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Bioavailability of isoflavones from soy products in equol producers and non-producers in Japanese women
Journal of Nutrition & Intermediary Metabolism 6 (2016) 41e47
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Bioavailability of isoflavones from soy products in equol producers and non-producers in Japanese women Ayako Miura a, b, Chitose Sugiyama b, Hiroyuki Sakakibara c, Kayoko Simoi a, Toshinao Goda a, * a b c
Graduate School of Nutritional and Environmental Sciences, The University of Shizuoka, Shizuoka 422-8526, Japan Department of Health and Nutritional Sciences, Faculty of Health Promotional Sciences, Tokoha University, Hamamatsu 431-2102, Japan Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2016 Received in revised form 25 August 2016 Accepted 31 August 2016 Available online 8 September 2016
Background: The estimated intake of soy isoflavones from a meal has been based on the content in a food, but the health effects of soy isoflavones are possibly affected by their bioavailability. In this study we have evaluated the isoflavone bioavailability after the intake of three kinds of soy foods and a commercial soy isoflavone supplement, and examined whether the isoflavone bioavailability is different between equol producers and non-producers. Methods: Healthy female subjects (n ¼ 20; 9 equol producers, 11 equol non-producers) aged between 20 and 26 y participated in this study. To assess the bioavailability of isoflavones, equol producers and nonproducers consumed three kinds of soy foods (tofu, fermented soybeans: natto in Japanese, soy milk) and a soy isoflavone supplement containing 75 mg isoflavones together with breakfast. Urine was collected for 24 h, and the amounts of urinary excretions of daidzein, genistein, and the metabolite equol were measured. The intra- and inter-individual variations in the bioavailability of isoflavones were also examined. Results: The bioavailability of daidzein following the consumption of tofu, natto, soy milk, and soy isoflavone supplement were 66.9%, 45.2%, 65.7%, and 57.9%, respectively, and the bioavailability of genistein following the consumption of these soy products were 33.7%, 24.4%, 31.2%, and 17.7%, respectively. The bioavailability of daidzein and genistein was significantly greater in equol non-producers than equol producers, especially following the consumption of soymilk. In the equol producers, the 24 h urinary excretion of equol was significantly greater after the intake of soy isoflavone supplement than after the intake of natto or soymilk. The analysis of relative reliability of intra- and inter-individual variations suggested that bioavailability of isoflavones is less variable following soymilk intake than soy isoflavone supplement. Conclusions: The results in this study suggest that bioavailability of isoflavones are different between equol producers and non-producers, because the 24 h urinary excretion of equol in the equol producers were significantly lower than those in the equol non-producers. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Isoflavone Bioavailability Soy product Equol producers Equol non-producers
1. Introduction Many studies have reported the beneficial health effects of isoflavones on estrogen-related cancer, cardiovascular disease, lipid profiles, climacteric symptoms, and osteoporosis in humans [1e3]. However, the health effects of soy isoflavones are dependent on the
* Corresponding author. Graduate School of Nutritional and Environmental Sciences, The University of Shizuoka, 52-1 Yada, Suruga-Ku, Shizuoka-Shi, Shizuoka 422-8526, Japan. E-mail address: [email protected] (T. Goda).
quantity of ingredient absorbed and its bioavailability [4]. There is a wide variation in the reported proportion of urinary isoflavone excretion that is not apparently dependent on the dose. Different factors have been suggested to influence the bioavailability of isoflavones; however, the results are inconsistent, probably due to the use of different study designs, isoflavone sources, or food matrices between and within studies [5]. Soybean and related products are one of the richest sources of isoflavones, which act as a phytoestrogen in humans [6]. The isoflavones genistein and daidzein are present as glucosides in soy and many unfermented foods. The isoflavone content of soybean has been reported as follows: genistein 50%: daidzein 40%: glycitein 10%
http://dx.doi.org/10.1016/j.jnim.2016.08.001 2352-3859/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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[7]. These estrogenic isoflavones are predominately found as glucosides and malonylglucosides, but also as acetylglucosides and aglycones [8,9]. In the human intestine, isoflavone glycosides present in soy products can be deglycosylated by b-glucosidases in the human small intestine [10]. Bacterial metabolism of soy isoflavones varies among individuals. Inter-individual differences in phytochemical metabolism and disposition may be affected by gut microbial identity and activity, stability, and variation in concentrations of endogenous compounds that modulate biotransformation pathways [11,12]. Gut bacterial modification of soy isoflavones produces metabolites that differ in biological activity from the parent compounds [13]. The predominant daidzein metabolites produced by human intestinal bacteria are dihydrodaidzein, equol, and o-desmethylangolensin (O-DMA) [8]. Observational studies suggest that the O-DMA-producer and equol-producer phenotypes are independent of each other, i.e., the capacity to harbor ODMAeproducing bacteria is not influenced by whether the individual has the capacity to harbor equol-producing bacteria [14]. The daidzein precursor is needed for the production of O-DMA, and the half-life of O-DMA in the body is short [15]. Equol is a potent antioxidant and its clinical importance has recently become a subject of interest. Among humans, interindividual differences in daidzein metabolism exist following soy or daidzein consumption; approximately 30%e50% of the human population produces equol and approximately 80%e90% produces O-DMA. Several large sample size studies have indicated that only 25e30% of the adult population of Western countries produces equol when they consume soy foods containing isoflavones [11,16]. This proportion is significantly lower than the reported proportion of 50e60% of equol producers in adults from Japan, Korea, and China or in Western adult vegetarians [12]. Notably, the source of isoflavones consumed is important for evaluating their physiological activities. Clinical studies on the bioavailability of various forms of soy isoflavones (as extractives, food additives, or natural soy foodstuffs) have been undertaken in different populations (e.g., Asians and Europeans) [17e21]. However, these results have been inconclusive, and it is difficult to draw a conclusion due to differences in study populations, isoflavone origin, form, dose, and the meals consumed concurrently [22]. Further, the majority of research did not provide sufficient explanation of the controlled meal consumed simultaneously with the isoflavones. While several studies reported bioavailability of isoflavones, data regarding physiological inter- or intra-individual variations of isoflavones availability are scarce. In dietary surveys, the intake of soy isoflavones from natural soy foods is estimated based on the reference values of isoflavone content [23e25] (available in food composition tables). Furthermore, a truer estimation of isoflavone intake, thereby enabling the determination of their health effects and the proper planning of the isoflavone intervention, is thought possible by taking intra- and inter-individual variations in bioavailability into account. Therefore, it is necessary to quantify inter- and intra-individual variations in isoflavone bioavailability under conditions of standardized daily food intake to fully consider the possible metabolic consequence of ingested isoflavones. In this study, we have evaluated the 24 h urinary excretion of isoflavones, in equol producers and non-producers, following the consumption of various soy products simultaneously with the meals in Japanese population.
with a body mass index of 18.6e29.1 kg/m2 (mean ± SD: 23.3 ± 3.7 kg/m2) were recruited to participate in the four studies. Before the start of the study, the subjects attended group orientations where the study purpose and protocol were explained. All volunteers provided written informed consent. All individuals reported no medication use and had no allergies to soybean. The study protocol was approved by the ethics committee of Tokoha University in accordance with the Helsinki Declaration and the Committee's own guidelines (1964 approval, modification).
