Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

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Factors Affecting the Bioavailability of Soy Isoflavones in Humans after. Ingestion of ... in soy isoflavone supplements or protein drinks (6–11) were studied, but ...
Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Factors Affecting the Bioavailability of Soy Isoflavones in Humans after Ingestion of Physiologically Relevant Levels from Different Soy Foods1 Aedin Cassidy,2 Jonathan E. Brown,* Anne Hawdon,* Marian S. Faughnan,* Laurence J. King,* Joe Millward,* Linda Zimmer-Nechemias,y Brian Wolfe,y and Kenneth D.R. Setchelly School of Medicine, University of East Anglia, Norwich, UK; * The Centre for Nutrition and Food Safety, School of Biomedical and Molecular Sciences, University of Surrey, Stag Hill, Guildford, Surrey, UK; and yClinical Mass Spectrometry, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

KEY WORDS:  Isoflavones  food matrix  soy foods  pharmacokinetics  humans

Dietary isoflavones, a subclass of flavonoids, are the focus of much of the recent interest in the nutritional benefits of soy foods because these phytoestrogens occur in relatively high concentrations in soybeans and purified soy proteins (1–3). The exact role that isoflavones play in the health-related effects of soy foods, and their potential for adverse effects have been controversial and difficult to discern. This may be due in part to a lack of basic knowledge regarding their bioavailability and metabolism particularly as it relates to the source of soy. The

pharmacokinetics of pure isoflavones (4), stable isotopically labeled isoflavones (5), and mixtures of isoflavones contained in soy isoflavone supplements or protein drinks (6–11) were studied, but with few exceptions, there is little information on whether there are differences in the bioavailability of isoflavones derived from natural soy foods consumed at physiologically relevant intakes as found in the typical Asian diet (12–15). Isoflavones exist primarily in soybeans and in most soy foods as a complex mixture of glucoside conjugates that are not bioavailable in this form (16). After ingestion, the isoflavone glucosides are hydrolyzed by both intestinal mucosal and bacterial b-glucosidases releasing the aglycons (16–18), which are then either absorbed directly or further metabolized by intestinal microflora in the large intestine into other metabolites, including equol (19–21) and O-desmethylangolensin (ODMA)3 (22,23). Recent studies suggested that the extent of

1

Supported by the Food Standards Agency (UK) grant number T05010. To whom correspondence should be addressed. E-mail: a.cassidy@uea. ac.uk. 3 Abbreviations used: Cmax, observed peak serum isoflavone concentration; CL/F systemic clearance normalized to bioavailability; ODMA, O-desmethylangolensin; t1/2, serum elimination half-life; tmax, the observed time taken to reach Cmax; TVP, textured vegetable protein; Vd/(Fkg), volume of distribution normalized to bioavailability and body weight. 2

0022-3166/06 $8.00 Ó 2006 American Society for Nutrition. Manuscript received 16 August 2005. Initial review completed 2 September 2005. Revision accepted 22 October 2005. 45

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ABSTRACT The precise role that isoflavones play in the health-related effects of soy foods, and their potential for adverse effects are controversial. This may be due in part to a lack of basic knowledge regarding their bioavailability and metabolism, particularly as it relates to the soy source. To date, there is little information concerning possible differences in the bioavailability of isoflavones derived from natural soy foods consumed at physiologically relevant intakes and whether age- or gender-related differences influence that bioavailability. In the current study of healthy adults [premenopausal (n ¼ 21) and postmenopausal (n ¼ 17) women and a group of men (n ¼ 21)], we examined the effect of age, gender, and the food matrix on the bioavailability of isoflavones for both the aglycon and glucoside forms that are naturally present in 3 different soy foods, soy milk, textured vegetable protein, and tempeh. The study was designed as a random crossover trial so that all individuals received each of the 3 foods. The dose of isoflavones administered to each individual as a single bolus dose was 0.44 mg/kg body weight. Pharmacokinetic parameters were normalized to mg of each isoflavone ingested per kilogram body weight to account for differences in daidzein and genistein content between the diets. Serum isoflavone concentrations in all individuals and groups increased rapidly after the ingestion of each soy food; as expected, genistein concentrations exceeded daidzein concentrations in serum. In this small study, gender differences in peak concentrations of daidzein were observed, with higher levels attained in women. Consumption of tempeh (mainly isoflavone aglycon) resulted in higher serum peak levels of both daidzein (P , 0.001) and genistein (P , 0.01) and the associated area under the curve (P , 0.001 and P , 0.03, respectively) compared with textured vegetable protein (predominantly isoflavone glucosides). However, soy milk was absorbed faster and peak levels of isoflavones were attained earlier than with the other soy foods. Only 30% of the subjects were equol producers and no differences in equol production with age or gender were observed. J. Nutr. 136: 45–51, 2006.

