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STRUCTURAL ELUCIDATION OF HYDROXYLATED METABOLITES OF THE ISOFLAVAN EQUOL BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

Institute of Nutritional Physiology, Federal Research Center for Nutrition and Food, Karlsruhe, Germany (C.E.R.); Department of Nutritional Toxicology, German Institute of Human Nutrition, Potsdam-Rehbrücke, Germany (H.R.G.); and Department of Food Chemistry, Institute of Nutritional Science, University of Potsdam, Potsdam-Rehbrücke, Germany (S.E.K.)

(Received March 31, 2005; Accepted September 28, 2005)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Equol has, as have other isoflavonoids, recently gained considerable interest due to its possible health effects. However, detailed studies on the metabolism of equol are scarce. Therefore, we investigated the phase I metabolism of equol using liver microsomes from Aroclor-treated male Wistar rats as well as from a male human. The identification of the metabolites formed was elucidated using high performance liquid chromatography (HPLC) with diode array detection, HPLC/atmospheric pressure ionization electrospray mass spectrometry, and gas chromatography-mass spectrometry, as well as reference compounds. (±)-Equol was converted to 11 metabolites by the liver microsomes from Aroclor-pretreated rats comprising three aromatic monohydroxylated and four aliphatic monohydroxylated as well as four dihydroxylated products. The main metabolite was identified as 3'-hydroxy-equol. Using human liver microsomes, equol was converted to six metabolites with 3'-hydroxy- and 6-hydroxy-equol as main products. Furthermore, the aliphatic hydroxylated metabolite 4-hydroxyequol, which was recently detected in human urine after soy consumption, was formed. On the basis of these findings, it is suggested that phase I metabolism of equol is part of a complex biotransformation of the soy isoflavone daidzein in humans in vivo.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Intestinal bacterial metabolism of the soy isoflavone daidzein to the isoflavan equol.

The metabolism of soy isoflavones is well documented. Daidzein is reduced to dihydrodaidzein by the gut microflora, which is further metabolized to equol and O-desmethylangolensin (Fig. 1) (Chang and Nair, 1995; Joannou et al., 1995). Despite the growing interest in the biological properties of equol, data on the pharmacokinetics and metabolism are scarce. There is only one study investigating the bioavailability of equol. Setchell et al. (2002) administered a single-bolus oral dose (25 mg) to one healthy adult. Maximal plasma concentrations were attained after 4 to 6 h, and the elimination half-life was determined to be 8.8 h. Equol exerted pharmacokinetic parameters similar to those of the other isoflavones; however, the plasma clearance in humans was much slower compared to its parent compound daidzein (CL/F = 6.9 l/h versus 17.5 l/h). The appearance of equol in plasma after ingestion of the parent compound daidzein is time-dependent on its production in the colon. Peak equol concentrations in humans occur 36 h post-ingestion of daidzein (Rowland et al., 2003).

To date, however, little is known about the metabolic fate of equol. So far, it is not known whether equol is a substrate for the hepatic cytochrome P450 (P450) enzymes or not. The goal of the present study is to elucidate the phase I metabolism by using liver microsomes from Aroclor-induced male Wistar rats and from a male human. The identification of the metabolites formed was elucidated using HPLC/diode array detection, HPLC/API-ES MS, GC/EI-MS, as well as reference compounds.

	Materials and Methods

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Chemicals. Daidzein was purchased from TCI (Tokyo, Japan). 6- and 3'-hydroxy-daidzein were obtained from Plantech (Reading, UK). (±)-Equol (7,4'-dihydroxyisoflavan), 6-hydroxy-equol (6,7,4'-trihydroxyisoflavan), and 3'-hydroxy-equol (7,3',4'-trihydroxyisoflavan) were synthesized in a microscale approach by reduction of the corresponding isoflavones (daidzein, 6-hydroxy-daidzein, and 3'-hydroxy-daidzein, respectively) as described by Wessely and Prillinger (1939) and Lamberton et al. (1978). The purity of daidzein and equol was >99% according to GC/MS analysis. NADP⁺ (ß-nicotinamide adenine dinucleotide phosphate), N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), deuterated N,O-bis-(trimethylsilyl)acetamide (d9-BSA), and dimethyl sulfoxide were obtained from Sigma-Aldrich (Taufkirchen, Germany). All other chemicals were of the highest grade available.