2. Materials and methods
2.4. Sample collection and urinary isoflavone analysis
2.1. Subjects
The subjects ate breakfast in the dining room on the test day, and ingested the soy foods or soy isoflavone supplement containing the identical amounts of isoflavones. Urine samples were collected at fixed time points (0, 2, 6, 12, 24 h) after ingestion of the test food.
The subjects were recruited from students in Tokoha University. Twenty healthy young adult Japanese women, aged 20e26 years
2.2. Study design Among the 20 subjects, 15 subjects participated in the study of the bioavailability of 4 soy products, and another 5 subjects participated in the three times repeated trial (soy milk and supplement) for intra-individual variation according to the same protocol. The study consisted of four feeding days, each of which was separated by more than one week as a washout period. All subjects were asked to avoid any soy foods and products, and complete a daily dietary record on the previous day. On the trial days, subjects were instructed to eat all of the basal test diet provided. The basal test diet consisted of a meal that did not include soy isoflavones. The other nutritive components were established according to the recommended dietary allowance (RDA) using the Japanese DRIs (Dietary References Intakes for Japanese, 2010). The soy isoflavone supplement (Otsuka Pharmaceutical Co., Ltd. Tokyo, Japan) contained 75 mg isoflavones, and other soy products also contained 75 mg isoflavone equivalent by references, i.e, 180 g of tofu, 100 g of fermented soybeans, hereafter referred to as natto, and 300 g of soy milk (Table 1). On the trial day, subjects consumed either one of each soy foods or soy isoflavone supplement together with the breakfast meal, which was served as a basal diet, and the consecutive meals were taken in a dining room at the designated time. 2.3. Quantitative analysis of soy foods, supplement, and the basal diet To determine isoflavones contents in soy products, samples of tofu, natto and soy milk were freeze-dried with the exception of the supplement, which was crushed and used as the analytical specimen. Three samples were analyzed, and the mean values were calculated. HPLC analysis of the flavonoid content of foods was performed according to the procedures of Sakakibara et al. [26]. Fifty milligrams of dried sample was placed in a test tube with ground glass stopper and mixed with 5 mL of citrate phosphate buffer (pH 4.6) containing 10 mg b-glucosidase (37 units). The sample was hydrolyzed at 37 C for 4 h, and 5 mL of ethanol was added. After centrifugation for 10 min, 8 mL of the supernatant was dried in a vacuum evaporator. Deconjugation was performed using b-glucosidase/sulfatase according to the modified method of King et al. [27]. The dietary isoflavones were analyzed using HPLC (SHIMADZU, Kyoto, Japan) equipped with a diode array detector (260 nm). The compounds were identified by retention times and spectra in comparison with standards and quantified by the peak area. The analyzed results indicated that soy isoflavones were absent in the basal diet.
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Table 1 Isoflavone contents of soy products. Soy products
Tested of food intake (g) Daidzein Total (mg) Aglycone (aglycone%) Genistein Total (mg) Aglycone (aglycone%) Total amount of isoflavones (mg)
Tofu
Fermented soybeans (natto)
Soymilk
Supplement
180
100
300
0.075
24.6 ± 2.69 33.8
41.1 ± 4.46 30.1
14.2 ± 2.99 3.8
23.3 ± 2.40 92.6
14.5 ± 1.75 29.6
22.5 ± 2.78 8.3
14.3 ± 2.38 2.7
29.6 ± 2.21 88.4
39.1 ± 4.44
63.6 ± 7.19
36.7 ± 4.10
52.9 ± 4.58
All values are presented as means of three replicates of each soy product analyzed.
Two trials were carried out on each trial day. The 24 h urine samples were collected using a U-Container (Akita Sumitomo Bakelite Co., Ltd., Akita, Japan). Following collection of the urine sample, the volume of the sample was measured. The urine samples were stored at 20 C until analysis. Urinary daidzein, genistein, and equol contents were determined using a modified version of the LC-MS methods previously described by Shimoi et al. [28]. Urine samples (0.5 mL) were mixed with 0.5 mL sodium acetate buffer (pH 4.5), preincubated at 37 C, and then incubated with bglucuronidase/sulfatase at 37 C for 20 min. The urine and incubation medium were acidified with the same volume of 0.01 M oxalic acid, and the solution was applied to a Sep-Pak C18 cartridge (Waters Co., Milford, MA). After washing the cartridge with 0.01 M oxalic acid, methanol/water/0.5 M oxalic acid (25:73:2, v/v), and distilled water, the methanol eluate was obtained. The eluate was evaporated to dryness, and the residue was dissolved in 200 mL of methanol. The solutions were analyzed chromatographically using a HPLC system (SHIMADZU). The LC conditions were as follows: column, Capcell Pak C18-UGI120 (250 4.6 mm I.D., Shiseido, Tokyo, Japan); detection, daidzein UV 250 nm, genistein UV 260 nm, equol UV 280 nm; flow rate, 0.7 mL/min; mobile phase, Solvent A: methanol with 0.03% trifluoroacetic acid, Solvent B: distilled water with 0.03% trifluoroacetic acid. The analysis was carried out at 50 C. Quantification of each isoflavone was carried out using peak areas according to calibration curves of the peak area of standards at various concentrations. HPLC standards were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan (daidzein, genistein) and Funakoshi Co., Ltd., Tokyo, Japan (equol). All solvents and chemicals used were of HPLC analytical grade. 2.5. Statistical analysis Data analysis was carried out using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA). Means and standard deviations were analyzed using descriptive statistics. Data distribution was expressed using quartile distributions. Analysis of variance test for normality was compared using the Shapiro-Wilk normality test. The distribution of each isoflavone was tested for normality before analysis, and skewed variables were log transformed. Comparison of urinary excretion among 4 soy products was analyzed using Kruskal-Wallis test, and comparison of the bioavailability in equol-producers and non-producers was analyzed using Mann-Whitney U test. Intra- and inter-individual variations were calculated with analysis of variance using mean ± standard deviation (SD). Intraindividual (within-individual) variations (CVw ¼ SDw/Mean of urinary excretion in 4 or 3 soy foods 100) and inter-individual (between-individual) variations (CVb ¼ SDb/Mean of urinary excretion in each same soy food 100) were calculated using the
formula provided by Beaton [29]. The CVw (within-individual variation) was calculated to estimate the true intake, dividing the subjects into equol-producers and non-producers. The relative reliability of intra- and inter-individual variations was evaluated using the intraclass correlation coefficient (ICC) calculated from the individual internal variance and the between individuals variance. The following formula was used to calculate ICC: s2B/(s2W þ s2B) [between-subject variance/(within-subject variance þ betweensubject variance)]. ICC was evaluated according to the valuation basis of Rosner [30] as follows: “0.75 ICC” is “excellent reproducibility” and “0.4 ICC<0.75” is “fair to good reproducibility”. 3. Results The isoflavone contents of tofu, natto, soy milk, and the soy isoflavone supplement used in this study are presented in Table 1. The soy isoflavone supplement contained a greater proportion of isoflavone aglycones than the other soy products. The 24 h urine excretion after soy food intake was collected at various time points. The total numbers of samples obtained for the 24 h urine analysis, excluding those subjects who failed urine collection or those who refused to consume natto, were 71 in total (15 for tofu, 16 for natto, 20 for soy milk, and 20 for soy isoflavone supplement). The urinary excretions of daidzein and genistein isoflavones were expressed as the proportion to those of respective isoflavones consumed as the soy products. Maximal excretions of daidzein and genistein were observed during the period of 6e12 h, and the urinary excretion of daidzein and genistein was much smaller during the period of 12e24 h in all groups following the consumption of soy products (Figs. 1 and 2). The proportions of 24 h urinary excretions of daidzein to the amounts of daidzein consumed from tofu, natto, soy milk, and soy isoflavone supplement were 66.9%, 45.2%, 65.7%, and 57.9%, respectively in average (Fig. 3). The proportions of 24 h urinary excretions of genistein to the amounts of genistein consumed from tofu, natto, soy milk, and soy isoflavone supplement were 33.7%, 24.4%, 31.2%, and 17.7%, respectively in average (Fig. 3). There was no significant difference in the proportion of daidzein recovered in 24 h urine among the groups following the consumption of different soy products. The proportion of genistein recovered in 24 h urine following the consumption of soymilk tended to be greater than those in 24 h urine following the consumption of other soy products, and it was significantly greater than that after the consumption of soy isoflavone supplement (p < 0.05; Fig. 3). The proportions of 24 h urinary excretions of daidzein and genistein to the amounts of respective isoflavone consumed from soymilk were significantly greater in equol non-producers than equol producers (p < 0.05), whereas no significant differences between equol non-producers and equol producers were observed in the urinary excretions of daidzein and genistein after
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Fig. 1. Distribution of urinary daidzein excretion levels after ingestion of soy foods and the supplement in the four soy product. Box-and-whisker plot indicates a quartile distribution (Upper ¼ 75th percentile, Lower ¼ 25th percentile, Middle ¼ 50th percentile). The lowest datum is within the 1.5 interquartile range of the lower quartile and the highest datum is within the 1.5 interquartile range of the upper quartile.
