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Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011
2To whom correspondence should be addressed. E-mail: shendric{at}iastate.edu.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • glucuronide • aglycone • isoflavone • humans
Since the detection and identification of isoflavones in animal and human diets and body fluids, many studies have been conducted on their biological roles in health and disease. The isoflavones daidzein, genistein, formononetin, biochanin A (1
) and glycitein (2
) are weak estrogens that may inhibit cancer cell growth by various mechanisms including stimulation of apoptosis and inhibition of angiogenesis (3
). Upon ingestion, isoflavone glucosides, the predominant forms of isoflavones in soybeans and traditional soy foods, undergo enzymatic hydrolysis in the intestine to aglycones before absorption. After absorption, glucuronide, sulfate and sulfoglucuronide conjugates of isoflavones are found in human urine (4
–6
). The aglycone forms are biologically active, as reported by many studies. But the activities of the major isoflavone sulfate and glucuronide conjugated metabolites have not been well studied.
Conjugation reactions are considered to be major detoxification processes, but conjugation products may also be biologically active. Weak estrogenic and human natural killer (NK)3 cell activation activities of daidzein and genistein glucuronides within the range of 0.1–10 µmol/L were noted in vitro (7
). Genistein at >5 µmol/L inhibited NK activity, but 50 µmol/L genistein glucuronide was required to inhibit NK activity. Peterson et al. (8
) detected genistein 7-sulfate in human breast cancer cell lines after incubation with [4-14C] genistein. But the hydroxylated and methylated metabolites, not the sulfate form, were shown to be the active forms of genistein in these cancer cell lines. The biological functions of isoflavone conjugates largely remain to be clarified.
The pattern of isoflavone metabolites produced in humans and animal models also needs clarification. Sfakianos et al. (9
) infused 14C-genistein into rat duodenum and recovered 70–75% of the dose from bile as 7-O-ß glucuronide conjugate. Recently, by feeding one man a single dose of soy protein isolate, Cimino et al. (10
) reported that 
86% of genistein and 75% of daidzein appearing in human urine were glucuronides. In general, glucuronides seem to predominate as isoflavone metabolites in vivo, but this requires confirmation.
Doerge et al. (11
) found that several UDP-glucuronyltransferase (UGT) isoforms catalyzed formation of 7- and 4'-glucuronides of isoflavones. The 1A10 isoform from human colonic microsomes was specific for genistein. UGT activity was similar with either daidzein or genistein as a substrate in human liver and kidney microsomes. The distribution of isoflavone glucuronides, sulfates and aglycones in vivo has not been well characterized. Few studies have been reported regarding conjugated isoflavone metabolites in women (12
). To investigate the proportion of glucuronide, sulfate and aglycone isoflavones in both plasma and urine after soy consumption and to determine whether isoflavone biotransformation differs with single vs. multiple doses, healthy women were fed soy milk powder with and without breakfast for 1 d or with breakfast for 6 d. To better assess intra-individual variation in isoflavone conjugation, six subjects were fed replicate soy milk doses in the early morning without breakfast; the replicates were separated by a 1-wk washout period. Such studies are important precursors to understanding the mechanisms of isoflavone action in vivo.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Soy milk powder (Now Foods, Glendale, IL) was purchased from a local grocery store. Isoflavone content was extracted and analyzed by HPLC at ISU, with 0.82 mg daidzein and 0.97 mg genistein/g, the total of all forms of each isoflavone normalized to the isoflavone aglycone (13
).
Venous blood samples were collected into EDTA-containing vacuum containers by a licensed medical technologist under stringent aseptic conditions. Blood samples of 10, 25 and 25 mL were collected at 3 h for Experiments 1–3, respectively. After soy milk dosing, urine was collected over 24 h from each subject for Experiments 1 and 2. In Experiment 3, urine was collected for 24 h on d 5, 6 and 7. The treatment of blood and urine samples was the same as described by Zhang et al. (14
).