Preparation and Characterization of Rat Liver Microsomes and HLMs. Microsomes were prepared and pooled from the livers of 10 Aroclor-treated male Wistar rats according to standard procedures as described by Lake (1987). Treatment with Aroclor 1254 (one intraperitoneal injection on day 1 of a dose of 500 mg/kg body weight dissolved in sesame oil at 100 mg/ml, killed on day 6) was carried out to induce hepatic P450 enzymes. HLMs were a friendly gift of Prof. M. Metzler, University of Karlsruhe (Karlsruhe, Germany). They were prepared from a normal liver tissue sample of a middle-aged white man in the same way as described above. The liver sample was obtained with appropriate human use approvals. Protein concentrations were determined using the Pierce Chemical (Rockford, IL) bicinchoninic acid reagent. P450 concentrations were measured as described by Omura and Sato (1964). The rat liver microsomes were further characterized by the specific activities of major P450 enzymes as determined by the method of Burke et al. (1994). Hepatic microsomes from Aroclor-treated rats were found to contain 1.69 nmol of P450 per mg of protein. 7-Ethoxycoumarin O-deethylase, 7-ethoxyresorufin O-deethylase, and 7-pentoxyresorufin O-dealkylase activity was found to be 23.5, 15.1, and 0.22 nmol/mg protein/min, respectively. HLMs contained 0.32 nmol of P450 per mg of protein.

Incubation with Microsomes and Extraction. Standard incubation mixtures with rat liver microsomes contained 2 mg of microsomal protein, 100 nmol of equol (dissolved in 40 µl of DMSO) in a final volume of 2 ml of 50 mM potassium phosphate buffer, pH 7.4. After 2 min of preincubation at 37°C in a shaking water bath the reaction was started by adding the NADPH-generating system (3 mM MgCl₂, 1 mM NADP⁺, 8 mM DL-isocitrate, and 0.5 unit of isocitrate dehydrogenase) and stopped after 60 min by extraction four times with 2 ml of ice-cold ethyl acetate. Controls were carried out by omitting the NADPH-generating system or by using heat-inactivated microsomes. The organic solvent was dried under a nitrogen stream at room temperature. The residue was dissolved in 50 µl of methanol and 3950 µl of water and applied to a reversed-phase C-18 cartridge (Waters, Eschborn, Germany). The column was first rinsed with 5 ml of 20% aqueous methanol and equol, and the metabolites were then eluted with 5 ml of 70% aqueous methanol. The eluate was dried under nitrogen and the residues were dissolved in 200 µl of aqueous 50% methanol. The samples were analyzed immediately by HPLC. All glass tubes used in this study were presilanized with 5% (v/v) dimethyldichlorosilane in toluene to avoid loss of hydroxylated equol metabolites due to adsorption to the glass surface. Incubations with HLMs were carried out in the same way with the exception that the incubation mixtures contained 4 mg of microsomal protein and 50 nmol of equol.

HPLC Analysis. HPLC separation of the oxidative metabolites was carried out on a Prontosil (250 mm x 4.6 mm i.d., particle size 3 µm) reversed-phase column (Bischoff, Leonberg, Germany). The solvent system consisted of 0.1% formic acid in water (pH 3) (A) and acetonitrile (B) with the following linear gradient: from 15% to 35% B in 50 min, from 35% to 50% in 10 min, and from 50% to 60% in 30 min. The flow rate was 0.9 ml/min and the eluate was recorded with a diode array detector at 280 nm. Observed peaks were scanned between 190 and 400 nm.