the consumption of tofu, natto, and soy isoflavone supplement (Table 2.). In the equol producers, the proportion of 24 h urinary excretions of equol to the amounts of daidzein consumed from soy isoflavone supplement was significantly greater than those following the consumption of natto or soymilk (p < 0.05; Table 2.). The inter-individual variations in the proportions of 24 h urinary excretions of daidzein and genistein to the amounts of respective isoflavone consumed from soy products were 45.3% and 53.8%, respectively in average. The intra-individual variations in the proportions of 24-h urinary excretions of daidzein and genistein to the amounts of respective isoflavone consumed from soy products were 36.1% and 38.4%, respectively in average. The intra-individual variations in the proportions of 24 h urinary excretions of daidzein and genistein in equol producers were 43.5% and 37.1%, respectively, whereas those in equol non-producers were 30.2% and 39.3%, respectively. The analysis of relative reliability of intra- and
inter-individual variations using ICC showed that daidzein and genistein values for the groups following the consumption of soymilk were 0.95 and 0.89, respectively, which indicated “excellent reproducibility”, while the daidzein and genistein values for the groups following the consumption of soy isoflavone supplement were 0.54 and 0.44, respectively, which indicated “fair to good reproducibility.” 4. Discussion The bioavailability of isoflavones from the daily diet may be variable, because most soy isoflavones in soy foods are bound with proteins, sugars, or other compounds. In this study, we attempted to estimate the bioavailability of isoflavones from three kinds of soy foods, which are popular and widely available in Japan, i.e., tofu, natto (fermented soybeans) and soymilk, by measuring the urinary
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Fig. 2. Distribution of urinary genistein excretion levels after ingestion of soy foods and the supplement. Box-and-whisker plot indicates a quartile distribution (Upper ¼ 75th percentile, Lower ¼ 25th percentile, Middle ¼ 50th percentile). The lowest datum is within the 1.5 interquartile range of the lower quartile and the highest datum is within the 1.5 interquartile range of the upper quartile.
excretions of the major soy isoflavones, i.e., daidzein and genistein, and an isoflavone metabolite equal during the 24-h period following the intake of soy products along with breakfast. It has been shown that urinary excretions of daidzein and genistein after intake of soy-based food and soya-based supplement are maximal between 6 and 12 h, and more than 70% of urinary excretions are completed by 24 h [31]. Indeed, our results showed that maximal excretions of daidzein and genistein were observed during the period of 6e12 h, and the urinary excretion of daidzein and genistein was much smaller during the period of 12e24 h in all groups following the consumption of soy products. Thus, we computed the proportions of 24 h urinary excretions of daidzein and genistein to the amounts of respective isoflavones consumed from tofu, natto, soy milk, and soy isoflavone supplement, as indices of bioavailability of daidzein and genistein from the soy products. In this study, we have demonstrated that the bioavailability of daidzein following the consumption of tofu, natto, soy milk, and soy
isoflavone supplement was relatively greater, ranging between 45% and 67% as assessed by the 24-h urinary excretion, and less variable than the bioavailability of genistein, which ranged between 17% and 34%. Isoflavones in soy products have antioxidant properties and act as phytoestrogens. Equol is a potent antioxidant in vitro (Wiseman et al. [29]), and its clinical importance has recently been of interest. The clinical importance of equol as an isoflavone metabolite has been reported, providing clues to the effectiveness of soy and its metabolites [11]. In this study we found that the 24 h urinary excretion of equol, as estimated from 24-h urinary excretion, was significantly greater after the intake of soy isoflavone supplement than after the intake of natto or soymilk. It is interesting to note that the bioavailability of daidzein and genistein was significantly greater in equol non-producers than equol producers, especially following the consumption of soymilk. Thus, it is likely that the 24 h urinary excretion of equol in the equol producers is variable among
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Fig. 3. Distribution of 24 h urinary isoflavone excretion levels after ingestion of soy foods and the supplement. Box-and-whisker plot indicates a quartile distribution (Upper ¼ 75th percentile, Lower ¼ 25th percentile, Middle ¼ 50th percentile). The lowest datum is within the 1.5 interquartile range of the lower quartile and the highest datum is within the 1.5 interquartile range of the upper quartile. a: Significant difference using Bonferroni's test with p < 0.05.
Table 2 24 h urinary isoflavone excretion of equol producers and non-producers. Tofu
Natto
Soymilk
Supplement
Equol
Daidzein (mg/mg intake) Genistein (mg/mg intake) Equol (mg/mg intake)
Producers (n ¼ 4)
Non-producers (n ¼ 11)
Producers (n ¼ 8)
Non-producers (n ¼ 8)
Producers (n ¼ 9)
Non-producers (n ¼ 11)
Producers (n ¼ 9)
Non-producers (n ¼ 11)
0.43 ± 0.63 (0.33e0.53)# 0.19 ± 0.04 (0.13e0.25) 0.50 ± 0.26a,b (0.28e0.71)
0.66 ± 0.14 (0.56e0.76) 0.40 ± 0.31 (0.17e0.62) Not detected
0.39 ± 0.12 (0.29e0.49) 0.19 ± 0.10 (0.11e0.28) 0.21 ± 0.14a (0.12e0.29)
0.51 ± 0.12 (0.42e0.61) 0.30 ± 0.11 (0.20e0.39) Not detected
0.54 ± 0.37 (0.31e0.76) 0.26 ± 0.16 (0.16e0.36) 0.27 ± 0.14a (0.14e0.39)
0.85 ± 0.29* (0.62e1.09) 0.40 ± 0.16* (0.27e0.53) Not detected
0.48 ± 0.19 (0.32e0.63) 0.16 ± 0.09 (0.08e0.23) 0.47 ± 0.09b (0.33e0.62)
0.66 ± 0.13 (0.56e0.76) 0.19 ± 0.06 (0.15e0.24) Not detected
Values are mean ± SD. * Equol producers vs equol non-producers after intake of each soy products using Mann-Whitney U test (P < 0.05). # Values are 95% confidence intervals. a-b Values not sharing a common alphabet are significantly different from each other at (P < 0.05) by Kruskal-Wallis test.