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metabolism of soy isoflavones may influence the overall efficacy and clinical outcome of ingesting diets containing isoflavones, and this seems to be the case when a propensity toward equol production exists (21). Furthermore, differences in the absorption rates between the glucosylated and aglycon forms of isoflavones were reported (4,10,13,14) suggesting that not all isoflavones can be considered the same if present in different types of foods. Whether age- or gender-related differences influence the bioavailability of isoflavones, and whether isoflavones are handled similarly when ingested in different foods is presently unclear, but is the focus of these studies. Therefore, in the current study of healthy adults, we examined the effect of age, gender, and the influence of the food matrix on the bioavailability of isoflavones for both the aglycon and glucoside forms that are naturally present in 3 different soy foods, i.e., soy milk, textured vegetable protein (TVP), and tempeh. SUBJECTS AND METHODS

RESULTS Subject characteristics. The premenopausal women and men were 18–53 and 18–55 y old, respectively; the postmenopausal women were 48–69 y old. The BMI ranged from 16 to 34 kg/m2 and the groups did not differ. The proportion of subjects that provided sufficient data from which to construct complete pharmacokinetic profiles was 95%. For each of the computed pharmacokinetic parameters, dot plots were constructed to evaluate the presence of extreme outliers. Individual values . 3 SD from the mean or within 5% of this value were removed. The proportion of these outliers was 1.4% of the total data set of pharmacokinetic data computed for serum daidzein and genistein concentrations. Isoflavone composition of the soy foods. The isoflavone composition of each of the 3 foods is presented in Table 1. The proportion of daidzein and genistein combined, present as aglycons, was ,15% in soy milk and TVP and ;50% in tempeh, thus demonstrating that the fermented soy product as expected contained a greater proportion of isoflavone aglycons. A variety of isoflavone glucosides were also present in each of the foods including their acetyl and malonyl derivatives. Serum kinetics of total daidzein and total genistein. Serum pharmacokinetic variables for daidzein and genistein in the 3 separate diet periods together with a summary of the statistical analyses are depicted in Tables 2 and 3. Results are expressed as genistein and daidzein aglycons, representing the composite of the conjugates and aglycons originally present in the serum.

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Foods. Three soy-containing foods were studied. To examine the effect of differences in the food matrix, a commercially available soy milk (So Good, Sanitarium Health Food Company) and a commercial brand of TVP (Goodness Foods) were purchased from local supermarkets. These two foods are representative of a dietary source of mainly isoflavone glucosides. The third food studied was the fermented soy protein, tempeh (Impulse Foods), which contained isoflavones mainly as aglycones. Over the 18-mo study, 2 batches of soy milk, 2 batches of tempeh, and 1 batch of TVP were utilized. Each batch of food was analyzed for isoflavone content and aglucon/glucoside profile using previously described methods (3). The amount of each food consumed by each subject was adjusted to ensure that the total intake of isoflavones was similar throughout the study. Soy milk was consumed directly from the carton in liquid form. TVP was prepared in the form of bread rolls, and provided a more solid matrix. These bread rolls were prepared in advance and stored (2208C) until required. Tempeh represented another solid matrix and was prepared as a burgers and stored (2208C) until required. Study Subjects. Single oral bolus dose food studies were conducted on individuals recruited from the staff and student population from the University of Surrey and from those living within the Guildford area (Surrey, UK). Premenopausal women (n 5 21; BMI 23.7 6 3.7 kg/m2), postmenopausal women (n 5 17; BMI 24.3 6 4.1 kg/m2) and 21 men (BMI 23.3 6 3.8 kg/m2) (aged 18–55 y) were enrolled in these studies. All individuals were healthy, reported no use of medications (including antibiotic use within the preceding 6 mo), and did not consume soy-rich foods or isoflavone supplements on a regular basis. All premenopausal women reported a history of regular menstrual cycles and were not currently using oral or dermal contraceptives. Postmenopausal women reported no menstrual bleeding for at least 2 y before the commencement of the study and were not taking any form of hormone replacement therapy. The study design and protocol were approved by the University of Surrey Ethics Committee and written informed consent was obtained from each subject after a detailed explanation of the study procedure. Design of study. Subjects were asked to refrain from eating foods containing soy products for at least 1 mo before the start of the study and for the duration of the study. After an overnight fast, each individual arrived at the Clinical Investigation Unit (University of Surrey) and was randomly assigned to 1 of the 3 isoflavone-rich foods, which were given as a single-meal intake on 3 separate occasions with a washout period of at least 2 wk between the soy foods. The soy milk, TVP rolls, and tempeh burgers each provided an intake equivalent to 0.44 mg isoflavones/kg body weight. A baseline 10-mL blood sample was collected before soy food ingestion. After the ingestion of the soy-rich food, taken over a period of a few minutes and under supervision, further blood samples were collected after 2, 4, 6, 8, 9, 10, 11, 12, 24, 36, 48, and 72 h. Blood was obtained via an indwelling cannula for samples up to 12 h, and thereafter by venipuncture. Blood samples were allowed to coagulate for . 30 min, centrifuged at 3000 3 g for 10 min at 48C and aliquots of serum (1 mL) were stored at 2808C.