Total daidzein and genistein concentrations in plasma and urine samples were analyzed by using ß-glucuronidase/sulfatase H-2 (EC 3.2.1.31, from Helix pomatia, Sigma Chemical, St. Louis, MO) as the deconjugation enzyme. To investigate the amount of isoflavone glucuronide alone, ß-glucuronidase B-3 (EC 3.2.1.31, from bovine liver, Sigma Chemical), which does not contain any detectable sulfatase activity, was used. The same number of glucuronidase units was used from both types of deconjugases in reaction mixes. For urine analysis, 5000 U of enzyme/2.5 mL sample was used; 2000 U/1.0 mL sample was used for plasma analysis. A preliminary analysis with enzyme at 7000 or 100,000 U/sample was tested on urine samples or 5000 U for plasma samples to verify by HPLC analysis (14
) that the reaction was complete with the amount selected. The preparation, extraction and HPLC methods were the same for both enzyme treatments, as stated (14
). Preparation of daidzein and genistein glucuronide standards for confirmation of those peak identities in samples that were not deconjugated before HPLC analysis was performed as described (7
).
For the study of isoflavone aglycone concentration in plasma and urine without deconjugation, 3-mL samples were mixed with 1.5 mL of 1.5 mol/L sodium acetate buffer (pH 5.5), internal standard 2,4,4'-trihydroxydeoxybenzoin (THB) and injected into a prewetted C18 Sep-Pak (Waters, Milford, MA) filter. After washing with 2 mL of 0.15 mol/L sodium acetate buffer (pH 3.0), plasma isoflavone aglycones were eluted with 4 mL methanol, dried under N2 and further dissolved with 0.25 mL of 80% methanol in water; urinary isoflavone aglycones were washed out with 2 mL of 80% methanol in water.
The percentages of daidzein and genistein as glucuronides were calculated by the following formula: [isoflavone aglycones present after B-3 (glucuronide only) hydrolysis - isoflavone aglycones present before hydrolysis]/total isoflavones (i.e., isoflavone aglycones present after H-2 glucuronidase-sulfatase hydrolysis) x 100. The percentages of isoflavone sulfates were calculated as: [isoflavone aglycones present after H-2 glucuronidase-sulfatase hydrolysis – isoflavones present after B-3 (glucuronide only) hydrolysis]/total isoflavones x 100.
Statistical analysis was applied with the SAS program (version 6.03, SAS Institute, Cary, NC). Across all three studies for plasma and urine (Tables 1
and 2
), differences between days were analyzed by ANOVA with Student’s t test. When the three studies were analyzed together, each study was treated as a block and analyzed by ANOVA with Student’s t test for differences between studies (Table 3
). When no significant differences were found between experiments, data were pooled for analysis of daidzein vs. genistein concentrations of urine and plasma. The differences between the percentage of glucuronide in urine and plasma and between daidzein and genistein were determined by Student’s t test. A P-value 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). All of these 24-h samples contained significantly more isoflavones than did urine samples collected on d 7 in Experiment 3 (P < 0.05). No significant differences in urinary isoflavone excretion (P = 0.12) were found between d 5 and 6 in Experiment 3. With soy milk intake at breakfast, the mean total excretions of daidzein and genistein were 58.2 ± 11.5% and 22.3 ± 11.6% of ingested doses, respectively, which were significantly different (P < 0.001). Isoflavone aglycones were also detected in the samples. No significant differences in 24-h urinary excretion of aglycones were found among the three studies. Total daidzein and genistein aglycones excreted in 24-h urine samples after soy milk was fed at breakfast were 2.5 ± 0.9 and 1.2 ± 0.6% of ingested dose, respectively.