the soy products. In the present study, the analysis of relative reliability of intraand inter-individual variations revealed that bioavailability of soy isoflavone supplement is more variable following soy isoflavone supplement isoflavones than soymilk intake. The greater intraindividual variations of bioavailability of isoflavones from soy isoflavone supplement may be related with the greater contents of isoflavone aglycones. Isoflavone aglycones are thought to be absorbed more efficiently than isoflavone glucosides. Wiseman et al. reported that although intra-individual variation in isoflavone metabolism was low, inter-individual variation was high [29]. It should be noted that the individual variation (CV%) of the 24 h urinary isoflavone excretion differed among the various soy foods and the supplement consumed by the subjects. In the present study, we attempted to evaluate the reproducibility of our data in this study using ICC (intraclass correlation coefficient). According to the evaluation criteria by Rosener et al. [31], values over 0.8 indicate excellent reproducibility, and values 0.7 indicate good reproducibility. The ICC of daidzein and genistein for soymilk were computed as 0.95e0.859. These results suggest that the data of soymilk are more reliable than the data of soy isoflavone supplement, with daidzein ICC values of 0.95 for soy milk and 0.54 for supplement, and with genistein ICC values of 0.89 for soy milk and 0.44 for supplement.
In conclusions, the results in this study suggest that bioavailability of isoflavones are different between equol producers and non-producers. Competing interest The authors declare that they have no competing interests. Authors' contributions AM and TG conceived and designed the study; AM conducted the trials and collected the data, wrote the manuscript, analyzed the data, and interpreted the data; TG supervised the conduct of the trial and data collection and provided advice on the manuscript; CS provided advice on the quantitative analysis and interpreted the data; KS supervised the conduct of the study, provided advice on the manuscript; HS provided advice on the analytical method. All authors contributed intellectually to the manuscript and reviewed the drafts. All authors have read and approved the final manuscript. Acknowledgments The author thanks Dr. Michiko Yasuda (Graduate School of Nutritional and Environmental Sciences, University of Shizuoka) for
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advice on the manuscript and Ms. Marina Suzuki (Faculty of Health Promotional Sciences, Tokoha University) for cooperation with the cooking of the standard meal. Finally, I greatly appreciate the assistance of Dr. Sachio Takase PhD (Professor Emeritus of the University of Shizuoka, Nagasaki Prefectural University) and Forte Science Communications for guidance in the translation of my manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] Yamori Y, Miura A, Taira K. Implications from and for food cultures for cardiovascular diseases: Japanese food, particularly Okinawan diets. 2001 Asia Pac J. Clin. Nutr. 2001;10:144e5. [2] Yamori Y, Moriguchi EH, Teramoto T, Miura A, Fukui Y, Honda KI, Fukui M, Nara Y, Taira K, Moriguchi Y. Soybean isoflavones reduce postmenopausal bone resorption in female Japanese immigrants in Brazil: a ten-week study. J. Am. Coll. Nutr. 2002;21:560e3. [3] Messina M, Watanabe S, Setchell KD. Report on the 8th international symposium on the role of soy in health promotion and chronic disease prevention and treatment. J. Nutr. 2009;139:796Se802S. [4] Hendrich S. Bioavailability of isoflavones. J. Chromatogr. B 2002;777:203e10. [5] IL1 Nielsen, Williamson G. Review of the factors affecting bioavailability of soy isoflavones in humans. Nutr. Cancer 2007;57:1e10. R, Petrlova J, Potesil D, Adam V, Stiborov [6] Klejdus B, Mikelova a M, Hodek P, Vacek J, Kizek R, Kub an V. Evaluation of isoflavone aglycon and glycoside distribution in soy plants and soybeans by fast column high-performance liquid chromatography coupled with a diode-array detector. J. Agric. Food Chem. 2005;27:5848e52. [7] Murphy PA, Song T, Buseman G, Barua K, Beecher GR, Trainer D, Holden J. Isoflavones in retail and institutional soy foods. J. Agric. Food Chem. 1999;47: 2697e704. [8] Wang HJ, Murphy PA. Isoflavone content in commercial soybean foods. J. Agric. Food Chem. 1994;42:1666e73. [9] Wang H, Murphy P. A. Isoflavone composition of American and Japanese soybeans in Iowa: effects of variety, crop year, and location. J. Agric. Food Chem. 1994;42:1674e7. [10] Day AJ, DuPont MS, Ridley S, Rhodes M, Rhodes MJ, Morgan MR, Williamson G. Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity. FEBS Lett. 1998;436:71e5. [11] Lampe JW, Chang JL. Inter individual differences in phytochemical metabolism and disposition. Semin Cancer Biol. 2007;17:347e53. [12] Lampe JW. Interindividual differences in response to plant-based diets: implications for cancer risk. Am. J. Clin. Nutr. 2009;89:1553Se7S. [13] Lampe JW. Is equol the key to the efficacy of soy foods? Am. J. Clin. Nutr. 2009;89:1664Se7S. [14] Frankenfeld CL, Atkinson C, Thomas WK, Goode EL, Gonzalez A, Jokela T, W€ ah€ al€ a K, Schwartz SM, Li SS, et al. Familial correlations, segregation analysis, and nongenetic correlates of soy isoflavone-metabolizing phenotypes. Exp. Biol. Med. (Maywood) 2004;229:902e13.