Serum isoflavone analysis. Daidzein, genistein, equol, ODMA, and glycitein concentrations were determined in serum samples (0.5 mL) by GC-MS with selected ion monitoring, after liquid-solid extraction, hydrolysis with a mixed glucuronidase/sulfatase, isolation of the aglycon isoflavones, and conversion to the tert-butyldimethylsilyl ether derivatives as described previously (15). Stable isotopically labeled internal standards were used for quantification (5). Determination of serum isoflavone pharmacokinetics. Pharmacokinetic analysis of serum isoflavone profiles was conducted using PK Solutions 2.0 software (Summit Research Services). The parameters determined were the terminal half-life, t1/2 (reflecting the rate of elimination), Cmax (the observed peak serum isoflavone concentration), tmax (the observed time taken to reach Cmax), area under the concentration-time curve, AUC(0–t) (reflecting the exposure of serum to isoflavone from time zero to time t when the serum concentration returned to baseline). These measured parameters were used to calculate CL/F (systemic clearance normalized to bioavailability) and Vd/(Fkg) (representing the systemic exposure to isoflavone normalized to bioavailability and body weight). To ensure that statistical comparisons were valid, Cmax and AUC values for daidzein and genistein were dose-adjusted to take into account the differences in the proportion of isoflavones within each food. As such, the values presented are adjusted to milligrams of each isoflavone ingested per kilogram body weight. AUC(0–t) was used rather than AUCinf because it was considered that concentration values for isoflavones and their metabolites would overestimate the latter variable. Vd/F was normalized to body weight due to the large variation in body weight among the 3 groups of subjects. For serum daidzein and genistein, a major peak and minor peak could often be observed in the time course profile. Furthermore a lag period was evident in some individuals. Statistical analyses. Statistical analyses were carried out using Minitab version 13.0. All data are expressed as means 6 SD. A 2-way ANOVA model was fitted to the data to account for the 2 fixed effects, which were subject group (i.e., premenopausal women, postmenopausal women, or men) and food (i.e., soy milk, TVP, or tempeh). A fixed effect relating to the interaction between these 2 fixed effects (i.e., subject group 3 food) was also included. TukeyKramer methods were used for post hoc multiple comparisons. Differences were considered significant at P , 0.05.

BIOAVAILABILITY OF SOY ISOFLAVONES

Isoflavone content of the 3 soy-rich foods Daidzein and genistein concentration Batch Total Daidzein Genistein Glycitein as aglycone Isoflavone concentration

Soy milk Soy milk TVP Tempeh Tempeh

1 2 1 1 2

63.9 50.3 476.0 234.4 345.6

mg aglycone 19.6 15.2 163.1 72.4 137.1

equivalents/g 22.3 22.0 28.3 6.9 312.9 0 140.3 21.7 186.4 22.1

g/100 g 14.1 5.9 8.2 50.0 48.1

After ingestion, soy isoflavones are readily absorbed from the gastrointestinal tract, attain maximal plasma levels within 5–8 h, and are then eliminated according to first-order kinetics. Effect of food matrix: comparison between soy milk and TVP. The appearance and disappearance curves for serum total daidzein and genistein are presented in Figure 1 for each of the subject groups and 3 food types. Serum isoflavone concentrations in all individuals increased rapidly reaching a maximum level (tmax) between 5 and 9 h after food ingestion. The peak serum daidzein and genistein concentrations were significantly higher after the consumption of soy milk than after TVP (Fig. 1). This difference was even more apparent when Cmax was adjusted for the proportion of dose of isoflavone ingested (Tables 2 and 3). Peak serum concentrations of both daidzein and genistein were also attained ;2 h later (P , 0.005) for TVP compared with soy

TABLE 2 Pharmacokinetic parameters of serum total daidzein in pre- and post menopausal women and men after consumption of 3 different soy products1,2 Cmax mmol/(Lmg dose)

tmax

t1/2

AUC (mmolh)/ (Lmg dose)