GC/MS Analysis of Hydroxylated Metabolites. GC/MS was carried out on a Finnigan MAT system (gas chromatograph model GCQ connected to an ion trap mass detector) (Thermo Electron Corporation, Bremen, Germany). The metabolites were isolated by HPLC and freeze-dried. For GC/MS analysis, the dry residues were derivatized with BSTFA or d9-BSA for at least 2 h at room temperature. The TMS derivatives were analyzed using a nonpolar capillary column (MDN-5S, 30 m x 0.25 mm i.d., 0.25-µm film thickness; Supelco, Taufkirchen, Germany) and a linear temperature gradient (60°C for 1 min, then 30°C/min to 250°C, hold for 10 min, then 1°C/min to 275°C and hold for 5 min). The injector port temperature was 50°C. Mass spectra were obtained by electron impact (EI) ionization at 70 eV and an ion source temperature of 150°C. Full-scan spectra (mass range 50–750 atomic mass units) were recorded at a rate of 2 spectra per second. Furthermore, the selected ion mode was used for the detection of equol and its metabolites.

HPLC/MS Analysis. HPLC/MS analysis was performed on an HP 1100 series HPLC apparatus (Agilent Technologies, Waldbronn, Germany) equipped with an autoinjector, quaternary HPLC pump, column heater, UV detector, and HP ChemStation for data collection and handling. The HPLC apparatus was interfaced to an HP series 1100 mass selective detector equipped with an API-ES chamber. For the analysis of oxidative metabolites, conditions in the positive mode were as follows: capillary voltage, 4 kV; fragmentor voltage, 80 V; nebulizing pressure, 50 psi; drying gas temperature, 350°C; drying gas flow, 10 l/min. Data were collected using both the scan mode and selected ion monitoring mode. Spectra were scanned over a mass range of m/z 100 to 500 at 1.03 s/cycle.

	Results

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Formation and Separation of Rat Microsomal Metabolites of Equol. Equol (Fig. 1) was incubated with hepatic microsomes of Aroclor-treated male Wistar rats. Aroclor 1254 induces P450 enzymes, especially of the 1A but also of the 2B subfamily (Burke et al., 1985; Correia, 1995). Therefore, the use of Aroclor-induced microsomes increases both the variety of the metabolites formed and their yield, thus facilitating the elucidation of the structures.

The complete organic extract of each incubation was analyzed by reversed-phase HPLC with diode array detection. Equol was extensively metabolized. Eleven metabolites were clearly detectable by HPLC analysis (Fig. 2). These products were not observed when NADPH was omitted or heat-inactivated microsomes were used.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Representative HPLC chromatogram of equol metabolites formed by the incubation of equol with liver microsomes of Aroclor-induced male rats. Peak numbers refer to Table 1.

Approach for the Elucidation of Metabolite Structures. Use of Reference Compounds. 6-Hydroxy-equol and 3'-hydroxy-equol were synthesized in a microscale approach (see Materials and Methods) and used as reference compounds for cochromatography and comparison with mass spectra. These standards proved to be helpful tools for the identification of equol metabolites; e.g., equol metabolites, which are also generated in the microsomal metabolism of 3'-hydroxy-equol, must contain an additional hydroxyl group in position C-3' of the B-ring.

HPLC/MS and GC/MS Analysis. Mass spectrometry can be used to determine the molecular weight of the metabolites and to clarify the distribution of hydroxyl groups between the A- and the B-ring of the isoflavan molecule. GC/MS with EI ionization (70 eV) of trimethylsilylated (TMS) derivatives of the metabolites gave, in most of the cases, mass spectra with an intense molecular ion or [M⁺ – 15] ion (loss of a CH₃ radical) as demonstrated in Fig. 3. EI mass spectra can thus be used to determine the molecular weight of the metabolites. Furthermore, in contrast to genistein and daidzein, equol showed a distinct fragmentation pattern using EI ionization. To minimize the uncertainty in the interpretation of the mass spectra, we used BSTFA as well as d9-BSA for the derivatization. With BSTFA, every proton in the hydroxyl groups is substituted by a trimethylsilyl group (TMS; m/z = 73), whereas per-deuterated TMS groups (d9-TMS; m/z = 82) are formed when using d9-BSA. By comparison of the mass spectra of the derivatives, information about the number of hydroxyl groups in the molecule and about the structure of the formed fragments is obtained. Figure 3 shows the EI mass spectra of equol, 3'-hydroxyequol, and 6-hydroxy-equol after derivatization with BSTFA and d9-BSA. In Fig. 4, the proposed fragmentation pathway of the TMS derivative of equol is depicted. Important fragments are formed via retro Diels-Alder (rDA) reaction in the C-ring. The molecule is fragmented into a dien- (a) and an en- (b) component. It depends on the molecule which component is the main product; e.g. for equol and 3'-hydroxy-equol, the en-component is formed more exclusively (m/z 192 and 280, respectively), whereas in the case of 6-hydroxy-equol, the en- and the dien-component are formed to the same extent (m/z 192 and 282) (Fig. 3). Furthermore, it must be taken into account that the dien-component a might react to c by migration of the TMS group to the keto group. The rDA-fragments provide important information about the degree of the substitution at each phenolic ring.