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[15] Watanabe S, Yamaguchi M, Sobue T, Takahashi T, Miura T, Arai Y, Mazur W, € h€ € K, Adlercreutz H. Pharmacokinetics of soybean isoflavones in plasma, Wa ala urine and feces of men after ingestion of 60 g baked soybean powder (kinako). J. Nutr. 1998;128. 1710e5. [16] 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;36:27e32. [17] Slavin JL, Karr SC, Hutchins AM, Lampe JW. Influence of soybean processing, habitual diet, and soy dose on urinary isoflavonoid excretion. Am. J. Clin. Nutr. 1998;68:1492Se5S. [18] 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;91:567e74. [19] Cassidy A, Brown JE, Hawdon A, Faughnan MS, King LJ, Millward J, ZimmerNechemias L, Wolfe B, Setchell KD. Factors affecting the bioavailability of soy isoflavones in humans after ingestion of physiologically relevant levels from different soy foods. J. Nutr. 2006;136:45e51. [20] Chanteranne B, Branca F, Kaardinal A, Wahala K, Braesco V, Ladroite P, Brouns F, Coxam V. Food matrix and isoflavones bioavailability in early post menopausal women: a European clinical study. Clin. Interv. Aging 2008;3: 711e8. [21] Chang Y, Choue R. Plasma pharmacokinetics and urinary excretion of isoflavones after ingestion of soy products with different aglycone/glucoside ratios in South Korean women. Nutr. Res. Pract. 2013;7:393e9. [22] Sugano M. Soy in health and disease prevention. In: Watanabe S, Zhuo XG, Melby MK, Ishikawa N, Kimira M, editors. Systematic review of intervention studies using isoflavone supplements and proposal for future studies. Taylor & Francis Group, Florida: CRC Press; 2006. p. 87e106. [23] Kimira Mitsuru, Arai Yusuke, Shimoi Kayako, Watanabe Shaw. Japanese intake of flavonoids and isoflavonoids from foods. J. Epidemiol. 1998;8:168e75. [24] Arai Y, Watanabe S, Kimira M, Shimoi K, Mochizuki R, Kinae N. Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration. J. Nutr. 2000;130:2243e50. [25] Yamamoto S, Sobue T, Sasaki S, Kobayashi M, Arai Y, Uehara M, Adlercreutz H, Watanabe S, Takahashi T, Itoi Y, Iwase Y, Akabane M, Tsugane S. J. Nutr. 2001;131:2741e7. [26] Sakakibara H, Viala D, Ollier A, Combeau A, Besle JM. Isoflavones in several clover species and in milk from goats fed clovers. Biofactors 2004;22:237e9. [27] King RA, Mano MM, Head RJ. Assessment of isoflavonoid concentrations in Australian bovine milk samples. J. Dairy Res. 1998;65:479e89. [28] Shimoi K, Okada H, Furugori M, Goda T, Takase S, Suzuki M, Hara Y, Yamamoto H, Kinae N. Intestinal absorption of luteolin and luteolin 7-O-betaglucoside in rats and humans. FEBS Lett. 1998;6:220e4. [29] Beaton GH, Milner J, Corey P, McGuire V, Cousins M, Stewart E, de Ramos M, Hewitt D, Grambsch PV, Kassim N, Little JA. Sources of variance in 24-hour dietary recall data: implications for nutrition study design and interpretation. Am. J. Clin. Nutr. 1979;32:2546e59. [30] Rosner B. Fundamentals of biostatistics. CA: Duxbury Press; 2006. p. 613e7. [31] Vergne S, Bennetau-Pelissero C, Lamothe V, Chantre P, Potier M, Asselineau J, Perez P, Durand M, Moore N, Sauvant P. Higher bioavailability of isoflavones after a single ingestion of a soya-based supplement than a soya-based food in young healthy males. Br. J. Nutr. 2008;99:333e44.
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Bioavailability of isoflavones from soy products in equol producers and non-producers in Japanese women Ayako Miura a, b, Chitose Sugiyama b, Hiroyuki Sakakibara c, Kayoko Simoi a, Toshinao Goda a, * a b c
Graduate School of Nutritional and Environmental Sciences, The University of Shizuoka, Shizuoka 422-8526, Japan Department of Health and Nutritional Sciences, Faculty of Health Promotional Sciences, Tokoha University, Hamamatsu 431-2102, Japan Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2016 Received in revised form 25 August 2016 Accepted 31 August 2016 Available online 8 September 2016
Background: The estimated intake of soy isoflavones from a meal has been based on the content in a food, but the health effects of soy isoflavones are possibly affected by their bioavailability. In this study we have evaluated the isoflavone bioavailability after the intake of three kinds of soy foods and a commercial soy isoflavone supplement, and examined whether the isoflavone bioavailability is different between equol producers and non-producers. Methods: Healthy female subjects (n ¼ 20; 9 equol producers, 11 equol non-producers) aged between 20 and 26 y participated in this study. To assess the bioavailability of isoflavones, equol producers and nonproducers consumed three kinds of soy foods (tofu, fermented soybeans: natto in Japanese, soy milk) and a soy isoflavone supplement containing 75 mg isoflavones together with breakfast. Urine was collected for 24 h, and the amounts of urinary excretions of daidzein, genistein, and the metabolite equol were measured. The intra- and inter-individual variations in the bioavailability of isoflavones were also examined. Results: The bioavailability of daidzein following the consumption of tofu, natto, soy milk, and soy isoflavone supplement were 66.9%, 45.2%, 65.7%, and 57.9%, respectively, and the bioavailability of genistein following the consumption of these soy products were 33.7%, 24.4%, 31.2%, and 17.7%, respectively. The bioavailability of daidzein and genistein was significantly greater in equol non-producers than equol producers, especially following the consumption of soymilk. In the equol producers, the 24 h urinary excretion of equol was significantly greater after the intake of soy isoflavone supplement than after the intake of natto or soymilk. The analysis of relative reliability of intra- and inter-individual variations suggested that bioavailability of isoflavones is less variable following soymilk intake than soy isoflavone supplement. Conclusions: The results in this study suggest that bioavailability of isoflavones are different between equol producers and non-producers, because the 24 h urinary excretion of equol in the equol producers were significantly lower than those in the equol non-producers. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Isoflavone Bioavailability Soy product Equol producers Equol non-producers
1. Introduction Many studies have reported the beneficial health effects of isoflavones on estrogen-related cancer, cardiovascular disease, lipid profiles, climacteric symptoms, and osteoporosis in humans [1e3]. However, the health effects of soy isoflavones are dependent on the
* Corresponding author. Graduate School of Nutritional and Environmental Sciences, The University of Shizuoka, 52-1 Yada, Suruga-Ku, Shizuoka-Shi, Shizuoka 422-8526, Japan. E-mail address: [email protected] (T. Goda).
quantity of ingredient absorbed and its bioavailability [4]. There is a wide variation in the reported proportion of urinary isoflavone excretion that is not apparently dependent on the dose. Different factors have been suggested to influence the bioavailability of isoflavones; however, the results are inconsistent, probably due to the use of different study designs, isoflavone sources, or food matrices between and within studies [5]. Soybean and related products are one of the richest sources of isoflavones, which act as a phytoestrogen in humans [6]. The isoflavones genistein and daidzein are present as glucosides in soy and many unfermented foods. The isoflavone content of soybean has been reported as follows: genistein 50%: daidzein 40%: glycitein 10%
http://dx.doi.org/10.1016/j.jnim.2016.08.001 2352-3859/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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[7]. These estrogenic isoflavones are predominately found as glucosides and malonylglucosides, but also as acetylglucosides and aglycones [8,9]. In the human intestine, isoflavone glycosides present in soy products can be deglycosylated by b-glucosidases in the human small intestine [10]. Bacterial metabolism of soy isoflavones varies among individuals. Inter-individual differences in phytochemical metabolism and disposition may be affected by gut microbial identity and activity, stability, and variation in concentrations of endogenous compounds that modulate biotransformation pathways [11,12]. Gut bacterial modification of soy isoflavones produces metabolites that differ in biological activity from the parent compounds [13]. The predominant daidzein metabolites produced by human intestinal bacteria are dihydrodaidzein, equol, and o-desmethylangolensin (O-DMA) [8]. Observational studies suggest that the O-DMA-producer and equol-producer phenotypes are independent of each other, i.e., the capacity to harbor ODMAeproducing bacteria is not influenced by whether the individual has the capacity to harbor equol-producing bacteria [14]. The daidzein precursor is needed for the production of O-DMA, and the half-life of O-DMA in the body is short [15]. Equol is a potent antioxidant and its clinical importance has recently become a subject of interest. Among humans, interindividual differences in daidzein metabolism exist following soy or daidzein consumption; approximately 30%e50% of the human population produces equol and approximately 80%e90% produces O-DMA. Several large sample size studies have indicated that only 25e30% of the adult population of Western countries produces equol when they consume soy foods containing isoflavones [11,16]. This proportion is significantly lower than the reported proportion of 50e60% of equol producers in adults from Japan, Korea, and China or in Western adult vegetarians [12]. Notably, the source of isoflavones consumed is important for evaluating their physiological activities. Clinical studies on the bioavailability of various forms of soy isoflavones (as extractives, food additives, or natural soy foodstuffs) have been undertaken in different populations (e.g., Asians and Europeans) [17e21]. However, these results have been inconclusive, and it is difficult to draw a conclusion due to differences in study populations, isoflavone origin, form, dose, and the meals consumed concurrently [22]. Further, the majority of research did not provide sufficient explanation of the controlled meal consumed simultaneously with the isoflavones. While several studies reported bioavailability of isoflavones, data regarding physiological inter- or intra-individual variations of isoflavones availability are scarce. In dietary surveys, the intake of soy isoflavones from natural soy foods is estimated based on the reference values of isoflavone content [23e25] (available in food composition tables). Furthermore, a truer estimation of isoflavone intake, thereby enabling the determination of their health effects and the proper planning of the isoflavone intervention, is thought possible by taking intra- and inter-individual variations in bioavailability into account. Therefore, it is necessary to quantify inter- and intra-individual variations in isoflavone bioavailability under conditions of standardized daily food intake to fully consider the possible metabolic consequence of ingested isoflavones. In this study, we have evaluated the 24 h urinary excretion of isoflavones, in equol producers and non-producers, following the consumption of various soy products simultaneously with the meals in Japanese population.
