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Baker Medical Research Institute, Melbourne, Australia and * Department of Clinical Dietetics and Human Nutrition, Josai University, Japan
2To whom correspondence should be addressed. E-mail: paul.nestel{at}baker.edu.au.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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25% of each isoflavone was recovered in urine, suggesting that similar amounts were absorbed irrespective of their glycoside/aglycone nature or the differing compositions of their sources (daidzein and genistein in soy and formononetin and biochanin in red clover). Although interindividual variability was high, there was less intraindividual variability; the amounts excreted when subjects consumed the two sources of isoflavone were correlated (r = 0.69; P = 0.007).
KEY WORDS: • isoflavones • absorption • red clover • soybeans • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 2
). This has been attributed in part to differences in the microbial population in the intestine because naturally occurring isoflavones are conjugated as glycosides that must be cleaved for absorption to take place. The differing composition of the preparations is a further issue because some supplements contain isolated isoflavones in their aglycone form, and the isoflavone content of soy products can also vary substantially. In soybeans, genistein, daidzein and glycetin are predominant, whereas in red clover, biochanin and formononetin (precursors of genistein and daidzein, respectively) may be the primary forms. Knowledge about their respective absorptions is therefore important; it is usually measured as the excretion in urine of the ingested forms and their major metabolites. The amounts excreted in urine correlate with isoflavone intake (3
,4
) although excreted isoflavones underestimate absorption because of the magnitude of unmeasured metabolites (2
).
Unconjugated isoflavones (aglycones) may be absorbed quantitatively to a greater extent than glycosides because the excretion of isoflavones after eating fermented soybean, in which most of the isoflavone is present as aglycones, has been reported to exceed that after the consumption of soybean glycosides (5
). Izumi et al. (6
) found higher levels of isoflavones in plasma after aglycone consumption than after glycoside ingested in pill form. By contrast, Setchell et al. (7
) reported greater bioavailability from glycosides than from aglycones on the basis of plasma areas under the curve kinetics.
The mix of isoflavones consumed may also influence absorption, metabolism or both because single-dose pharmacokinetics suggest that the bioavailability of genistein and daidzein differs in different mixes of the two isoflavones (8
). Intraindividual, as contrasted with interindividual variability has not been assessed adequately, although isoflavone excretion on two consecutive days gave values that were not significantly different in subjects drinking soy milk once daily (9
).
We conducted experiments comparing aglycones from red clover and glycosides from soy that also differed in their isoflavone composition. In a randomized, single-blind, crossover design, the two preparations were consumed for 2 wk each, separated by a 2-wk low isoflavone period that served as control and washout. Urinary excretion of the parent isoflavones was measured at the end of the three periods and the intraindividual correlation between the two active periods assessed.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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At the end of each 2-wk period, 3-d dietary records were obtained and analyzed with a computer-based program developed by the Anti-Cancer Council of Victoria (Melbourne, Australia). Approval to conduct the study was given by the Human Ethics Committee of The Alfred Group of Hospitals; all subjects gave consent.
Foods and isoflavones.
A major cereal company (Uncle Tobys, Rutherglen, Victoria, Australia) prepared three breakfast cereals of similar composition, odor and taste; these were kindly supplied by Dr. Richard Tupper. Two different types of isoflavone, one from soybean and the other from red clover, were added to each of two batches of cereal; the third batch served as control. A survey revealed that as many of the subjects differentiated the control from the isoflavone-enriched cereal incorrectly as correctly, justifying the single-blind categorization. Each 25-g portion of cereal (the daily serving) comprised 415 kJ, 2.5 g dietary fiber (equally divided between soluble and insoluble), 3.05 g protein, 1.65 g fat and 19 g carbohydrate (2.7 g sugar). The cereal was eaten once daily at 0800 ± 1 h, with fat-reduced milk.