View larger version (15K): [in this window] [in a new window]   FIG. 3. GC/EI-MS spectra of the TMS and per-deuterated d9-TMS derivatives of equol, 3'-, and 6-hydroxy-equol obtained after derivatization with BSTFA and d9-BSA. The mass shift caused by the deuterium labeling is a helpful tool for a detailed interpretation of the fragmentation pattern. Further information is given under Results (HPLC/MS and GC/MS Analysis). A detailed explanation of the EI-MS spectra of the TMS and the d9-TMS derivative of equol is shown in Fig. 4.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Proposed fragmentation pathways of equol after trimethylsilylation with BSTFA (a) and d9-BSA (b). The percentage of the fragment ion formed is given in parentheses.

Besides the rDA reaction, further fragmentation is observed in the C-ring. The fragment at m/z 206 in the mass spectrum of equol is assigned to be the alkene ionic radical d, which is stabilized by the +I-effect of the methyl and phenyl groups (Fig. 4). Mass spectra of A-ring-substituted equol derivatives show a fragment ion at m/z 295, which is proposed to occur via the fragmentation pathway shown in Fig. 5.

View larger version (10K): [in this window] [in a new window]   FIG. 5. Conceivable formation pathway of the fragment ion m/z 295, which is observed in A-ring hydroxylated equol derivatives. The m/z values of the TMS derivative of 6-hydroxy-equol (a) and the corresponding d9-TMS derivative (b) are given.

Valuable information about the location of the hydroxyl groups can also be obtained by HPLC/MS with positive API-ES if a relatively low fragmentor voltage of 80 V is used (see Materials and Methods). A higher fragmentor voltage leads to the complete breakdown of the molecule. In Fig. 6, the fragmentation pathway of equol and aromatic hydroxylated equol derivatives is depicted. The fragmentation pattern can be used to determine the number of hydroxyl groups in the A- or the B-ring. The loss of a hydroxystyrene derivative results in the ionic dien component , which gives information about the hydroxylation degree in the A-ring. The loss of a hydroxyphenol derivative leads to the fragment ß, which gives information about the number of hydroxyl groups in the B-ring.

View larger version (19K): [in this window] [in a new window]   FIG. 6. HPLC/API-ES MS fragmentation pattern of hydroxylated isoflavans. The fragment ions m/z 123 and 139 give an indication to the number of hydroxyl groups at the A-ring (fragment ), and the fragment ions m/z 133 and 149 to the number of hydroxyl groups in the B-ring (fragment ß).

Theoretical Considerations about the Stereochemistry of Aliphatic Hydroxylated Metabolites. Possible positions for the aromatic hydroxylation of equol by P450 enzymes are the positions C-8 and C-6 in the A-ring as well as C-3' (equivalent to C-5') in the B-ring. Possible positions for the aliphatic hydroxylation are C-2, C-3, and C-4 in the C-ring.

In contrast to genistein and daidzein, equol possesses a chiral center at C-3 in the C-ring. The synthesis of equol results in the mixture of the two enantiomers, R-(+)- and S-(–)-equol (Lamberton et al., 1978). With respect to the hydroxylation at the aromatic positions C-6, C-8, and C-3', as well as at the aliphatic position C-3, the corresponding R- and S-enantiomers are formed. Hydroxylation at the aliphatic positions C-2 and C-4 leads to the formation of a second asymmetric center in the C-ring, and four stereoisomers can theoretically be formed. Figure 7 depicts the four possible stereoisomers of 4-hydroxy-equol. In analogy, the corresponding products can be postulated after introduction of a hydroxyl group at C-2.

