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Nutrition and Cancer

Plasma Phytoestrogens Are Not Altered by Probiotic Consumption in Postmenopausal Women with and without a History of Breast Cancer¹

Jennifer A. Nettleton, Kristin A. Greany, William Thomas^*, Kerry E. Wangen, Herman Adlercreutz and Mindy S. Kurzer²

Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108; the ^* Division of Biostatistics, University of Minnesota, Minneapolis, MN 55407; and Folkhälsan Research Center and the Division of Clinical Chemistry, Biomedicum, 00014 University of Helsinki, Helsinki, Finland

²To whom correspondence should be addressed. E-mail: mkurzer{at}umn.edu.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Soy phytoestrogens were suggested to reduce the risk of a number of diseases including breast cancer. Given that these compounds are metabolized by bacteria, alteration of intestinal bacteria and enzymes may affect phytoestrogen metabolism. We hypothesized that probiotics, when consumed with soy protein, would increase plasma isoflavones, as well as equol producer frequency, in postmenopausal women. We further hypothesized that these effects would differ between women who have had breast cancer and women who have not. To test these hypotheses, 20 breast cancer survivors and 20 controls completed four 6-wk treatments in a randomized, crossover design: supplementation with soy protein (S) (26.6 ± 4.5 g protein, 44.4 ± 7.5 mg isoflavones/d); soy + probiotics (S+P) (10⁹ colony-forming units Lactobacillus acidophilus DDS+1 and Bifidobacterium longum, 15–30 mg fructooligosaccharide/d); milk protein (M) (26.6 ± 4.5 g protein/d); and milk + probiotics (M+P). Plasma phytoestrogen concentrations did not differ between controls and survivors, although genistein tended to be lower in survivors at baseline (P = 0.15), and during soy (P = 0.16) and milk protein (P = 0.16) consumption. As expected, soy consumption increased plasma phytoestrogen concentrations (P < 0.0001). Plasma phytoestrogen concentrations and the number of equol producers did not differ between the S and S+P diets. At the same time, plasma equol concentrations as well as urinary equol excretion in 2 subjects were more than 7-fold different between the 2 diets. These results indicate that this particular probiotic supplement does not generally affect plasma isoflavones, although the large differences between plasma and urinary equol in some subjects suggest that equol producer status may be modifiable in some individuals.

KEY WORDS: • soy • probiotic • plasma isoflavones

Isoflavones are suggested to be among the constituents responsible for many of the purported health benefits of soy. Most soyfoods contain isoflavones in glycosylated form, predominantly as genistin and daidzin, and in lesser amounts, glycitin (1). Intestinal and mucosal enzymes remove the glucose moiety and release the aglycones, genistein, daidzein, and glycitein, thus facilitating their absorption (2,3). Genistein and daidzein can be further metabolized to other nonsteroidal compounds such as p-ethyl phenol, O-desmethylangolensin (O-dma),³ and equol. Considerable interindividual variability exists in intestinal bacteria (4) and, subsequently, isoflavone metabolism and absorption (5–8).

Specific isoflavone metabolites are hypothesized to have important biological effects and thus may impart greater health benefits. Equol, one of the daidzein metabolites, was associated with lower breast cancer risk in pre- and postmenopausal women (9) and with a hormone profile that is consistent with reduced breast cancer risk in premenopausal women (10). Intestinal bacteria are essential to the metabolism of daidzein to equol, as demonstrated by studies in germ-free animals that do not excrete equol in their urine (11–13). When evaluated in Western populations, only about one third of the population excretes large quantities of equol (5,6,14–17), likely due to the large interindividual variability in intestinal flora (7,10). It was suggested that equol itself may directly exert cancer preventive effects and that differences in breast cancer risk are due in part to the interindividual variability in intestinal metabolism of dietary phytoestrogens. It was also hypothesized that the ability to produce equol may be a marker for a breast cancer-protective profile of gut bacteria (10).

Because diet influences the composition of intestinal flora, it may secondarily influence equol production. Researchers investigated whether certain components of the diet are associated with equol production (10,14–16). These studies did not reach a uniform conclusion, with some reporting no association with diet (10,15), and others reporting associations between equol and dietary fat or meat intake (18,19) and also between equol and dietary fiber (14,16). However, the addition of wheat bran to the diet of premenopausal women did not increase equol production or equol producer frequency, leading to the conclusion that the ability to produce notable quantities of equol is a characteristic that is relatively static and resistant to change (15).

