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Human Nutrition and Metabolism
Research Communication

In Vitro Incubation of Human Feces with Daidzein and Antibiotics Suggests Interindividual Differences in the Bacteria Responsible for Equol Production¹

Cancer Prevention Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 and ^* Bastyr University, Kenmore, WA 98028

²To whom correspondence should be addressed. E-mail: jlampe{at}fhcrc.org.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Daidzein can be metabolized to equol, dihydrodaidzein (DHD), and O-desmethylangolensin (ODMA) by intestinal bacteria. Only one third to one half of individuals produce equol, and evidence exists to support potential cancer-protective effects of equol production. We investigated the in vitro metabolism of daidzein by fecal bacteria and assessed the effect of several antibiotics on metabolism. Fresh or previously frozen feces from 7 equol producers and 6 nonproducers were incubated with daidzein, with or without antibiotics, for 5 d at 37°C. With the exception of one previously frozen sample, fecal inoculates from equol producers converted daidzein to equol. Conversion occurred under anaerobic, but not aerobic conditions. Fecal inoculates from equol nonproducers did not produce equol, but some produced ODMA and DHD. Between-subject differences in the effects of antibiotics on daidzein metabolism were apparent. Some antibiotics inhibited the production of equol but had no effect on DHD production. These results suggest that several bacteria may be involved in daidzein metabolism, and that they may differ among subjects. This simple in vitro system can facilitate the study of factors influencing equol production and minimize the need for animal models or human interventions. Furthermore, these analyses can be conducted on fecal samples that have been frozen and stored.

KEY WORDS: • antibiotic • bacteria • equol • isoflavone • soy

Daidzein, a soy isoflavone, can be metabolized to equol, dihydrodaidzein (DHD),³ and O-desmethylangolensin (ODMA). When presented with a soy challenge, 33–50% of healthy adults produce equol (1–3), and 80–90% produce ODMA (4,5). The clinical significance of these daidzein-metabolizing phenotypes remains to be established fully, but some data suggest that equol production can be beneficial, particularly in relation to markers of breast cancer risk (6–9).

Interindividual variability in equol production may be unique to humans; of the animals tested systematically, including rats, mice, and chimpanzees, all excrete equol (3). Intestinal bacteria play a key role in daidzein metabolism; young infants with underdeveloped gut microflora do not produce equol (10), germ-free animals do not produce equol or ODMA (11,12), and, in vitro, intestinal bacteria from equol-producers convert daidzein to equol (13–15). However, the bacteria involved in equol and ODMA production, and determinants of the ability of humans to harbor these bacteria, remain to be established.

Intestinal microfloral populations are influenced by many factors, including antibiotic use. The in vitro metabolism of daidzein was described previously (13–15), but no studies have investigated the impact of antibiotics within such a system. In addition, studies to date involved small numbers of subjects, and none investigated the use of fecal samples stored before incubation.

The aims of the present study were to investigate further the in vitro metabolism of daidzein, to test the effects of several classes of antibiotics on daidzein-metabolizing bacteria in vitro, and to determine the viability of daidzein-metabolizing bacteria after being frozen at -180°C.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The equol- and ODMA-producer status of study participants was determined using a 3-d soy challenge; briefly, participants consumed a serving of soy protein on 3 consecutive days, and collected a first-void urine sample on d 4 (1). Urinary equol and ODMA excretion (16) was used to classify participants as equol producers or nonproducers and as ODMA producers or nonproducers. Thirteen individuals (10 women and 3 men), comprising 7 equol producers and 6 nonproducers, provided fecal samples. Individuals were ineligible if they had taken antibiotics in the 3 mo before urine and fecal collections. One equol producer who provided fecal samples over a 2-y period took a course of sulfonamide antibiotics during this time. We verified that the fecal bacteria were still able to convert daidzein and DHD to equol in vitro before conducting any experiments for this study. Institutional Review Boards of the Fred Hutchinson Cancer Research Center and Bastyr University approved all study procedures, and all participants provided written informed consent.

Two equol producers and 1 nonproducer provided fresh fecal samples on several different occasions over 2 y; for experiments with fresh feces, samples were used within 1 h of collection. Samples from 7 equol producers (including the 2 who provided fresh samples on several different occasions) and 5 equol nonproducers were frozen and stored as follows: 1 part feces was added to 4 parts 10% glycerol in PBS and mixed on a vortex. Aliquots (1–2 mL) were stored in cryovials in a liquid nitrogen freezer (MVE) at -180°C. Mean time interval between sample collection and processing was 1 h (range 5 min to 2 h and 35 min). Samples were stored for at least 6 mo before use in the in vitro experiments.