View larger version (22K): [in this window] [in a new window]   FIG. 7. The four possible stereoisomers of 4-hydroxy-equol. 4-Hydroxy-equol possesses two chiral centers (C-3 and C-4).

View larger version (15K): [in this window] [in a new window]   FIG. 8. Proposed fragmentation pathway of 3-hydroxy-equol. a, TMS derivative; b, d9-TMS derivative.

The GC/MS analysis of the TMS derivative of the major microsomal metabolite (peak 11, Fig. 2) shows a molecular ion at m/z 474 and a fragment ion m/z 280 indicating a monohydroxylated equol structure with two hydroxyl groups in the B-ring. The positive HPLC/API-ES mass spectrum displays a quasimolecular ion [M + 1]⁺ at m/z 256, indicating a monohydroxylation, and a diagnostic fragment ion at m/z 123, implying an unchanged A-ring. Furthermore, metabolite 11 coelutes with the synthesized reference compound 3'-hydroxy-equol. Taking these findings together, metabolite 11 can be identified as 3'-hydroxy-equol. It is the main product of the microsomal metabolism of equol and accounts for up to 60%, calculated on the basis of the peak area at = 280 nm.

Peak 10 was identified by cochromatography in GC/MS with the synthesized reference compound as 6-hydroxy-equol. The positive API-ES mass spectrum displays a quasimolecular ion [M + 1]⁺ at m/z 256 —which confirms the monohydroxylation—and a diagnostic fragment ion at m/z 139, implying a hydroxylated A-ring (Fig. 6).

Metabolite 9 represents an A-ring monohydroxylated isoflavan according to its mass spectral data (Table 1). The EI and API-ES mass spectra are found to have the same fragment ions as 6-hydroxy-equol (metabolite 10), which has already been identified. The spectra of these two metabolites only differ in the intensities of the fragment ions. The remaining possible positions for the introduction of an additional hydroxyl group in the A-ring are C-5 and C-8. Hydroxylation at C-5 would mean a hydroxylation in the meta position relative to the existing hydroxy group at C-7, which is very unusual and is, to our knowledge, so far not described. Therefore, we assign peak 9 to be 8-hydroxy-equol.

View larger version (9K): [in this window] [in a new window]   FIG. 9. GC/EI-MS spectra of 4-hydroxy-equol after trimethylsilylation with BSTFA (A) and d9-BSA (B). An explanation of the mass fragmentation pattern is given in Fig. 10 and is described in detail under Results.

View larger version (17K): [in this window] [in a new window]   FIG. 10. Proposed fragmentation pathway of 4-hydroxy-equol. a, TMS derivative; b, d9-TMS derivative.

The GC mass spectrum of the TMS derivatives of metabolite 4 lacks the ions m/z 192 and 177, respectively, or 179, which are minor fragments of all other metabolites and are derived by the formation of the en-component or a hydroxybenzylium ion (m/z 179). Furthermore, peak 4 additionally exhibits strong fragment ions at m/z 280 and m/z 268, which are formed by the introduction of a hydroxyl group at C-3. On the basis of these data, peak 4 is assigned to be 3-hydroxy-equol. Fig. 8 shows the proposed fragmentation pathway.

View larger version (10K): [in this window] [in a new window]   FIG. 11. HPLC/API-ES mass spectrum of metabolite 6. The most intense ion at m/z 137 (base peak) is based on the formation of an ionic 3-phenylethanal as shown in Fig. 12.

View larger version (17K): [in this window] [in a new window]   FIG. 12. Proposed fragmentation pathway of metabolite 6, identified as 2-hydroxy-equol according to the fragment ions derived by HPLC/API-ES MS analysis. The mass fragmentation spectrum is shown in Fig. 11.