Probiotic supplements and probiotic-containing food products are gaining great interest in the United States, Europe, and Japan (20). Probiotic formulations of bacteria are designed to increase the populations of bacteria that benefit host health, while minimizing pathogenic bacteria. Oral consumption of probiotic strains was shown to alter fecal enzyme activity in animals (21,22) and humans (23–25), including enzymes involved in isoflavone metabolism. For example, both Lactobacilli and Bifidobacteria have significant ß-glucosidase activity (7), and studies showed that specific strains of Escherichia coli metabolize isoflavone glycosides (26). Specific strains of probiotic bacteria such as Bifidobacterium longum, Lactobacillus casei, and L. helveticus were also shown to reduce populations of Clostridia (27–29), which are known to cleave the C-ring of isoflavones and other flavonoids. This could potentially alter the biological activity of these compounds (7,30,31).

For this study, we hypothesized that probiotic consumption together with soy protein would increase bioavailability and plasma concentrations of phytoestrogens, in particular the isoflavone metabolite equol. We further hypothesized that plasma phytoestrogen levels and equol producer frequency would be significantly lower in breast cancer survivors than in women with no history of breast cancer. To test this hypothesis, we conducted a randomized, crossover intervention in 40 postmenopausal women, 20 women with no cancer history and 20 breast cancer survivors, who consumed soy and milk protein with and without probiotic capsules, each for 6 wk.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Postmenopausal women with and without a history of breast cancer were recruited from Minneapolis/St. Paul, MN and surrounding communities. Telephone and in-person interviews were conducted to identify qualified individuals. Exclusionary criteria were as follows: use of hormone replacement therapy, selective estrogen receptor modulators, or antibiotics within the previous 3 mo; current use of medications or herbal therapies hypothesized to alter hormone metabolism; current tobacco use; BMI < 18 or > 36 kg/m²; family history of breast cancer or personal history of other cancer (women in control group); past or current chemotherapy treatment; regular consumption of soy-containing foods. All subjects were postmenopausal, defined as cessation of menstruation at least 1 y before enrollment and follicle-stimulating hormone concentration > 35 µg/L. Potential subjects underwent a health screen to verify satisfactory health status and gave documentation of a negative mammogram within the previous 12 mo. Twenty women without a history of cancer (controls) and 20 women successfully treated for breast cancer (survivors) participated in the study.

    Experimental design and dietary treatments. The University of Minnesota Institutional Review Board: Human Subjects Committee and the Institutional Review Board of the United States Army Medical Research and Materiel Command approved the study protocol. Subjects were asked to complete four 42-d diet treatments in random order: soy protein isolate (S) (Solae); milk protein isolate (M) (Solae); soy protein isolate + 3 probiotic capsules (DDS Plus, UAS Laboratories)/d (S+P); milk protein isolate + 3 probiotic capsules/d (M+P). The diet periods were separated by 2-wk washout periods. Soy and milk protein isolates provided 0.38 g protein/(kg body weight · d) (26.6 ± 4.5 g protein/d), and the soy protein provided 0.64 mg isoflavones/(kg body weight · d) (44.4 ± 7.5 mg isoflavones/d), expressed as aglycone equivalents (34.4% daidzein, 57.1% genistein, 8.5% glycitein, analyzed by Dr. Pat Murphy, Department of Food Science and Human Nutrition, Iowa State University). Probiotic capsules contained 10⁹ colony-forming units of L. acidophilus DDS+1 and B. longum and 15–30 mg fructooligosaccharide/d (analyzed by Dr. Joellen Feirtag, Department of Food Science and Nutrition, University of Minnesota). Quality assurance checks of all supplements were completed periodically during the study.

Subjects were free living and incorporated study supplements into their habitual diets. During each diet period, subjects were asked to exclude from their habitual diets alcohol, probiotic supplements, flaxseed, soy-containing products, and fermented dairy foods, and also to limit their consumption of legumes and nondairy fermented foods to <1 serving/wk. To maintain uniform background nutrient intakes, only vitamin and mineral supplements containing 100% Recommended Dietary Allowance for any nutrient were permitted.