with a body mass index of 18.6e29.1 kg/m2 (mean ± SD: 23.3 ± 3.7 kg/m2) were recruited to participate in the four studies. Before the start of the study, the subjects attended group orientations where the study purpose and protocol were explained. All volunteers provided written informed consent. All individuals reported no medication use and had no allergies to soybean. The study protocol was approved by the ethics committee of Tokoha University in accordance with the Helsinki Declaration and the Committee's own guidelines (1964 approval, modification).
2. Materials and methods
2.4. Sample collection and urinary isoflavone analysis
2.1. Subjects
The subjects ate breakfast in the dining room on the test day, and ingested the soy foods or soy isoflavone supplement containing the identical amounts of isoflavones. Urine samples were collected at fixed time points (0, 2, 6, 12, 24 h) after ingestion of the test food.
The subjects were recruited from students in Tokoha University. Twenty healthy young adult Japanese women, aged 20e26 years
2.2. Study design Among the 20 subjects, 15 subjects participated in the study of the bioavailability of 4 soy products, and another 5 subjects participated in the three times repeated trial (soy milk and supplement) for intra-individual variation according to the same protocol. The study consisted of four feeding days, each of which was separated by more than one week as a washout period. All subjects were asked to avoid any soy foods and products, and complete a daily dietary record on the previous day. On the trial days, subjects were instructed to eat all of the basal test diet provided. The basal test diet consisted of a meal that did not include soy isoflavones. The other nutritive components were established according to the recommended dietary allowance (RDA) using the Japanese DRIs (Dietary References Intakes for Japanese, 2010). The soy isoflavone supplement (Otsuka Pharmaceutical Co., Ltd. Tokyo, Japan) contained 75 mg isoflavones, and other soy products also contained 75 mg isoflavone equivalent by references, i.e, 180 g of tofu, 100 g of fermented soybeans, hereafter referred to as natto, and 300 g of soy milk (Table 1). On the trial day, subjects consumed either one of each soy foods or soy isoflavone supplement together with the breakfast meal, which was served as a basal diet, and the consecutive meals were taken in a dining room at the designated time. 2.3. Quantitative analysis of soy foods, supplement, and the basal diet To determine isoflavones contents in soy products, samples of tofu, natto and soy milk were freeze-dried with the exception of the supplement, which was crushed and used as the analytical specimen. Three samples were analyzed, and the mean values were calculated. HPLC analysis of the flavonoid content of foods was performed according to the procedures of Sakakibara et al. [26]. Fifty milligrams of dried sample was placed in a test tube with ground glass stopper and mixed with 5 mL of citrate phosphate buffer (pH 4.6) containing 10 mg b-glucosidase (37 units). The sample was hydrolyzed at 37 C for 4 h, and 5 mL of ethanol was added. After centrifugation for 10 min, 8 mL of the supernatant was dried in a vacuum evaporator. Deconjugation was performed using b-glucosidase/sulfatase according to the modified method of King et al. [27]. The dietary isoflavones were analyzed using HPLC (SHIMADZU, Kyoto, Japan) equipped with a diode array detector (260 nm). The compounds were identified by retention times and spectra in comparison with standards and quantified by the peak area. The analyzed results indicated that soy isoflavones were absent in the basal diet.
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Table 1 Isoflavone contents of soy products. Soy products
Tested of food intake (g) Daidzein Total (mg) Aglycone (aglycone%) Genistein Total (mg) Aglycone (aglycone%) Total amount of isoflavones (mg)
Tofu
Fermented soybeans (natto)
Soymilk
Supplement
180
100
300
0.075
24.6 ± 2.69 33.8
41.1 ± 4.46 30.1
14.2 ± 2.99 3.8
23.3 ± 2.40 92.6
14.5 ± 1.75 29.6
22.5 ± 2.78 8.3
14.3 ± 2.38 2.7
29.6 ± 2.21 88.4
39.1 ± 4.44
63.6 ± 7.19
36.7 ± 4.10
52.9 ± 4.58
All values are presented as means of three replicates of each soy product analyzed.
Two trials were carried out on each trial day. The 24 h urine samples were collected using a U-Container (Akita Sumitomo Bakelite Co., Ltd., Akita, Japan). Following collection of the urine sample, the volume of the sample was measured. The urine samples were stored at 20 C until analysis. Urinary daidzein, genistein, and equol contents were determined using a modified version of the LC-MS methods previously described by Shimoi et al. [28]. Urine samples (0.5 mL) were mixed with 0.5 mL sodium acetate buffer (pH 4.5), preincubated at 37 C, and then incubated with bglucuronidase/sulfatase at 37 C for 20 min. The urine and incubation medium were acidified with the same volume of 0.01 M oxalic acid, and the solution was applied to a Sep-Pak C18 cartridge (Waters Co., Milford, MA). After washing the cartridge with 0.01 M oxalic acid, methanol/water/0.5 M oxalic acid (25:73:2, v/v), and distilled water, the methanol eluate was obtained. The eluate was evaporated to dryness, and the residue was dissolved in 200 mL of methanol. The solutions were analyzed chromatographically using a HPLC system (SHIMADZU). The LC conditions were as follows: column, Capcell Pak C18-UGI120 (250 4.6 mm I.D., Shiseido, Tokyo, Japan); detection, daidzein UV 250 nm, genistein UV 260 nm, equol UV 280 nm; flow rate, 0.7 mL/min; mobile phase, Solvent A: methanol with 0.03% trifluoroacetic acid, Solvent B: distilled water with 0.03% trifluoroacetic acid. The analysis was carried out at 50 C. Quantification of each isoflavone was carried out using peak areas according to calibration curves of the peak area of standards at various concentrations. HPLC standards were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan (daidzein, genistein) and Funakoshi Co., Ltd., Tokyo, Japan (equol). All solvents and chemicals used were of HPLC analytical grade. 2.5. Statistical analysis Data analysis was carried out using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA). Means and standard deviations were analyzed using descriptive statistics. Data distribution was expressed using quartile distributions. Analysis of variance test for normality was compared using the Shapiro-Wilk normality test. The distribution of each isoflavone was tested for normality before analysis, and skewed variables were log transformed. Comparison of urinary excretion among 4 soy products was analyzed using Kruskal-Wallis test, and comparison of the bioavailability in equol-producers and non-producers was analyzed using Mann-Whitney U test. Intra- and inter-individual variations were calculated with analysis of variance using mean ± standard deviation (SD). Intraindividual (within-individual) variations (CVw ¼ SDw/Mean of urinary excretion in 4 or 3 soy foods 100) and inter-individual (between-individual) variations (CVb ¼ SDb/Mean of urinary excretion in each same soy food 100) were calculated using the