View larger version (15K): [in this window] [in a new window]   FIG. 13. GC-selected ion chromatogram of the incubation of equol with HLM. *, 4-hydroxy-equol is overlapped by an unknown compound having the same retention time.

View larger version (18K): [in this window] [in a new window]   FIG. 14. Microsomal metabolites of equol (racemate) formed through aromatic and aliphatic hydroxylation catalyzed by P450 enzymes. Refer to Table 1 for the percentages formed of each metabolite under the incubation conditions used in this study. In the case of 2- and 4-hydroxy-equol, four possible stereoisomers can be formed due to the introduction of an additional chiral center at C-2 and C-4, respectively (see Fig. 7).

Metabolites 1, 2, and 3 are formed only in small yields. According to the API-ES mass spectra, they correspond to dihydroxylated equol derivatives. The isolated HPLC peaks degraded during GC/MS analysis. Therefore, the structures have not yet been identified. These three metabolites are also formed in the microsomal metabolism of 3'-hydroxy-equol, implying that peaks 1, 2, and 3 are monohydroxylated 3'-hydroxy-equol derivatives.

Human Microsomal Metabolites of Equol. HLMs have been used to gain information about the metabolites formed in vitro by humans in comparison to the rat. Equol is converted to six metabolites by HLMs. The main metabolites are the aromatic monohydroxylated products 3'- and 6-hydroxy-equol. Further metabolites are 8-hydroxyequol and both diastereomeric pairs of 4-hydroxy-equol (Fig. 13). In trace amounts, two monohydroxylated 3'-hydroxy-equol derivatives are found.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we have investigated the in vitro phase I metabolism of equol using pooled Aroclor-induced rat liver microsomes. Eleven metabolites were formed, separated by HPLC and GC, and identified with the help of their HPLC/API-ES and GC/EI-MS spectra (Fig. 9) and by comparison with reference compounds. Our results clearly indicate that equol is a substrate for P450 enzymes. A scheme of the identified metabolites is shown in Fig. 14. Preliminary experiments with HLM from one male individual suggest that the oxidative metabolism of equol may also be relevant to humans, although HLMs caused, in our experiments, compared with the Aroclor-induced rat liver microsomes, a less complex metabolite profile. This is not very surprising since Aroclor 1254 induces a variety of different P450 enzymes including, also, P450 enzymes that are usually not expressed in the liver; e.g., CYP 1A1. Furthermore, the amount of P450 enzyme per nanomole of equol substrate was higher in incubation mixtures with rat liver microsomes. However, in both cases, the rat and the human microsomal metabolism, 3'-hydroxy-equol, as well as 6- and 8-hydroxy-equol, and the aliphatic hydroxylated metabolite 4-hydroxy-equol are the main products. Further studies with pooled HLMs or with a larger number of different HLMs as well as with human recombinant P450 enzymes are needed to get additional information on the interindividual variation of the metabolite pattern in humans and on the P450 enzymes involved in the formation of the different metabolites.

4-Hydroxy-equol has already been identified by Kelly at al. (1993), Joannou et al. (1995), and Heinonen et al. (1999) in urine of human volunteers after the consumption of soy. The in vivo formation of 4-hydroxy-equol is proposed to occur via the reductive metabolism of daidzein to dihydrodaidzein and a further reduction step of the keto group in the C-ring of dihydrodaidzein to 4-hydroxy-equol (Fig. 1). Our in vitro experiments with rat liver microsomes and HLMs show that the formation of 4-hydroxy-equol can also occur via hydroxylation of equol by P450 enzymes. Perhaps both pathways are relevant for the in vivo situation. The same may hold for the aromatic hydroxylated equol derivatives formed by human and rat liver microsomes. Kulling et al. (2000, 2001) have been able to show that the soy isoflavones daidzein and genistein are good substrates for P450 enzymes in vitro. Furthermore, several mono- and dihydroxylated daidzein and genistein as well as monohydroxylated equol metabolites have been identified in vivo (Kulling et al., 2001; Heinonen et al., 2003). Therefore, the formation of the aromatic hydroxylated equol metabolites may occur via reduction of the corresponding hydroxylated isoflavones or via hydroxylation of equol by P450 enzymes as shown in this study. Further studies are needed to clarify the importance of these two metabolic pathways. It seems to be likely that both pathways are of relevance for the in vivo formation of the hydroxylated equol metabolites. Therefore, our findings might have a substantial impact on the understanding of the biotransformation of isoflavones.