In vitro experiments were based on the method of Chang and Nair (13) with minor modifications. Briefly, 100 mL autoclaved brain heart infusion (BHI) medium (37 g/L deionized water; Becton Dickinson) was supplemented with phylloquinone (0.05 mg; Sigma-Aldrich), heme (0.5 mg; Sigma-Aldrich), and L-cystine (50 mg; Sigma-Aldrich). Fresh feces (1–2 g) or 1–2 mL thawed feces was added to 10 or 5 mL BHI medium, respectively, and inverted to mix. This bacterial broth (100 µL) was added to 5 mL BHI media containing 39.4 µmol/L daidzein (Indofine Chemical) or DHD (generous gift from K. Wähälä, University of Helsinki, Finland). We tested several concentrations of daidzein (range 39.4–393.7 µmol/L); compared with higher concentrations, 39.4 µmol/L was most reproducible in terms of complete conversion of daidzein to equol after 5 d (data not shown). Experiments were carried out in duplicate or triplicate, and tubes were incubated at 37°C. To verify bacterial growth after incubation, the absorbance (490 nm) of 200 µL incubated media containing fecal inoculate was compared with the absorbance of 200 µL nonincubated media containing fecal inoculate.

    Initial and time-course experiments. To determine whether equol could be produced in this system, and to determine optimal time to production, we carried out a series of experiments using fresh fecal samples from 2 equol producers (subjects 1 and 2) and 1 nonproducer. Feces from subjects 1 and 2 were incubated with daidzein under aerobic and anaerobic conditions, and with DHD under anaerobic conditions. Feces from the equol nonproducer were incubated with daidzein and DHD under anaerobic conditions. The Gas Pak system (Becton Dickinson) was used to create an anaerobic environment. Aerobic conditions were achieved by shaking tubes at 220 rpm. Tubes were incubated for 5 d, with the aim of achieving maximal daidzein metabolism. For time-course experiments, fecal samples from subjects 1 and 2 were used in Expt. 1, and tubes were removed from the anaerobic chamber daily for 5 d. In Expt. 2, a fecal sample from subject 2 was used, and tubes were removed from the anaerobic chamber at 3, 6, 9, 12, 18, 24, and 30 h.

    Stored frozen samples. Frozen fecal samples from 7 equol-producers and 5 nonproducers were thawed and incubated with daidzein under anaerobic conditions for 5 d.

    Antibiotic experiments. Antibiotic solutions were prepared according to manufacturer instructions (Sigma-Aldrich and ICN Biomedicals). Concentrations were based primarily on minimum inhibitory concentrations for a wide range of bacterial species (17) (Tables 1 and 2). Antibiotics were added to the BHI media containing daidzein before the addition of the fecal inoculate. Fresh feces from subjects 1 and 2 were used to test the effects of 10 antibiotics on daidzein metabolism. The effects of Colistin (30 mg/L) and Kanamycin (100 mg/L) on daidzein metabolism by thawed feces from 5 equol producers and 5 nonproducers also were tested; the 30 mg/L Colistin was used as an antibiotic-treated positive control for equol production and was tested for its effects on ODMA production. An untreated positive control (feces and daidzein without antibiotics) was also included in each experiment. These experiments were carried out for 5 d under anaerobic conditions. Ampicillin and penicillin are labile; therefore, 48-h incubations also were carried out for these antibiotics.

    Data analysis. Paired t tests (PROC MEANS; SAS version 8.02; SAS Institute) were used to determine effects of antibiotic treatment on daidzein metabolism by thawed fecal samples. Differences with P 0.05 were considered significant.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Initial and time-course experiments. Feces from subjects 1 and 2 converted daidzein to equol under anaerobic, but not aerobic conditions. Under anaerobic conditions, feces from these subjects also converted DHD to equol. Feces from an equol nonproducer did not metabolize daidzein or DHD to equol or ODMA, despite the detection of ODMA in the urine (data not shown). Feces from subject 1 converted some daidzein to DHD and equol after 2 d (Fig. 1A), whereas feces from subject 2 rapidly converted daidzein to equol within 1 d (Fig. 1B). When a more thorough timed investigation was carried out with an additional fecal sample from subject 2, DHD and equol were present already at 9 h (data not shown). In both subjects, the initial disappearance of daidzein was not accompanied by an increase in either DHD or equol.

View larger version (20K): [in this window] [in a new window]   FIGURE 1 Anaerobic metabolism of daidzein by human fecal bacteria from two known equol producers, subject 1 (panel A) and subject 2 (panel B).

    Antibiotic experiments. Interindividual differences in effects of antibiotics on DHD and equol production were observed; for example, several antibiotics inhibited equol production by fresh feces from subject 1 but not subject 2 (Table 1). Similar differences were observed for the effects of Colistin and Kanamycin on thawed feces from equol producers (Table 2). Mean recovery of daidzein plus metabolites ranged from 31.4 to 33.9 µmol/L across treatments, and differences between treatments were not significant. Compared with no antibiotic treatment, the production of equol by thawed feces from equol producers tended to be lower with Colistin (P = 0.15) whereas, with Kanamycin, both equol production in equol producers (P = 0.05) and ODMA production in equol nonproducers (P = 0.03) were lower. Feces from subjects 5 (equol producer) and 11 (equol nonproducer) did not produce equol or ODMA even in the absence of antibiotics (Table 2).