formula provided by Beaton [29]. The CVw (within-individual variation) was calculated to estimate the true intake, dividing the subjects into equol-producers and non-producers. The relative reliability of intra- and inter-individual variations was evaluated using the intraclass correlation coefficient (ICC) calculated from the individual internal variance and the between individuals variance. The following formula was used to calculate ICC: s2B/(s2W þ s2B) [between-subject variance/(within-subject variance þ betweensubject variance)]. ICC was evaluated according to the valuation basis of Rosner [30] as follows: “0.75 ICC” is “excellent reproducibility” and “0.4 ICC<0.75” is “fair to good reproducibility”. 3. Results The isoflavone contents of tofu, natto, soy milk, and the soy isoflavone supplement used in this study are presented in Table 1. The soy isoflavone supplement contained a greater proportion of isoflavone aglycones than the other soy products. The 24 h urine excretion after soy food intake was collected at various time points. The total numbers of samples obtained for the 24 h urine analysis, excluding those subjects who failed urine collection or those who refused to consume natto, were 71 in total (15 for tofu, 16 for natto, 20 for soy milk, and 20 for soy isoflavone supplement). The urinary excretions of daidzein and genistein isoflavones were expressed as the proportion to those of respective isoflavones consumed as the soy products. Maximal excretions of daidzein and genistein were observed during the period of 6e12 h, and the urinary excretion of daidzein and genistein was much smaller during the period of 12e24 h in all groups following the consumption of soy products (Figs. 1 and 2). The proportions of 24 h urinary excretions of daidzein to the amounts of daidzein consumed from tofu, natto, soy milk, and soy isoflavone supplement were 66.9%, 45.2%, 65.7%, and 57.9%, respectively in average (Fig. 3). The proportions of 24 h urinary excretions of genistein to the amounts of genistein consumed from tofu, natto, soy milk, and soy isoflavone supplement were 33.7%, 24.4%, 31.2%, and 17.7%, respectively in average (Fig. 3). There was no significant difference in the proportion of daidzein recovered in 24 h urine among the groups following the consumption of different soy products. The proportion of genistein recovered in 24 h urine following the consumption of soymilk tended to be greater than those in 24 h urine following the consumption of other soy products, and it was significantly greater than that after the consumption of soy isoflavone supplement (p < 0.05; Fig. 3). The proportions of 24 h urinary excretions of daidzein and genistein to the amounts of respective isoflavone consumed from soymilk were significantly greater in equol non-producers than equol producers (p < 0.05), whereas no significant differences between equol non-producers and equol producers were observed in the urinary excretions of daidzein and genistein after
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Fig. 1. Distribution of urinary daidzein excretion levels after ingestion of soy foods and the supplement in the four soy product. Box-and-whisker plot indicates a quartile distribution (Upper ¼ 75th percentile, Lower ¼ 25th percentile, Middle ¼ 50th percentile). The lowest datum is within the 1.5 interquartile range of the lower quartile and the highest datum is within the 1.5 interquartile range of the upper quartile.
the consumption of tofu, natto, and soy isoflavone supplement (Table 2.). In the equol producers, the proportion of 24 h urinary excretions of equol to the amounts of daidzein consumed from soy isoflavone supplement was significantly greater than those following the consumption of natto or soymilk (p < 0.05; Table 2.). The inter-individual variations in the proportions of 24 h urinary excretions of daidzein and genistein to the amounts of respective isoflavone consumed from soy products were 45.3% and 53.8%, respectively in average. The intra-individual variations in the proportions of 24-h urinary excretions of daidzein and genistein to the amounts of respective isoflavone consumed from soy products were 36.1% and 38.4%, respectively in average. The intra-individual variations in the proportions of 24 h urinary excretions of daidzein and genistein in equol producers were 43.5% and 37.1%, respectively, whereas those in equol non-producers were 30.2% and 39.3%, respectively. The analysis of relative reliability of intra- and
inter-individual variations using ICC showed that daidzein and genistein values for the groups following the consumption of soymilk were 0.95 and 0.89, respectively, which indicated “excellent reproducibility”, while the daidzein and genistein values for the groups following the consumption of soy isoflavone supplement were 0.54 and 0.44, respectively, which indicated “fair to good reproducibility.” 4. Discussion The bioavailability of isoflavones from the daily diet may be variable, because most soy isoflavones in soy foods are bound with proteins, sugars, or other compounds. In this study, we attempted to estimate the bioavailability of isoflavones from three kinds of soy foods, which are popular and widely available in Japan, i.e., tofu, natto (fermented soybeans) and soymilk, by measuring the urinary
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Fig. 2. Distribution of urinary genistein excretion levels after ingestion of soy foods and the supplement. Box-and-whisker plot indicates a quartile distribution (Upper ¼ 75th percentile, Lower ¼ 25th percentile, Middle ¼ 50th percentile). The lowest datum is within the 1.5 interquartile range of the lower quartile and the highest datum is within the 1.5 interquartile range of the upper quartile.
excretions of the major soy isoflavones, i.e., daidzein and genistein, and an isoflavone metabolite equal during the 24-h period following the intake of soy products along with breakfast. It has been shown that urinary excretions of daidzein and genistein after intake of soy-based food and soya-based supplement are maximal between 6 and 12 h, and more than 70% of urinary excretions are completed by 24 h [31]. Indeed, our results showed that maximal excretions of daidzein and genistein were observed during the period of 6e12 h, and the urinary excretion of daidzein and genistein was much smaller during the period of 12e24 h in all groups following the consumption of soy products. Thus, we computed the proportions of 24 h urinary excretions of daidzein and genistein to the amounts of respective isoflavones consumed from tofu, natto, soy milk, and soy isoflavone supplement, as indices of bioavailability of daidzein and genistein from the soy products. In this study, we have demonstrated that the bioavailability of daidzein following the consumption of tofu, natto, soy milk, and soy
isoflavone supplement was relatively greater, ranging between 45% and 67% as assessed by the 24-h urinary excretion, and less variable than the bioavailability of genistein, which ranged between 17% and 34%. Isoflavones in soy products have antioxidant properties and act as phytoestrogens. Equol is a potent antioxidant in vitro (Wiseman et al. [29]), and its clinical importance has recently been of interest. The clinical importance of equol as an isoflavone metabolite has been reported, providing clues to the effectiveness of soy and its metabolites [11]. In this study we found that the 24 h urinary excretion of equol, as estimated from 24-h urinary excretion, was significantly greater after the intake of soy isoflavone supplement than after the intake of natto or soymilk. It is interesting to note that the bioavailability of daidzein and genistein was significantly greater in equol non-producers than equol producers, especially following the consumption of soymilk. Thus, it is likely that the 24 h urinary excretion of equol in the equol producers is variable among
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Fig. 3. Distribution of 24 h urinary isoflavone excretion levels after ingestion of soy foods and the supplement. Box-and-whisker plot indicates a quartile distribution (Upper ¼ 75th percentile, Lower ¼ 25th percentile, Middle ¼ 50th percentile). The lowest datum is within the 1.5 interquartile range of the lower quartile and the highest datum is within the 1.5 interquartile range of the upper quartile. a: Significant difference using Bonferroni's test with p < 0.05.