The aim was to deliver 30 mg isoflavone/d; the final analysis showed that 30 mg/d was eaten as red clover aglycones and 28.5 mg/d from soybean glycosides (calculated as the aglycone). The isoflavone composition from soy provided 22.5 mg daidzein and 6 mg genistein, which resembles that in the hypocotyledon. Daily consumption of red clover isoflavones comprised mainly formononetin (16.9 mg) and biochanin (11.5 mg); very small amounts of daidzein (1.2 mg) and genistein (0.4 mg) were also included. Because formononetin and biochanin give rise to daidzein and genistein, respectively, the two isoflavone mixtures differed in the proportions of daidzein and genistein, although both contained relatively more daidzein. The red clover aglycones were prepared by Novogen, North Ryde, NSW, Australia and kindly supplied by Professor Alan Husband, Research Director.
Experimental design.
The study began with a 2-wk run-in period during which the subjects began a legume-free diet and were taught how to complete food diaries. Most of the subjects had been eating prudent fat-reduced diets and their habitual eating patterns were established as the basis of background diets. Regular physical activity was encouraged. Then followed the two test periods and the isoflavone-free period that was slotted between the test periods and served as control and washout. The two isoflavone periods occurred in random order.
Body weights were measured at the end of each 2-wk period. Glass containers with ascorbate as preservative were provided on the penultimate day with instructions to collect a precise 24-h urine sample beginning ± 1 h from the final intake of the cereal preferably at 0800 h. Uneaten packets of cereal were to be returned but this occurred rarely.
Laboratory analyses.
The four primary isoflavones, formononetin, biochanin, daidzein and genistein were measured in urine. Although this underestimates the clearance of total isoflavones, it serves adequately for the main purpose of the study, i.e., the comparison of excretion of the two sources of isoflavones. The isoflavones were measured by modification of published methods (10
,11
). Aliquots (10 mL) of urine were mixed with 100 mL glucuronidase and the mixture incubated for 24 h at 37°C. Isoflavones were eluted with 3 mL methanol from a C-18 solid phase extraction column (Waters, Sydney, Australia) and separated by HPLC. The HPLC system consisted of a 25 cm, 5 nm C-18 stationary phase column (Symmetry, Waters) and a gradient acetonitrile/water mobile phase. The limit of detection of the assay for each isoflavone was 5 ng/mL. The interassay CV was <15%.
Statistical analysis.
Medians were compared using Kruskal-Wallis ANOVA on ranks. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The amounts of isoflavones excreted during the final 24 h of the three periods are shown in Table 1
. At the end of the run-in period, only two subjects still showed measurable amounts of isoflavones in urine. Only small amounts were measured in the same two subjects after the control or washout phase (0.24 ± 0.6 mg/d). These individuals claimed to have avoided legumes but it might have reflected the widespread use of soy protein in processed foods. Total excretions did not differ when the subjects consumed the two test isoflavone mixtures; they were 6.85 ± 3.49 mg/d for soy and 7.82 ± 3.78 mg/d for red clover. Because the values were not normally distributed, the means with the 25–75% range are also shown.
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). After red clover consumption, all subjects showed some formononetin in the urine, whereas only 8 subjects excreted biochanin. However, there had been considerable conversion from the two parent isoflavones to daidzein and genistein. Although there was little daidzein and genistein in the ingested red clover, the ratios in the urine showed far greater amounts of daidzein than of formononetin (formononetin:daidzein, 0.19) and of genistein than of biochanin (biochanin:genistein, 0.27). As occurred with soy, more daidzein than genistein was excreted. The ratio of daidzein:genistein in the urine was 4.37 and that of formononetin:biochanin in the food, 1.47. The amounts excreted represented 25% of the ingested isoflavones. However, as discussed above, these represent minimal values.