Soy and milk protein isolates were similar in nutritional content, 80% energy as protein and 16–20% as carbohydrate, providing 557 ± 96 kJ/d (mean ± SD). The protein isolate allotment for each day was prepackaged and based on each subject’s most recently recorded weight. Subjects were encouraged to consume half of the day’s protein supplement during their morning meal and the remainder before their evening meal. Probiotic capsules were taken before breakfast each day. Capsule bottles were taken to each appointment (d 22 and 43 of each diet period) where remaining capsules were counted and the number recorded. As an additional measure of compliance, subjects independently documented their adherence to the study protocol on individualized recording calendars.

    Study procedures. Dietary records were maintained during the 3 d previous to the start of the study (baseline) and on d 40–42 of each diet period. Subjects were given oral and written instruction on how to properly detail their daily food consumption. Study coordinators reviewed all records with each subject after each 3-d series. Body weight was measured on study d 1 (baseline) and on d 22 and 43 of each diet period. Body composition was measured at baseline for descriptive purposes only. Skinfold thicknesses (triceps, biceps, subscapular, and suprailiac sites) were used in an age-specific equation to predict the percentage of body fat (32). A single dietitian performed measurements on all subjects. Fasting blood samples were drawn by venipuncture at a consistent time for each subject between 0630 and 1030 h at baseline and on d 43 of each diet period. Samples were drawn into heparinized tubes, centrifuged at 2000 x g, and plasma was separated. Sodium azide and ascorbic acid were added to reach a final concentration of 0.1% each. Samples were stored at –70°C. A single, 1-mL aliquot from each sample was later thawed, and 400 µL was divided into aliquots and lyophilized for subsequent phytoestrogen quantification. Three 24-h urine samples were collected on the last 3 d of each diet period. Collections from women consuming the soy protein-containing diets were later thawed, proportionally pooled, and 200 µL was divided into aliquots and lyophilized for subsequent equol analysis.

    Analytical methods. Diet records were analyzed for total energy, protein, carbohydrate, total fat, saturated fat, monounsaturated fat, polyunsaturated fat, cholesterol, and fiber (Nutritionist V, version 2.1, The Hearst Corporation). Plasma genistein, daidzein, O-dma, equol, and enterolactone and urinary equol were analyzed by competitive time-resolved fluoroimmunoassay (TR-FIA) as previously described (33–37). A GC-MS conversion factor was applied to the urinary equol TR-FIA values before data analysis (33). Plasma sample values falling below the lowest standard were assigned the value of the lowest standard (0.5 nmol/L for daidzein; 1.0 nmol/L for genistein, equol, and O-dma; 0.4 nmol/L for enterolactone). The intra-assay CV were 6.3, 7.1, 8.1, and 7.5%, and the interassay CV were 9.5, 9.9, 15.2, and 8.2% for genistein, daidzein, equol, and enterolactone, respectively. The intra-assay CV for plasma O-dma and urinary equol, which were assayed in 1 batch, were 15.8 and 8.4%, respectively.

    Statistical methods. Unpaired t tests were used to compare baseline characteristics and nutrient intakes between breast cancer survivors and controls. Unpaired t tests were also used to compare baseline characteristics and nutrient intakes between high-equol producers and low-equol producers. Standard repeated-measures ANOVA was used to assess the effects of the intervention. In this ANOVA, F-tests based on the error term for within-subject variability were used in determining effects of diet order, interactions between soy protein and probiotic supplements, and interactions between cancer status and diet treatment. F-tests based on the between-subject variability were employed to compare survivors and controls. Nutrient intake during the study was compared to baseline by linear contrasts within the repeated-measures ANOVA. Spearman correlation coefficients were used in determining the associations among plasma isoflavones and between plasma and urinary equol.

Subjects completing at least 2 diet periods were included in the final analyses. Data from only 2 diet periods were available for 1 subject and data from 3 diet periods for 3 other subjects (all breast cancer survivors). Therefore, the phytoestrogen concentrations of 195 (of 200 planned) samples were measured (baseline and intervention diet periods). A model including all diet treatments was used to compare both soy protein-containing diets (S and S+P) vs. both milk protein-containing diets (M and M+P) (155 observations). A reduced model including only soy diets was used to assess the effect of probiotic consumption on phytoestrogen concentrations (77 observations). Only subjects with data from both soy diets (n = 37) were included in statistical analyses assessing the effects of probiotic consumption on mean equol and when comparing equol producers and nonproducers.

Baseline characteristics and nutritional information are presented as least-squares mean (lsmean) ± SD. Due to the wide range of phytoestrogen values, data were analyzed on the log₁₀ scale and reported on the original scale as geometric means and 95% CI. Differences were considered significant at P < 0.05. Data were analyzed using SAS Proc GLM (SAS version 8.2, SAS Institute) (38).