At 3 h after soy milk feeding, the plasma concentrations of total daidzein and genistein after glucuronidase-sulfatase hydrolysis were significantly greater (P < 0.05) in Experiment 1 than in Experiments 2 or 3 (Table 2
). No significant differences in plasma isoflavone concentrations were found between d 5 and 6 in Experiment 3 or between Experiments 2 and 3. In all three studies, plasma daidzein was significantly greater than plasma genistein (P < 0.001) at 3 h after soy milk consumption. In Experiment 1, the total amount of plasma obtained was not enough for aglycone isoflavone analysis without prior enzymatic hydrolysis by our method. In the other two experiments, we were able to analyze aglycone isoflavone concentration by increasing blood sample volume. Again, no significant differences were found between plasma drawn on d 5 and 6 in Experiment 3 and between Experiments 2 and 3, and the amounts of daidzein and genistein found were significantly different (P < 0.001). The mean plasma daidzein and genistein aglycone concentrations were 0.47 ± 0.09 and 0.11 ± 0.05 µmol/L, respectively.
The percentage of isoflavone glucuronide was calculated by subtracting isoflavone aglycones present without enzymatic hydrolysis from isoflavone measured after enzyme hydrolysis with B-3 sulfatase-free glucuronidase. The amount obtained from this calculation was divided by that seen after hydrolysis by glucuronidase/sulfatase and then multiplied by 100. With no significant differences among the three studies, percentages of urinary isoflavone glucuronide or aglycone were combined (Table 3)
. For plasma, only Experiments 2 and 3 were combined. In both plasma and urine, percentages of total daidzein as glucuronide (DG, 62 and 73% respectively) were significantly greater (P < 0.01) than genistein glucuronide (GG, 53 and 71% respectively). The percentage of genistein aglycone (26%) was significantly greater (P < 0.05) than that of daidzein aglycone (18%) in plasma, but not in urine (5 and 4%, respectively). Comparing plasma and urine, the percentages of both DG and GG in plasma were significantly less (P < 0.05) than in urine. Conversely, the percentages of daidzein and genistein aglycone in plasma were greater than in urine.
Dihydrodaidzein and dihydrogenistein were detected in urine only as minor metabolites (Fig. 1
). Equol was detected only in one subject’s urine in Experiment 3 and also as a minor metabolite.
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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in urine samples from women detected by HPLC with UV detection were similar to those (75 and 86%) detected by Cimino et al. (10
) in one man using HPLC-mass spectrometry (MS). However, in the same paper (10
), in the urine of female Sprague-Dawley rats, the proportions of genistein, genistein glucuronide and genistein sulfate were 48, 26 and 26%, respectively, of total isoflavones detected and daidzein, daidzein glucuronide and daidzein sulfate were present as 33, 17 and 50%, respectively, of the total isoflavones detected. The rats were fed 19 mg genistein and 9 mg daidzein/kg body. By HPLC/electrospray-MS, Holder et al. (15
) detected >90% of total isoflavones as genistein glucuronide in the blood in female Sprague-Dawley rats, 90–100 d old, fed 20–100 mg total isoflavones/kg body. (This estimate of isoflavone intake was based on our assumptions of a presumed body weight of 250 g and food intake of 20 g/d in rats fed 250-1250 mg total isoflavones/kg diet.) Cimino et al. (10
) did not report plasma isoflavone concentrations or specify the ages or body weights of the rats and Holder et al. (15
) did not report urinary isoflavones in their studies. Our glucuronidation results and calculated daidzein and genistein sulfates were in the same range as those reported for one man (10
), as stated above. Holder et al. (15
) reported no differences in glucuronidation between male and female rats. Thus, limited data in rats and humans suggest that there is little gender difference in isoflavone biotransformation. But two studies of rat urine and plasma isoflavone conjugation (10
,15
) differed markedly in their findings. Our results in women show a somewhat increased proportion of isoflavone conjugates in urine compared with plasma (Table 3)
. This is to be expected due to the conjugation of circulating isoflavones by the kidney en route to urinary excretion. There is no clear explanation of the difference between the two rat studies. Isoflavone doses were not markedly different. Perhaps an unreported effect of age was a factor. Also, somewhat different extraction and analysis methods were applied by the two research groups. The results of Holder et al. (15
) are fairly consistent with those of Sfakianos et al. (9
), who reported that 75% of genistein in blood and bile of rats measured within a few hours after an oral genistein dose was genistein glucuronide.