Vd/F L/kg body weight

CL/F L/h

Daidzein Milk Premenopausal Postmenopausal Men

2.19 6 0.72 2.55 6 1.07 1.79 6 0.63

6.1 6 1.7 5.9 6 1.5 6.5 6 2.2

8.0 6 1.2 8.7 6 1.3 7.5 6 1.4

22.09 6 4.29 28.94 6 10.02 22.08 6 9.25

1.53 6 0.57 1.31 6 0.67 1.65 6 0.71

8.47 6 2.54 6.59 6 2.36 11.49 6 5.32

TVP Premenopausal Postmenopausal Men

1.09 6 0.39 1.37 6 0.37 1.21 6 0.35

8.4 6 1.3 7.8 6 2.0 8.0 6 1.6

9.4 6 2.8 10.9 6 3.3 8.3 6 2.1

15.28 6 3.76 15.41 6 4.02 16.29 6 4.65

2.07 6 0.84 2.44 6 1.27 1.66 6 0.58

9.86 6 2.08 10.04 6 3.38 10.97 6 3.32

Tempeh Premenopausal Postmenopausal Men

2.33 6 1.23 2.24 6 0.91 1.32 6 0.33

8.4 6 0.8 7.5 6 1.9 8.0 6 2.0

7.8 6 1.3 8.6 6 2.3 7.3 6 1.4

27.13 6 8.99 33.99 6 16.66 19.79 6 7.87

1.33 6 0.61 1.40 6 0.77 1.73 6 0.80

6.55 6 1.69 7.12 6 2.87 14.87 6 9.36

Statistics (ANOVA) Diet effect Subject effect Diet 3 group

, .0010 ,0.0010 0.0260

,0.0010 0.2190 0.8750

,0.0010 ,0.0010 0.7090

,0.0010 ,0.0010 0.0009

,0.0010 0.9150 0.0180

0.3090 ,0.0010 0.0090

Tukey-Kramer Soy milk vs. TVP TVP vs. tempeh Soy milk vs. tempeh Pre- vs. postmenopausal Premenopausal vs. male Postmenopausal vs. male

,0.0001 ,0.0001 0.3246 0.4288 0.0103 0.00001

,0.0005 0.9705 0.0005 0.2233 0.9080 0.3839

0.0012 0.0004 0.9393 0.0748 0.1800 0.0001

,0.0001 ,0.0001 0.2977 0.0283 0.4374 0.0004

0.0022 0.0022 0.9966 0.9067 0.9734 0.9713

0.2774 0.6995 0.7778 0.9250 0.0001 0.0005

1 2

h

P-value

Values are means 6 SD, n ¼ 21 premenopausal women, 17 postmenopausal women, and 21 men. Cmax and AUC were adjusted to take into account the differences in the proportion of isoflavones within each food. As such the values presented are adjusted to milligrams of each isoflavone ingested per kilogram body weight.

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milk. Bioavailability as measured by AUC(0–t) was significantly greater for genistein and daidzein in the soy milk-fed group compared with the TVP-fed group. Neither age nor gender affected the mean tmax values for either isoflavone; tmax ranged from 5.9–8.4 h for daidzein and 5.5–7.8 h for genistein for the group as a whole during all 3 diet periods. Bioavailability was modestly increased in the postmenopausal group because there was a significant difference in the AUC(0–t) for daidzein (P 5 0.0283), although this age-related difference was not apparent for genistein. The terminal elimination t1/2 of daidzein and genistein was similar among the groups as expected and was 8.5 6 2.3 h for daidzein and 10.4 6 2.5 h for genistein for all subjects. Effect of chemical composition: comparison between TVP and tempeh. The chemical composition of TVP, which contains mainly isoflavone glucosides, differs from that of tempeh, which contains primarily aglycons even though both foods represent solid matrices. Over the 3 subject groups as a whole, Cmax was significantly greater after the ingestion of tempeh compared with TVP for both genistein and daidzein (Tables 2 and 3). The AUC(0–t) for serum total daidzein was 20–120% higher when tempeh was ingested compared with TVP (P , 0.0001). Similarly, the chemical composition of the soy food increased the AUC(0–t) serum total genistein (P 5 0.0217). However, the time to reach peak serum concentrations, tmax, did not differ for the pharmacokinetic profiles of these 2 solid foods. The terminal elimination t1/2 for genistein and daidzein were similar for the 2 groups, as expected. Effect of age: comparison between pre- and postmenopausal women. There was an increase in AUC(0–t) for daidzein in postmenopausal women (P 5 0.0283) but the bioavailability of

TABLE 1

Food

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TABLE 3 Pharmacokinetic parameters of serum total genistein in pre- and post menopausal women and men after consumption of 3 different soy products1,2 Cmax mmol/ (Lmg dose)

tmax

AUC (mmolh)/ (Lmg dose)