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Baseline characteristics and dietary intakes did not differ between controls and breast cancer survivors, although controls tended to be younger than breast cancer survivors (P = 0.06) (Table 1). Cancer treatment varied within the group of breast cancer survivors. Six had lumpectomies plus radiation; 2 had lumpectomies plus radiation followed by a course of tamoxifen; 1 had a mastectomy followed by radiation treatment; and 11 had mastectomies with no further treatment. None had been treated with chemotherapy. At the time of diagnosis, 6 of the breast cancer survivors were premenopausal, 4 were perimenopausal, and 10 were postmenopausal. At baseline, there were no differences in plasma phytoestrogen concentrations between controls and survivors, although there was a trend toward lower genistein concentrations in survivors than in controls (P = 0.15) (Table 2).

During the soy diets (S and S+P combined), there were no significant differences in plasma phytoestrogen concentrations or urinary equol excretion between controls and breast cancer survivors, although plasma genistein concentrations tended to be lower (P = 0.16) and plasma O-dma concentrations tended to be greater (P = 0.12) in survivors (Table 3). There were also no significant differences in plasma phytoestrogens between the 2 groups during the milk protein diets (M and M+P combined). However, again there were trends toward lower plasma genistein (P = 0.16) and greater plasma equol concentrations in survivors than in controls during milk protein diets (P = 0.06) (Table 3).

View larger version (11K): [in this window] [in a new window]   FIGURE 1 Plasma and urinary equol of postmenopausal breast cancer survivors and controls after consumption of the S and S+P diets. Subjects’ 24-h urinary equol excretion and fasting plasma equol concentration are shown. Data from both S and S+P diets are displayed for subjects with values from both S and S+P diets (n = 37; 74 total observations shown). Equol producer during S diet only (); equol producer during S+P diet only (); equol producer = plasma equol > 15 nmol/L and 24-h urinary equol > 1500 nmol/L.

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
This is the first study to assess plasma phytoestrogen concentrations in breast cancer survivors and healthy control subjects under standardized dosing conditions. Interestingly, although there were no significant differences between controls and survivors, there appeared to be a pattern of lower levels of most phytoestrogens, especially genistein, in the survivors under all 3 conditions. Although the small sample size may have prevented us from detecting statistical significance, these results suggest that differences in phytoestrogen metabolism, independent of phytoestrogen consumption, may exist between cancer survivors and women who have never had cancer. Epidemiologic studies have generally reported an inverse association between urinary phytoestrogen excretion and breast cancer risk (9,39–41), perhaps due to differences in phytoestrogen consumption, although 1 study reported no significant associations (42). The overall pattern of lower phytoestrogen concentrations in cancer survivors may reflect differences in gut microflora that alter phytoestrogen metabolism and thus influence cancer risk. However, it must be noted that this was not a prospective study, and observations in the survivor group may not have preceded breast cancer development but instead may be a result of breast cancer or its treatment.

The numbers of controls and breast cancer survivors who were equol producers and nonproducers did not differ, and there were no differences in equol concentrations between controls and survivors at baseline or during consumption of the soy diets. These observations are similar to those of a study that found no difference in equol excretion between cases and controls (43), but it is in contrast to the study by Ingram et al. (9), which reported that women with breast cancer excreted significantly less equol than controls. The different nature of our study and theirs (intervention vs. epidemiologic) makes comparison of the studies difficult.

Previous studies that evaluated equol producer status have used differing levels of urinary and plasma concentrations to define equol producer status. In general, cutoff values were based on the unique distribution of data in each study, which likely depends on several factors such as the population studied, type of soy food, the study duration, and measurement methods. Although our study reported a lower equol producer frequency than other studies (10,14,44,45) (22%), this number was confirmed by both plasma and urinary data. These data suggest that fasting plasma samples requiring less subject burden and storage space can determine equol producer status as effectively as 24-h excretion determined from 3-d urine pools.

Probiotic supplementation did not affect plasma phytoestrogen concentrations or urinary equol excretion, although analyses of fecal microflora suggested that probiotic supplementation did significantly alter gut flora, most notably in increasing counts of Bifidobacteria (personal communication, Dr. Joellen Feirtag, Department of Food Science and Nutrition, University of Minnesota). Lampe et al. (15) made a similar, but more indirect, attempt to alter isoflavone metabolism by adding wheat bran to the diets of 26 women. They also reported no significant effects on metabolism. Our results, and those of Lampe et al. (15), suggest that isoflavone metabolism is likely to be quite consistent within an individual and difficult to change.