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The large standard deviations and the 25–75% ranges reflected the relatively large interindividual variability in excretion (and presumably in absorption) of isoflavones. However, there was a significant intraindividual correlation between the amounts excreted during the two test periods (r = 0.69; P = 0.007), demonstrating that low and high excreters showed consistent metabolic responses to eating isoflavones.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The greater absorption of isoflavones from tempeh than from unfermented soybean reported by Hutchins (5
) may result from causes other than cleavage of glycosides through fermentation. Nevertheless, Izumi et al. (6
) also reported higher plasma concentrations of isoflavones consumed as aglycones than as glycosides in the form of pills rather than food. On the other hand, a single-dose, pharmacokinetic study suggested the opposite, i.e., the area under the curve for the plasma concentrations of genistein and daidzein showed greater bioavailability with the glycosides than the aglycones (7
). By contrast, our study showed equal apparent bioavailability from both sources of isoflavones. The issue is clearly not yet resolved but may be a function of the duration of isoflavone intake. The nature of the food in which the isoflavone is delivered also does not appear to influence absorption (12
).
We compared two moderately dissimilar mixes of primary isoflavones because they represented two commonly consumed sources, soy and red clover. These differences did not appear to influence excretion (absorption). Zhang et al. (13
) examined the different daidzein:genistein ratios in soy milk and soy germ and found that the patterns in plasma 6 h after the meals resembled that in the foods. This may not be the case, however, with single meals because Busby et al. (8
) observed significantly different excretion rates for both genistein and daidzein from a predominantly genistein-rich mixture vs. a 2:1 mix of the two isoflavones, a difference that the authors could not explain.
The total amounts excreted cannot be fully assessed. In this study, two common metabolites, equol and O-desmethylangloensin, were not measured. In this, as in our previous studies (3
,14
), 25% was recovered in urine, which is consistent with the amount (> 30%) reported by Setchell (2
). This may reflect in part a saturation effect at higher doses, although this would not apply at the present 30 mg/d dose. The true amounts that are absorbed are probably twice the 30% recovered in urine by routine measurements. Only small amounts are recovered in feces. Some individuals, the so-called high equol excreters, convert substantial amounts of daidzein to equol, leading to an inverse correlation between daidzein and equol excretions (12
). Although we did not measure equol, a potentially major metabolite, this is unlikely to have altered the conclusion that absorption of the aglycone and glycoside forms did not differ. Karr et al. (15
) found a linear relationship between soy isoflavone intake and excretion that was similar in high and low equol excreters. As in the present study, proportionately more daidzein than genistein was excreted for pharmacokinetic reasons that are not fully understood (16
).
The second aim of the study was to explore the intraindividual consistency of isoflavone absorption (excretion). Table 1
shows the large standard deviations for the group of 14 subjects, confirming substantial interindividual variability (1
). Xu et al. (9
) reported urinary excretion of 15–18% after a single dose of soybean milk and a 2.5-fold difference in isoflavone absorption among healthy individuals. An even greater range of responses (eightfold) was reported by Zhang et al. (13
) among only 13 subjects.
On the other hand, it was interesting to find a strong correlation in isoflavone excretion between the two treatments (r = 0.069). Subjects who excreted more isoflavone with one mix of isoflavones did so also with the other. This suggests that bioavailability and biological action will vary among individuals but will be more consistent for an individual.
Although the health benefits of isoflavones are not fully understood or validated, strategies that might increase absorption deserve exploration. The precise fractional absorption of ingested isoflavones is unknown but as shown here, is clearly low in many individuals. Individual isoflavones are likely to confer different health benefits but at least, as suggested by the present study, bioavailability differs only modestly.
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FOOTNOTES |
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Manuscript received 10 April 2002. Initial review completed 3 May 2002. Revision accepted 15 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Kelly, G. E., Joannou, G. E., Reeder, A.Y., Nelson, C. & Waring, M. A (1995) The variable metabolic response to dietary isoflavones in humans. Proc. Soc. Exp. Biol. Med. 208:40-43.[Medline]
2. Setchell, K. D. (1998) Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. Am. J. Clin. Nutr. 68(Suppl.):1333S-1346S.[Abstract]
3. Nestel, P. J., Pomeroy, S., Kay, S., Komesaroff, P., Behrsing, J., Cameron, J. D. & West, L. (1999) Isoflavones from red clover improve systemic arterial compliance but not plasma lipids in menopausal women. J. Clin. Endocrinol. Metab. 84:895-898.