In addition to the conjugated isoflavones and isoflavone aglycones, we also detected dihydrodaidzein and dihydrogenistein in every participant in our studies (Fig. 1)
. Heinonen et al. (16
) detected both compounds in all subjects when they fed 3 soy bars/d for 2 wk; they found that dihydrodaidzein was the main metabolite of daidzein and dihydrogenistein was the major metabolite of genistein. Kelly et al. (17
) identified dihydrogenistein as a minor metabolite of genistein in urine in only 1 of 12 subjects after 40 g soy flour was consumed for two consecutive days. In our study, both dihydrodaidzein and dihydrogenistein appeared as minor metabolites of daidzein and genistein, respectively. These metabolites appeared in the urine after a single feeding as well as during six consecutive days of feeding. The greatest amount observed in all the studies was <0.3 and 0.1 µmol/L for dihydrodaidzein and dihydrogenistein, respectively. Apparently, the formation of dihydrodaidzein and dihydrogenistein differed from that of equol, which appeared after repeated isoflavone feeding as an adaptive metabolite from gut microflora in one subject (data not shown). Feeding length did not make any difference for the appearance of either dihydrodaidzein or dihydrogenistein in the current study. The different results (dihydroisoflavones as minor or major metabolites) observed by three different researchers may be due to differences in gut microflora among human populations. These three studies were performed in three different countries [Australia (17
), Finland (16
) and the United States, current study]. Isoflavone metabolism might well vary among people in different regions. However, no direct evidence on this point is available at present.
In Experiment 3, we did not see any significant differences in either urinary isoflavone excretion or 3-h plasma isoflavone concentrations between d 5 and 6. Xu (18
) also observed that urinary excretions of isoflavones on d 6 and 7 of continuous soy milk feeding were similar. With prolonged feeding, daily constancy seems to be achieved for isoflavone excretion, hence apparent absorption. We observed that 2 of 5 women consistently excreted a relatively greater proportion of isoflavones from urine on d 7 than did other subjects (data not shown). But we did not see large differences in urinary excretion and plasma concentrations of isoflavones among these three feeding protocols, which indicated that isoflavone metabolism was not affected by the minor differences in the three protocols used.
We noticed that in Experiment 1, greater plasma isoflavone concentrations were seen than in the other two experiments. This may be due to the feeding protocol. Subjects were limited to only soy milk at breakfast before the 3-h blood samples in Experiment 1, but had free access to breakfast foods along with soy milk in Experiments 2 and 3. As the only available energy source, soy milk may be digested rapidly and more isoflavones would enter the circulation earlier. However, comparing 24-h urine excretion, there were no significant differences among the three studies. Either the food consumption after 3 h blood sampling in Experiment 1 slowed isoflavone excretion, or food consumption limited isoflavone conjugation and excretion. The metabolism of isoflavones by gut microflora seemingly was not influenced by meal intake patterns, which should be further studied.