Vd/F L/kg body weight

CL/F L/h

Genistein Milk Premenopausal Postmenopausal Men

4.07 6 1.89 3.82 6 1.90 3.47 6 1.95

5.6 6 1.7 5.5 6 1.3 6.6 6 2.1

9.9 6 2.2 10.5 6 2.1 9.8 6 1.6

50.01 6 21.31 54.06 6 32.68 51.26 6 37.00

0.72 6 0.37 1.39 6 0.82 1.04 6 0.67

3.31 6 1.73 3.90 6 1.85 5.24 6 2.78

TVP Premenopausal Postmenopausal Men

1.36 6 0.56 1.42 6 0.65 1.38 6 0.59

7.8 6 1.0 7.7 6 1.9 7.2 6 2.1

10.9 6 2.9 11.5 6 3.1 10.5 6 2.1

18.35 6 6.42 18.64 6 8.36 22.98 6 14.12

1.90 6 0.86 2.05 6 1.03 1.83 6 1.07

7.81 6 3.26 8.22 6 2.99 7.90 6 3.76

Tempeh Premenopausal Postmenopausal Men

2.35 6 1.03 2.63 6 1.41 1.59 6 0.58

7.2 6 1.3 6.6 6 2.6 7.5 6 1.8

9.4 6 2.1 11.6 6 3.8 9.6 6 1.4

32.28 6 14.26 35.02 6 14.52 26.91 6 13.50

1.12 6 0.46 1.37 6 0.77 1.55 6 0.66

6.58 6 3.07 5.55 6 2.35 8.73 6 4.05

Statistics (ANOVA) Diet effect Subject effect Diet-subject interaction

,0.0010 0.1160 0.5050

,0.0010 0.3970 0.2680

0.1540 0.0310 0.6620

,0.0010 0.8250 0.7720

,0.0010 0.3420 0.4790

,0.0010 0.0290 0.1910

Tukey-Kramer Soy milk vs. TVP TVP vs. tempeh Soy milk vs. tempeh Pre- vs. postmenopausal Premenopausal vs. male Postmenopausal vs. male

,0.0001 0.0084 ,0.0001 0.9944 0.2035 0.1548

0.0001 0.4904 0.3627 0.7702 0.8053 0.0048

0.1657 0.3072 0.9493 0.0858 0.9766 0.0393

,0.0001 0.0217 ,0.0001 0.8486 0.9991 0.8556

,0.0005 0.0172 0.0712 0.3081 0.7232 0.7199

,0.0005 0.2305 0.0005 0.9999 0.0623 0.5640

h

1 2

Values are means 6 SD, n ¼ 21 premenopausal women, 17 postmenopausal women, and 21 men. Cmax and AUC were adjusted to take into account the differences in the proportion of isoflavones within each food. As such the values presented are adjusted to milligrams of each isoflavone ingested per kilogram body weight.

genistein did not differ between the 2 age groups. Age had no effect for either daidzein or genistein on the t1/2, tmax, Cmax, CL/F, or Vd/F. Effect of gender: comparison between pre-menopausal women and men. Cmax was higher in women than in men for serum daidzein (P 5 0.0103), and CL/F of daidzein was consistently lower in women (P 5 0.0001). However, there were no gender differences for t1/2, tmax, AUC(0–t), and Vd/(Fkg) for either daidzein or genistein. Serum kinetics of the intestinal metabolite of daidzein, equol. Of the subjects in this study population, 30% were capable of converting daidzein into equol. This proportion of equol producers was relatively low and somewhat similar among the study groups allowing for the small sample size of the groups. Equol production did not differ with age or gender, although this study was not powered sufficiently to address this issue (Fig. 2). In examining the equol producers as a group, the mean Cmax for equol ranged from 0.04 to 0.13 mmol/L and was attained between 8 and 24 h (median 24 h) after intake of the foods, consistent with its production in the distal intestine. The mean equol AUC(0–t) ranged from 1.04 to 3.12 (mmolh)/L; .80% of the subjects were capable of producing ODMA. This proportion varied little across the 3 subject groups, but it was apparent that a slightly greater proportion of ODMA producers consumed tempeh than consumed either soy milk or TVP. DISCUSSION Numerous studies showed that when soy isoflavones are ingested, they are readily absorbed from the gastrointestinal