It is also possible that probiotic bacteria other than those used in this study would be successful in altering phytoestrogen metabolism. Efforts were made to determine which bacteria are responsible for isoflavone metabolism and equol production (13,26,31,46–50), but conclusive results have yet to be published. Human consumption of L. acidophilus and B. longum was shown to greatly increase ß-glucosidase activity (25), a change that would theoretically increase the release of aglycones and intestinal absorption of isoflavones. However, introduction of specific bacteria may change the overall composition of the intestinal flora and enzymatic activity in a manner that cannot be predicted and may differ by individual. Furthermore, even germ-free rats absorb isoflavones after hydrolysis of the glucosides by intestinal mucosal enzymes and in higher amounts than normal rats, presumably due to less bacterial destruction of the isoflavone molecules (13). Additionally, although at high doses hydrolysis of glycones to aglycones appears to be a rate-limiting step in availability (2), at levels given here (45 mg), hydrolysis is not likely to limit availability and absorption (51).

Although probiotic consumption did not alter plasma equol concentrations, urinary equol excretion, or the number of equol producers, there were 2 subjects whose equol values varied substantially. The plasma concentrations and urinary excretion of 1 subject increased approximately 9-fold and the concentrations and excretion of another subject decreased approximately 7-fold with probiotic consumption. In the subject whose equol concentrations decreased with probiotic, it appeared that the change represented a decrease in the conversion of daidzein to equol rather than an increase in equol degradation because both daidzein and O-dma concentrations increased relative to the soy without probiotic diet. It was also interesting that within the group of equol producers, 67% increased equol excretion when consuming the probiotic, although there were not consistent changes in daidzein or O-dma to explain how isoflavone metabolism as a whole was being affected. These results indicate that although equol production is quite consistent in most individuals, there may be a small subgroup whose ability to metabolize equol is highly variable, likely due to the unique nature of each individual’s indigenous gut microflora.

In this study, baseline characteristics or nutrient intakes did not differ between equol producers and nonproducers after consumption of each soy diet. Although studies reported fat or meat (18,19) and dietary fiber intake (14,16) to be positively associated with the ability to produce equol, other studies reported no associations with diet (10,15,43). The lack of agreement among these studies suggests that factors other than diet are most important in determining an individual’s ability to metabolize daidzein to equol.

This study is the first to report the effects of probiotic consumption on isoflavone metabolism. Mean isoflavone concentrations and equol producer frequency were unaffected by probiotic consumption, although probiotic consumption changed equol producer status in 2 of 37 subjects. In addition, 67% of the equol producers increased equol excretion when consuming probiotics. There were no differences in equol concentrations or number of equol producers between breast cancer survivors and healthy controls. However, there appeared to be a pattern toward lower phytoestrogen concentrations in survivors, suggesting the possibility that individual differences in phytoestrogen metabolism may influence breast cancer risk. These results indicate that fasting plasma equol concentrations and 24-h urinary equol excretion are equally useful in identifying equol producers. Studies of larger populations and with greater statistical power are warranted to verify differences in phytoestrogen metabolism between controls and survivors and increased equol excretion in equol producers consuming probiotic.

	ACKNOWLEDGMENTS

 
We are grateful to the 40 postmenopausal women who participated in our study and the staff at the General Clinical Research Center. We also thank Adile Samaletdin for her laboratory expertise and the companies Solae and UAS Laboratories for generously donating the study supplements.

	FOOTNOTES

 
¹ Supported by the U.S. Army Department of Defense Grant DAMD17-99-1-9297, General Clinical Research Center Grant MO1-RR00400 from the National Center for Research Resources, and the Minnesota Agricultural Experiment Station. The protein powders were generously donated by the Solae Company, St. Louis, Missouri. The probiotic capsules were generously donated by UAS Laboratories, Minnetonka, MN.

³ Abbreviations used: M, milk protein isolate; M+P, milk protein isolate plus probiotic; O-dma, O-desmethylangolensin; S, soy protein isolate; S+P, soy protein isolate plus probiotic.

Manuscript received 4 November 2003. Initial review completed 4 December 2003. Revision accepted 6 May 2004.

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TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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