The soy milk powder we used contained slightly higher genistein (0.97 mg/g soy) than daidzein (0.82 mg/g soy). In our previous study (14
), 6 h after a soy-containing meal, the plasma isoflavone concentrations paralleled the concentrations in the soy foods, with genistein concentration higher than that of daidzein. Also, during in vitro synthesis of isoflavone glucuronides (7
), genistein was a better substrate for UGT than was daidzein. Watanabe et al. (19
) also found that plasma concentration of daidzein was always lower than that of genistein over 72 h after feeding 60 g kinako (103 µmol daidzein and 112 µmol genistein). There is no evidence that biotransformation activity changes over the course of a day given normal dietary conditions. In our studies, plasma genistein concentrations were lower than daidzein 3 h after dosing (Table 2)
. This plasma data paralleled the urinary excretion pattern over 24 h (the time course for near total elimination of a single dose of isoflavones). Thus either urinary isoflavone excretion or 3-h plasma isoflavone concentrations may be a good reflection of overall isoflavone absorption. But detailed studies of plasma isoflavone concentrations over time showed that for purified genistein and daidzein glucosides, peak plasma concentrations were achieved at 9 h after ingestion, and both isoflavones had similar total plasma concentrations over time of 18–20 µmol(L · h) (20
). It may be that plasma kinetics after feeding isoflavones in a protein matrix as in our studies differed from feeding purified isoflavones, or that our subjects differed from those studied by Setchell et al. (20
) in gut microbial composition or other characteristics that might alter isoflavone bioavailability (21
).
Genistein was rapidly glucuronidated in the gastrointestinal mucosa in rats (9
). The proportion of both isoflavone glucuronides and aglycones in plasma differed significantly from urine in the current studies, with relatively higher percentages of aglycone but lower glucuronides found in plasma than in urine, showing that other tissues were also important glucuronidation sites for isoflavones.
Two recent studies of human biotransformation of isoflavones examined the reaction of isoflavone conjugates with specific deconjugases, sulfatase (12
) and glucuronidase (the present study). Shelnutt et al. (12
) noted that measuring the sulfates after specific sulfatase reaction rather than estimating sulfates by difference between total glucuronides and isoflavone glucuronides (our method) gave a more accurate picture of the pharmacokinetics of the isoflavone sulfates. But the difference in half-life (one third faster with measured sulfates vs. calculated sulfates) would not greatly change the overall picture of isoflavone metabolism. Direct measurement of urine and plasma isoflavone glucuronides and sulfates, which would be the "gold standard" for understanding isoflavone metabolism, will depend upon synthesis of the appropriate standards. We have accomplished synthesis of genistein and daidzein glucuronides (7
) but not isoflavone sulfates.
In this study, with three different feeding protocols, we demonstrated that women metabolize isoflavones relatively consistently. Glucuronides are the predominant metabolites of isoflavones. Given our previous findings of biological effects of isoflavone glucuronides, these metabolites deserve further study with respect to the mechanisms of cholesterol lowering (22
), anticancer (23
) and antiosteoporotic effects (24
) of isoflavones. However, considerable amounts of aglycone isoflavones appeared in plasma as well. In studying biological effects of isoflavones, nutritionally relevant concentrations of isoflavones and their major metabolites should be considered.
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FOOTNOTES |
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3 Abbreviations used: BMI, body mass index; DG, daidzein glucuronide; GG, genistein glucuronide; MS, mass spectrometry; NK, natural killer cell; THB, 2,4,4'-trihydroxydeoxybenzoin; UGT, UDP-glucuronosyltransferase.
Manuscript received 11 June 2002. Initial review completed 26 July 2002. Revision accepted 4 November 2002.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Kuiper, G.G.J.M., Lemmen, J. G., Carllson, B., Corton, J. C., Safe, S. H., van der Saag, P. T., van der Burg, B. & Gustafsson, J.-A. (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 139:4252-4263.
2. Song, T. T., Hendrich, S. & Murphy, P. A. (1999) Estrogenic activity of glycitein, a soybean isoflavone. J. Agric. Food Chem. 47:1607-1610.[Medline]
3. Zhou, J.-R., Mukherjee, P., Gugger, E. T., Tanaka, T., Blackburn, G. L. & Clinton, S. K. (1998) Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis. Cancer Res. 58:5231-5328.
4. Adlercreutz, H., Fotsis, T., Lampe, J., Wähälä, K., Mäkelä, T., Brunow, G. & Hase, T. (1993) Quantitative determination of lignans and isoflavonoids in plasma of omnivorous and vegetarian women by isotope dilution gas chromatography-mass spectrometry. Scand. J. Clin. Lab. Investig. 53(suppl. 215):5-18.