tract after hydrolysis, attain maximal plasma concentrations within 4–8 h, and are then eliminated from the body through the bile and kidneys with a mean terminal elimination t1/2 of ;8 h (4–7,9,10,12–15). Most of the previously published studies on the serum pharmacokinetics of soy isoflavones have focused on a single food type (12,13,15,24), purified isoflavone aglycons or glucosides (4,6,7,9,10,14), or stable isotopically labeled tracers (5). Several reports measured only urinary isoflavone excretion over a short time period and attempted to deduce pharmacokinetics (25–27). As a result, several erroneous conclusions have appeared concerning the apparent bioavailability of isoflavones. We now present one of the first studies to examine how age, gender, and differences in the food matrix influence the serum pharmacokinetics of daidzein and genistein in healthy adults fed ‘‘normal’’ amounts of soy foods delivering physiologically relevant intakes of isoflavones. Three defined soy foods and 3 subject groups were selected so that the effects of differences in the food matrix, food chemical composition, age, and gender on the pharmacokinetics of serum daidzein and genistein as well as the daidzein metabolites, equol and ODMA, could be examined. These studies were performed according to a classic single-bolus oral intake pharmacokinetic design, using a level of isoflavone intake that was reflective of habitual intake in Asia; they complement our previously published studies, which used stable isotopes of the pure compounds (5). The pharmacokinetic data resulting from this study agree with limited information available from previous studies of isoflavones in foods (12,13,15,16). Specifically, Cmax, tmax, t1/2, and AUC(0–t) were all within the range of values obtained from

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t1/2

BIOAVAILABILITY OF SOY ISOFLAVONES

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Downloaded from jn.nutrition.org by guest on September 29, 2017 FIGURE 1 Appearance and disappearance curves for total serum daidzein (diamonds) and total serum genistein (squares) in premenopausal women (n ¼ 21, A panels), postmenopausal women (n ¼ 17, B panels), and men (n ¼ 21, C panels) after consumption of soy milk, TVP, or tempeh. Values are means 6 SD.

other studies using similar dietary intakes of isoflavones (12,13,15,16,24). These data show that humans absorb isoflavones from a range of different soy-rich foods in a similar manner, but that the food matrix can alter the pharmacokinetic profiles. To our knowledge, the effect of food matrix on serum isoflavone pharmacokinetics was not evaluated previously, although one study purported to show no effect of diet background or soy food type

on isoflavone bioavailability based only on urine excretion data in a small number of subjects (27). Several studies used a liquid matrix, such as soy milk (24) or specific soy protein drinks (8,12,13,25,28). In the present study, after soy milk consumption, the time taken to reach peak plasma isoflavone concentrations was 6.2 and 5.9 h for daidzein and genistein, respectively. This is in agreement with the above-mentioned studies of liquid soy foods. Solubility of a substance in the

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FIGURE 2 Proportion of premenopausal women (black), postmenopausal women (white), and men (gray) who had equol and ODMA in their serum after consumption of the soy foods.

LITERATURE CITED 1. Murphy PA. Phytoestrogen content of processed soybean products. Food Technol. 1982;36:60–4. 2. Coward L, Barnes NC, Setchell KDR, Barnes S. Genistein and daidzein, and their b-glucoside conjugates: anti-tumor isoflavones in soybean foods from American and Asian diets. J Agric Food Chem. 1993;41:1961–7. 3. Setchell KD, Cole SJ. Variations in isoflavone levels in soy foods and soy protein isolates and issues relating to isoflavones databases and food labelling. J Agric Food Chem. 2003;51:4146–55. 4. Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, Kirschner AS, Cassidy A, Heubi JE. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr. 2001;131:1362S–75. 5. Setchell KD, Faughnan MS, Avades T, Zimmer-Nechemias L, Brown NM, Wolfe BE, Brashear WT, Desai P, Oldfield MF, 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. 6. Bloedon LT, Jeffcoat AR, Lopaczynski W, Schell MJ, Black TM, Dix KJ, Thomas BF, Albright C, Busby MG, et al. Safety and pharmacokinetics of purified soy isoflavones: single-dose administration to postmenopausal women. Am J Clin Nutr. 2002;76:1126–37.

