nguyen BL, Vella C. Importance of estrogen sulfates in breast cancer. J Steroid Biochem 1989;34:155–63.[Medline]
  54. Rowland IR, Wiseman H, Sanders TA, Adlercreutz H, Bowey EA. Interindividual variation in metabolism of soy isoflavones and lignans: influence of habitual diet on equol production by the gut microflora. Nutr Cancer 2000;36:27–32.[Medline]
Received for publication October 14, 2007. Accepted for publication January 4, 2008.




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