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7. Zhang, Y., Song, T. T., Cunnick, J. E., Murphy, P. A. & Hendrich, S. (1999) Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells at nutritional relevant concentrations. J. Nutr. 129:399-405.
8. Peterson, T. G., Ji, G. P., Kirk, M., Coward, L., Falany, C. N. & Barnes, S. (1998) Metabolism of the isoflavones genistein and biochanin A in human breast cancer cell lines. Am. J Clin. Nutr. 68:1505S-1511S.[Abstract]
9. Sfakianos, J., Coward, L., Kirk, M. & Barnes, S. (1997) Intestinal uptake and biliary excretion of the isoflavone genistein in rats. J. Nutr. 127:1260-1268.
10. Cimino, C. O., Shelnutt, S. R., Ronis, M. J. J. & Badger, T. M. (1999) An LC-MS method to determine concentrations of isoflavones and their sulfate and glucuronide conjugates in urine. Clin. Chim. Acta 287:69-82.[Medline]
11. Doerge, D. R., Chang, H. C., Churchwell, M. I. & Holder, C. L. (2000) Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry. Drug Metab. Dispos. 28:298-307.
12. Shelnutt, S. R., Cimino, C. O., Wiggins, P. A. & Badger, T. M. (2000) Urinary pharmacokinetics of the glucuronide and sulfate conjugates of genistein and daidzein. Cancer Epidemiol. Biomark. Prev. 9:413-419.
13. Wang, H. J. & Murphy, P. A. (1994) Isoflavone content in commercial soybean foods. J. Agric. Food Chem. 42:1666-1673.
14. Zhang, Y., Wang, G.-J., Song, T. T., Murphy, P. A. & Hendrich, S. (1999b) Urinary disposition of the soybean isoflavones daidzein, genistein and glycitein differs among humans with moderate fecal isoflavone degradation activity. J. Nutr. 129:957-962.
15. Holder, C. L., Churchwell, M. I. & Doerge, D. R. (1999) Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS. J. Agric. Food Chem. 47:3764-3770.[Medline]
16. Heinonen, S., Wähälä, K. & Adlercreutz, H. (1999) Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6'-OH-O-DMA, and cis-4-OH-equol in human urine by gas chromatography-mass spectroscopy using authentic reference compounds. Anal. Biochem. 274:211-219.[Medline]
17. Kelly, G. E., Nelson, C., Waring, M. A., Joannou, G. E. & Reeder, A. Y. (1993) Metabolites of dietary (soya) isoflavones in human urine. Clin. Chim. Acta 223:9-22.[Medline]
18. Xu, X. (1994) Human isoflavone bioavailability and natural killer cell activity after seven day soymilk feeding. Human Bioavailability and Health Protective Effects of Soy Isoflavones 1994:106-128 Doctoral thesis, Iowa State University Ames, Iowa.
19. Watanabe, S., Yamaguchi, M., Sobue, T., Takahashi, T., Miura, T., Arai, Y., Mazur, W., Wähälä, K. & Adlercreutz, H. (1997) Pharmacokinetics of soybean isoflavones in plasma, urine and feces of men after ingestion of 60 g baked soybean powder (kinako). J. Nutr. 128:1710-1715.
20. Setchell, K.D.R., Brown, N. M., Desai, P., Zimmer-Nechemias, L., Wolfe, B. E., 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.
21. Xu, X., Harris, K., Wang, H.-J., Murphy, P. & Hendrich, S. (1995) Bioavailability of soybean isoflavones depends upon gut microflora in women. J. Nutr. 125:2307-2315.
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23. Barnes, S., Peterson, G., Grubbs, C. & Setchell, K. (1994) Potential role of dietary isoflavones in the prevention of cancer. Jacobs, M. M. eds. Diet and Cancer: Markers, Prevention and Treatment 1994:135-147 Plenum Press New York, NY. .
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