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intestine influences the rate of absorption, and because the isoflavones in soy milk are mainly hydrophilic b-glycoside conjugates and therefore in solution, faster absorption rates and earlier peak serum concentrations are expected for all subjects given soy milk compared with the solid soy food matrix of TVP. Stomach emptying occurs later after the ingestion of solid foods compared with liquid food matrices (29). On average, the peak serum concentrations of daidzein and genistein occurred ;2 h later for TVP and tempeh than for soy milk, and stomach emptying may explain in part the differences in observed tmax. When the 2 solid food matrices were compared, differences were observed that could be attributed to differences in chemical composition of the isoflavones in the foods. The main finding was that the Cmax and AUC(0–t) were significantly higher for tempeh than for TVP for both daidzein and genistein. Tempeh is a fermented food that contains mainly aglycons of daidzein and genistein, whereas TVP contains mainly the glucoside conjugates. In previous studies of a fermented soy food (14), daidzein and genistein were absorbed more quickly, as evidenced by the higher Cmax and earlier tmax, although conclusions regarding the relative bioavailability of aglycon and glucosides could not be drawn. This more rapid rate of absorption is consistent with the findings comparing the behavior of single purified forms of isoflavones (4), which showed that the bioavailability of the glucosides was greater than that of aglycons. A 50-mg dose of daidzein or daidzin (as aglycon equivalents) resulted in a serum AUC of 11.6 6 0.90 and 17.80 6 1.90 mmol/(Lh), respectively; after genistein or genistin ingestion, the AUC was 16.8 6 5.2 and 18.30 6 3.8 mmol/(Lh), respectively (4). However, in a recent study of 2 extracts of soy, 1 that contained mainly aglycons and 1 with mainly glucosides, the pharmacokinetics did not differ, although that study used very high doses (10). These differences may be due to interactions between the mixtures of isoflavones when soybean extracts are used; this could yield differences in the pharmacokinetics compared with the administration of a purified single compound (4,5). However, Richelle et al. (28) compared the absorption of isoflavones in liquid matrices, and their data suggested that earlier hydrolysis of glucosides to aglycons does not enhance the bioavailability of isoflavones in humans, whereas in a previous study using pure compounds, our data suggested that isoflavone glycosides were more bioavailable than aglycones. The effects of isoflavones did not differ between the pre- and postmenopausal women groups although there was a trend toward a lower Cmax and AUC(0–t) for serum daidzein in the younger women. It is unlikely that these differences were the result of body compositional differences because Vd/(Fkg) did not differ between the 2 groups. The older women tended to have a higher t1/2 for the elimination of daidzein and genistein, but the physiological significance of this finding is difficult to interpret. Whether this may be due to reduced renal function,

a well-recognized feature of aging (30), is not known. Renal clearance is a major route of elimination of conjugated isoflavones from the body, and patients with renal disease have a significantly longer plasma elimination half-life for daidzein and genistein (11). Few studies have examined the effect of gender on isoflavone absorption and metabolism. Lu and Anderson (31) reported longer elimination half-lives for daidzein and genistein in women, whereas Zhang et al. (24) found no gender differences; however, because these studies were based on urinary excretion, the findings are difficult to interpret due to the invalidity of using only urinary measurements for calculating pharmacokinetics. We previously reported the urinary isoflavone levels in an earlier study (32) and the current plasma data complement those data. We showed that urinary genistein excretion was influenced by gender and the food matrix, with the highest genistein recovery occurring after tempeh consumption in premenopausal women. Gut microflora play an important role in the metabolism of isoflavones, in particular the degradation of daidzein, which is converted to the highly active metabolite, equol (19–21) and the inactive metabolite, ODMA (22). In our study, differences were observed in the proportion of subjects who were equol producers and those in whom ODMA was identified; this is consistent with their divergent pathways of synthesis. It is not possible to determine with accuracy the pharmacokinetics of the metabolites, but their relatively late appearance in serum is consistent with their colonic production. In conclusion, these studies show that the bioavailability and pharmacokinetics of isoflavones are influenced mainly by the type of food matrix or form in which they are ingested. A liquid matrix, such as soy milk, yields a faster absorption rate and higher peak plasma concentrations than a solid matrix, whereas aglycones in a fermented food such as tempeh are absorbed more rapidly than glucoside conjugates. Although these data are suggestive of an influence of gender, there was no major influence of age. The assessment of the pharmacokinetic characteristics of both daidzein and genistein after the consumption of different foods helps to provide valuable data that can improve our understanding of isoflavone bioavailability. These findings will help formulate food composition/matrix characteristics of foods to enhance their bioavailability after ingestion. This will ensure that the most appropriate soy food can be employed in studies designed to assess the risks and benefits of these compounds for human health.