4. Arai, Y., Uehara, M., Kimira, M., Eboshida, A., Adlercreutz, H. & Watanabe, S. (2000) Comparison of isoflavones among dietary intake, plasma concentration and urinary excretion for accurate estimation of phytoestrogen intake. J. Epidemiol. 10:127-135.[Medline]
5. Hutchins, A. M., Slavin, J. L. & Lampe, J. W. (1995) Urinary isoflavanoid phytoestrogen and lignan excretion after consumption of fermented and unfermented soy products. J. Am. Diet. Assoc. 95:545-551.[Medline]
6. Izumi, T., Piskula, M. K., Osawa, S., Obata, A., Tobe, K., Saito, M., Kataoka, S., Kubota, Y. & Kikuchi, M. (2000) Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J. Nutr. 130:1695-1699.
7. Setchell, K. D., Brown, N. M., Desai, P., Zimmer-Nechemias, L., Brashear, W.T., Kirschner, A. S., Cassidy, A. & Heubi, J. E. (2001) Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J. Nutr. 131:1362S-1375S.
8. Busby, M. G., Jeffcoat, A. R., Bloedon, L.T., Koch, M. A., Black, T., Dix, K. J., Heizer, W. D., Thomas, B. F., Hill, J. M., Crowell, J. A. & Zeisel, S. H. (2002) Clinical characteristics and pharmacokinetics of purified soy isoflavones: single-dose administration to healthy men. Am. J. Clin. Nutr. 75:126-136.
9. Xu, X., Wang, H-J., Murphy, P. A., Cook, L. & Hendrich, S. (1994) Daidzein is a more bioavailable soybean isoflavone than is genistein in adult women. J. Nutr. 124:825-832.
10. Franke, A. A., Custer, L. J., Cerna, C. M. & Narala, K. (1995) Rapid HPLC analysis of phytoestrogens from legumes and from human urine. Proc. Soc. Exp. Biol. Med. 208:18-26.[Medline]
11. Setchell, K. D., Welsh, M. B. & Lim, C. K. (1987) High-performance liquid chromatographic analysis of phytoestrogens in soy protein preparations with ultraviolet, electrochemical and thermospray mass spectrometric detection. J. Chromatogr. 386:315-323.[Medline]
12. Xu, X., Wang, H-J., Murphy, P. A. & Hendrich, S. (2000) Neither background diet nor type of soy food affects short-term isoflavone bioavailability in women. J. Nutr. 130:798-801.
13. Zhang, Y., Wang, H-J., Song, T. T., Murphy, P. A. & Hendrich, S. (1999) Urinary disposition of the soybean isoflavones daidzein, genistein and glycetin differs among humans with moderate fecal isoflavone degradation activity. J. Nutr. 129:957-962.
14. Nestel, P. J., Yamashita, T., Sasahara, T., Pomeroy, S., Dart, A., Komesaroff, P., Owen, A. & Abbey, M. (1997) Soy isoflavones improve arterial compliance but not plasma lipids in menopausal and perimenopausal women. Arterioscler. Thromb. Vasc. Biol. 17:3392-3398.
15. Karr, S. C., Lampe, J. W., Hutchins, A. M. & Slavin, J. L. (1997) Urinary isoflavonoid excretion in humans is dose dependent at low to moderate levels of soy-protein consumption. Am. J. Clin. Nutr. 66:46-51.
16. King, R. A. & Bursill, D. A. (1998) Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. Am. J. Clin. Nutr. 67:867-872.[Abstract]
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