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Using a simple in vitro system, we demonstrated that fresh, as well as previously frozen fecal samples from equol producers convert daidzein to equol, and that samples from equol nonproducers do not. We also showed between-subject differences in the effects of selected antibiotics on equol and DHD production, and that some antibiotics (e.g., Rifampicin and Kanamycin) inhibit the production of equol but do not affect the production of DHD by fecal bacteria from some subjects. Together, these findings suggest that the conversion of daidzein to DHD, and of DHD to equol, might be carried out by different bacteria, and that the bacteria involved may differ among individuals. These findings are in agreement with Hur et al. (18), who reported that an isolated strain of human fecal bacteria was able to convert daidzein to DHD, but not equol.

Our experiments were carried out over a period of 2 y, and several fecal samples were provided by 2 equol producers during this time. All samples converted daidzein to equol in vitro, irrespective of when they were collected. This is in keeping with our previous observations in which equol-producer status remained constant over a period of at least several months (1). In contrast, Rafii et al. (19) observed inconsistent equol production in vitro using fecal samples collected from the same individual over a period of 9 mo.

We observed interindividual differences in the time taken for fecal bacteria to metabolize daidzein to equol, i.e., bacteria from one subject took <24 h to completely convert daidzein to equol. In contrast, Rafii et al. (19) reported that 6 or 9 d were needed for complete metabolism of daidzein by fecal bacteria. Other studies, with smaller sample sizes, have reported either the disappearance of daidzein, or production of equol within 72 h (13,15,20). The lack of equol or ODMA production by thawed feces from subjects 5 and 11 might have been because the 5-d incubation was too short. Alternatively, processing and storage might have affected the viability of the bacteria, although turbidity data indicated good bacterial growth after incubation. Our time-course studies also revealed that the initial disappearance of daidzein was not accompanied by an initial increase in DHD or equol, and that levels of equol remained relatively stable after reaching a plateau. These findings suggest that other, yet to be identified metabolites might be produced in the pathway to equol, and that once equol has been produced, it is not further metabolized.

To our knowledge, this is the first study to show that daidzein-metabolizing bacteria remain viable after storage at -180°C. The ability to store and maintain the viability of these bacteria will enable investigators to carry out in vitro incubations while minimizing the burden on study participants. Furthermore, this in vitro system could be used to study the effects of bacterial populations on the metabolism of certain endogenous and exogenous components to which the gut is exposed.

In our study, equol production under anaerobic, but not aerobic conditions, and inhibition of DHD and equol production by Metronidazole [an antibiotic that targets primarily anaerobic bacteria (17,21)] suggest that the bacteria involved are strictly anaerobic. The inhibition of equol production by Metronidazole and Kanamycin is in agreement with recent data published by Blair et al. (22) who reported that administration of these antibiotics in monkeys significantly reduced plasma levels of equol.

With the exception of subjects 2 and 7, all participants had detectable levels of ODMA in their urine after the 3-d soy challenge. Thawed feces from 4 equol nonproducers, but no equol producers, converted some or all of the daidzein to ODMA. These findings are in agreement with Bowey et al. (12); rats inoculated with human fecal flora from an equol producer/ODMA producer excreted equol but not ODMA, and rats inoculated with fecal flora from an equol nonproducer/ODMA producer excreted ODMA but not equol. These findings suggest that in the presence of equol-producing bacteria, conversion of daidzein to ODMA under these experimental conditions is impaired. The reasons for this are unclear, but it is possible that the production of equol may inhibit the growth of the ODMA-producing bacteria, or that there may be competition for substrates between the equol- and ODMA-producing bacteria. Thus, a potential limitation of this in vitro system, as well as the rat model, is that it does not exactly represent daidzein metabolism in humans, especially in relation to ODMA production.

In conclusion, our findings provide insight into the complexity of determining the bacteria responsible for equol production in humans. The lack of daidzein metabolism under aerobic conditions, in combination with the effects of select antibiotics, supports a role of strictly anaerobic bacteria. Our data also suggest that equol is a final product of daidzein metabolism in individuals who harbor the equol-producing bacteria, and that several bacteria are likely involved in daidzein metabolism.

	ACKNOWLEDGMENTS

 
The authors thank Wendy Thomas and Heather Skor for their assistance with the isoflavone analyses.

	FOOTNOTES

 
¹ Supported by grants from the Fred Hutchinson Cancer Research Center and Bastyr University.

³ Abbreviations used: BHI, brain heart infusion; DHD, dihydrodaidzein; ODMA, O-desmethylangolensin.

Manuscript received 22 August 2003. Initial review completed 30 September 2003. Revision accepted 15 December 2003.

	LITERATURE CITED

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Atkinson, C. Articles by Lampe, J. W. Search for Related Content PubMed PubMed Citation Articles by Atkinson, C. Articles by Lampe, J. W.