BIOAVAILABILITY OF SOY ISOFLAVONES

19. Axelson M, Kirk DN, Farrant RD, Cooley G, Lawson AM, Setchell KD. The identification of the weak oestrogen equol [7-hydroxy-3-(49-hydroxyphenyl)chroman] in human urine. Biochem J. 1982;201:353–7. 20. Axelson M, Sjovall J, Gustafsson BE, Setchell KD. Soya - a dietary source of the non-steroidal oestrogen equol in man and animals. J Endocrinol. 1984; 102:49–56. 21. Setchell KDR, 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. 22. Bannwart C, Adlercreutz H, Fotsis T, Wa¨ha¨la¨ K, Hase T, Brunow G. Identification of O-desmethylangolensin, a metabolite of daidzein and of matairesinol, one likely plant precursor of the animal lignan enterolactone in human urine. Finn Chem Lett. 1984;4–5:120–5. 23. 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. 24. Zhang Y, Wang GJ, Song TT, Murphy PA, Hendrich S. Urinary disposition of the soybean isoflavones daidzein, genistein and glycitein differs among humans with moderate fecal isoflavone degradation activity. J Nutr. 1999;129:957–62. 25. Xu X, Wang HJ, Murphy PA, Cook L, Hendrich S. Daidzein is a more bioavailable soymilk isoflavone than is genistein in adult women. J Nutr. 1994; 124:825–32. 26. Tew BY, Xu X, Wang HJ, Murphy PA, Hendrich S. A diet high in wheat fiber decreases the bioavailability of soybean isoflavones in a single meal fed to women. J Nutr. 1996;126:871–7. 27. Xu X, Wang HJ, Murphy PA, Hendrich S. Neither background diet nor type of soy food affects short-term isoflavone bioavailability in women. J Nutr. 2000; 130:798–801. 28. Richelle M, Pridmore-Merten S, Bodenstab S, Enslen M, Offord EA. Hydrolysis of isoflavone glycosides to aglycones by b-glycosidase does not alter plasma and urine isoflavone pharmacokinetics in postmenopausal women. J Nutr. 2002;132:2587–92. 29. Cadwallader DE. Biopharmaceutics and drug interactions. 3rd ed. New York: Raven Press, 1983. 30. Siegel EB. Drugs and the aging. Regul Toxicol Pharmacol. 1982;2: 287–95. 31. Lu LJ, Anderson KE. Sex and long-term soy diets affect the metabolism and excretion of soy isoflavones in humans. Am J Clin Nutr. 1998;68:1500S–4. 32. Faughnan MS, Hawdon A, Ah-Singh E, Brown J, Millward DJ, Cassidy A. Urinary isoflavone kinetics: the effect of age, gender, food matrix and chemical composition. Br J Nutr. 2004;91:567–74.

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7. Busby MG, Jeffcoat AR, Bloedon LT, Koch MA, Black T, Dix KJ, Heizer WD, Thomas BF, Hill JM, et al. Clinical characteristics and pharmacokinetics of purified soy isoflavones: single-dose administration to healthy men. Am J Clin Nutr. 2002;75:126–36. 8. Shelnutt SR, Cimino CO, Wiggins PA, Ronis MJ, Badger TM. Pharmacokinetics of the glucuronide and sulfate conjugates of genistein and daidzein in men and women after consumption of a soy beverage. Am J Clin Nutr. 2002; 76:588–94. 9. Takimoto CH, Glover K, Huang X, Hayes SA, Gallot L, Quinn M, Jovanovic BD, Shapiro A, Hernandez L, et al. Phase I Pharmacokinetic and pharmacodynamic analysis of unconjugated soy isoflavones administered to individuals with cancer. Cancer Epidemiol Biomarkers Prev. 2003;12:1213–21. 10. Zubik L, Meydani M. Bioavailability of soybean isoflavones from aglycone and glucoside forms in American women. Am J Clin Nutr. 2003;77:1459–65. 11. Fanti P, Sawaya BP, Custer LJ, Franke AA. Serum levels and metabolic clearance of the isoflavones genistein and daidzein in hemodialysis patients. J Am Soc Nephrol. 1999;10:864–71. 12. King RA, Bursill DB. Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. Am J Clin Nutr 1998; 67:867–72. 13. Watanabe S, Yamaguchi M, Sobue T, Takahashi T, Miura T, Arai Y, Mazur W, Wa¨ha¨la¨ K, Adlercreutz H. 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. 14. Izumi T, Piskula MK, Osawa S, Obata A, Tobe K, Saito M, Kataoka S, Kubota Y, Kikuchi M. Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J Nutr. 2000;130:1695–9. 15. Setchell KDR, Brown NM, Desai PB, Zimmer-Nechimias L, Wolfe B, Jakate AS, Creutzinger V, Heubi JE. Bioavailability, disposition, and doseresponse effects of soy isoflavones when consumed by healthy women at physiologically typical dietary intakes. J Nutr. 2003;133:1027–35. 16. Setchell KD, Brown NM, Zimmer-Nechemias L, Brashear WT, Wolfe BE, Kirschner AS, Heubi JE. 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. 17. Day AJ, DuPont MS, Ridley S, Rhodes M, Rhodes MJC, Morgan MRA, Williamson G. Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver b-glucosidase activity. FEBS Lett. 1998;436:71–5. 18. McMahon LG, Nakano H, Levy MD, Gregory JF III. Cytosolic pyridoxineb-D-glucoside hydrolase from porcine jejunal mucosa. Purification, properties and comparison with broad specificity b-glucosidase. J Biol Chem. 1997;272: 32025–33.

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