Table 2 24 h urinary isoflavone excretion of equol producers and non-producers. Tofu
Natto
Soymilk
Supplement
Equol
Daidzein (mg/mg intake) Genistein (mg/mg intake) Equol (mg/mg intake)
Producers (n ¼ 4)
Non-producers (n ¼ 11)
Producers (n ¼ 8)
Non-producers (n ¼ 8)
Producers (n ¼ 9)
Non-producers (n ¼ 11)
Producers (n ¼ 9)
Non-producers (n ¼ 11)
0.43 ± 0.63 (0.33e0.53)# 0.19 ± 0.04 (0.13e0.25) 0.50 ± 0.26a,b (0.28e0.71)
0.66 ± 0.14 (0.56e0.76) 0.40 ± 0.31 (0.17e0.62) Not detected
0.39 ± 0.12 (0.29e0.49) 0.19 ± 0.10 (0.11e0.28) 0.21 ± 0.14a (0.12e0.29)
0.51 ± 0.12 (0.42e0.61) 0.30 ± 0.11 (0.20e0.39) Not detected
0.54 ± 0.37 (0.31e0.76) 0.26 ± 0.16 (0.16e0.36) 0.27 ± 0.14a (0.14e0.39)
0.85 ± 0.29* (0.62e1.09) 0.40 ± 0.16* (0.27e0.53) Not detected
0.48 ± 0.19 (0.32e0.63) 0.16 ± 0.09 (0.08e0.23) 0.47 ± 0.09b (0.33e0.62)
0.66 ± 0.13 (0.56e0.76) 0.19 ± 0.06 (0.15e0.24) Not detected
Values are mean ± SD. * Equol producers vs equol non-producers after intake of each soy products using Mann-Whitney U test (P < 0.05). # Values are 95% confidence intervals. a-b Values not sharing a common alphabet are significantly different from each other at (P < 0.05) by Kruskal-Wallis test.
the soy products. In the present study, the analysis of relative reliability of intraand inter-individual variations revealed that bioavailability of soy isoflavone supplement is more variable following soy isoflavone supplement isoflavones than soymilk intake. The greater intraindividual variations of bioavailability of isoflavones from soy isoflavone supplement may be related with the greater contents of isoflavone aglycones. Isoflavone aglycones are thought to be absorbed more efficiently than isoflavone glucosides. Wiseman et al. reported that although intra-individual variation in isoflavone metabolism was low, inter-individual variation was high [29]. It should be noted that the individual variation (CV%) of the 24 h urinary isoflavone excretion differed among the various soy foods and the supplement consumed by the subjects. In the present study, we attempted to evaluate the reproducibility of our data in this study using ICC (intraclass correlation coefficient). According to the evaluation criteria by Rosener et al. [31], values over 0.8 indicate excellent reproducibility, and values 0.7 indicate good reproducibility. The ICC of daidzein and genistein for soymilk were computed as 0.95e0.859. These results suggest that the data of soymilk are more reliable than the data of soy isoflavone supplement, with daidzein ICC values of 0.95 for soy milk and 0.54 for supplement, and with genistein ICC values of 0.89 for soy milk and 0.44 for supplement.
In conclusions, the results in this study suggest that bioavailability of isoflavones are different between equol producers and non-producers. Competing interest The authors declare that they have no competing interests. Authors' contributions AM and TG conceived and designed the study; AM conducted the trials and collected the data, wrote the manuscript, analyzed the data, and interpreted the data; TG supervised the conduct of the trial and data collection and provided advice on the manuscript; CS provided advice on the quantitative analysis and interpreted the data; KS supervised the conduct of the study, provided advice on the manuscript; HS provided advice on the analytical method. All authors contributed intellectually to the manuscript and reviewed the drafts. All authors have read and approved the final manuscript. Acknowledgments The author thanks Dr. Michiko Yasuda (Graduate School of Nutritional and Environmental Sciences, University of Shizuoka) for
A. Miura et al. / Journal of Nutrition & Intermediary Metabolism 6 (2016) 41e47
advice on the manuscript and Ms. Marina Suzuki (Faculty of Health Promotional Sciences, Tokoha University) for cooperation with the cooking of the standard meal. Finally, I greatly appreciate the assistance of Dr. Sachio Takase PhD (Professor Emeritus of the University of Shizuoka, Nagasaki Prefectural University) and Forte Science Communications for guidance in the translation of my manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] Yamori Y, Miura A, Taira K. Implications from and for food cultures for cardiovascular diseases: Japanese food, particularly Okinawan diets. 2001 Asia Pac J. Clin. Nutr. 2001;10:144e5. [2] Yamori Y, Moriguchi EH, Teramoto T, Miura A, Fukui Y, Honda KI, Fukui M, Nara Y, Taira K, Moriguchi Y. Soybean isoflavones reduce postmenopausal bone resorption in female Japanese immigrants in Brazil: a ten-week study. J. Am. Coll. Nutr. 2002;21:560e3. [3] Messina M, Watanabe S, Setchell KD. Report on the 8th international symposium on the role of soy in health promotion and chronic disease prevention and treatment. J. Nutr. 2009;139:796Se802S. [4] Hendrich S. Bioavailability of isoflavones. J. Chromatogr. B 2002;777:203e10. [5] IL1 Nielsen, Williamson G. Review of the factors affecting bioavailability of soy isoflavones in humans. Nutr. Cancer 2007;57:1e10. R, Petrlova J, Potesil D, Adam V, Stiborov [6] Klejdus B, Mikelova a M, Hodek P, Vacek J, Kizek R, Kub an V. Evaluation of isoflavone aglycon and glycoside distribution in soy plants and soybeans by fast column high-performance liquid chromatography coupled with a diode-array detector. J. Agric. Food Chem. 2005;27:5848e52. [7] Murphy PA, Song T, Buseman G, Barua K, Beecher GR, Trainer D, Holden J. Isoflavones in retail and institutional soy foods. J. Agric. Food Chem. 1999;47: 2697e704. [8] Wang HJ, Murphy PA. Isoflavone content in commercial soybean foods. J. Agric. Food Chem. 1994;42:1666e73. [9] Wang H, Murphy P. A. Isoflavone composition of American and Japanese soybeans in Iowa: effects of variety, crop year, and location. J. Agric. Food Chem. 1994;42:1674e7. [10] Day AJ, DuPont MS, Ridley S, Rhodes M, Rhodes MJ, Morgan MR, Williamson G. Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity. FEBS Lett. 1998;436:71e5. [11] Lampe JW, Chang JL. Inter individual differences in phytochemical metabolism and disposition. Semin Cancer Biol. 2007;17:347e53. [12] Lampe JW. Interindividual differences in response to plant-based diets: implications for cancer risk. Am. J. Clin. Nutr. 2009;89:1553Se7S. [13] Lampe JW. Is equol the key to the efficacy of soy foods? Am. J. Clin. Nutr. 2009;89:1664Se7S. [14] Frankenfeld CL, Atkinson C, Thomas WK, Goode EL, Gonzalez A, Jokela T, W€ ah€ al€ a K, Schwartz SM, Li SS, et al. Familial correlations, segregation analysis, and nongenetic correlates of soy isoflavone-metabolizing phenotypes. Exp. Biol. Med. (Maywood) 2004;229:902e13.
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