View larger version (21K): [in this window] [in a new window]   FIG. 15. Proposed stereospecific formation of 4-hydroxy-equol. Daidzein is exclusively converted to the S-(–)-equol enantiomer by the gut microflora (Setchell et al., 2005). We assume that cis-4-hydroxy-equol, described as the intermediate metabolite in the gut bacterial metabolism, can also be formed via hydroxylation catalyzed by P450 enzymes. For further explanation, see Discussion.

The hydroxylated metabolites exert interesting biological properties: the anticancer activities of both 4-hydroxy-equol isomers have been studied by Wahala et al. (1997). Both isomers inhibit the growth of prostate cells (LNCaP 90) much more effectively than the parent compound daidzein. The activities were comparable to that of genistein; however, cis- and trans-4-hydroxy-equol were less cytotoxic. Regarding the antioxidant activity, it has been shown that equol is more potent than genistein or daidzein and that the hydroxylated metabolites of genistein and daidzein exert an even higher antioxidant efficacy than equol (Arora et al., 1998; Rimbach et al., 2003; Turner et al., 2004). Therefore, studies on the antioxidant capacity of the hydroxylated equol metabolites seem to be interesting. Reference compounds are needed to investigate the biological effects in detail.

In conclusion, this is the first study, to our knowledge, to show that equol is a substrate for P450 enzymes and is therefore not inevitably the metabolic end product of daidzein. It is well established that the biological activities of isoflavones are dramatically altered with the chemical structure. On the basis of these findings, it can be assumed that the hydroxylated products have biological properties other than those of the parent compound. Recent studies show that the formation of equol through the action of intestinal bacteria might play a central role regarding the chemopreventive potential of isoflavones (Frankenfeld et al., 2004). Therefore, it seems necessary to completely understand the metabolism pathways of isoflavones and to identify all metabolites that were formed during this process, even if these metabolites might appear in small quantities.

	Footnotes

 
This work was funded by the Deutsche Forschungsgemeinschaft (Grant Ku 1079/6-1).

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.004929.

ABBREVIATIONS: P450, cytochrome P450; API-ES, atmospheric pressure electrospray ionization; BSTFA, N,O-bis-(trimethylsilyl)trifluoroacetamide; d9-BSA, deuterated N,O-bis-(trimethylsilyl)acetamide; EI-MS, electron impact ionization mass spectrometry; GC, gas chromatography; GC/MS, gas chromatography/mass spectrometry; HLM, human liver microsome; HPLC, high performance liquid chromatography; rDA, retro Diels-Alder; TMS, trimethylsilyl.

Address correspondence to: Sabine E. Kulling, Arthur-Scheunert-Allee 114-116, Institute of Nutritional Science, Department of Food Chemistry, University of Potsdam, 14558 Nuthetal, Germany. E-mail: kulling{at}rz.uni-potsdam.de

	References

Top Abstract Materials and Methods Results Discussion References

 


Arora A, Nair MG, and Strasburg GM (1998) Antioxidant activities of isoflavones and their biological metabolites in a liposomal system. Arch Biochem Biophys 356: 133–141.[CrossRef][Medline]

Axelson M, Kirk DN, Farrant RD Cooley G, Lawson AM, and Setchell KD (1982) The identification of the weak oestrogen equol [7-hydroxy-3-(4'-hydroxyphenyl)-chroman] in human urine. Biochem J 201: 353–357.[Medline]

Burke MD, Thompson S, Elcombe CR, Halpert J, Haaparanta T, and Mayer RT (1985) Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochrome P-450. Biochem Pharmacol 34: 3337–3345.[CrossRef][Medline]

Burke MD, Thompson S, Weaver R, Wolf C, and Mayer R (1994) Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem Pharmacol 48: 923–936.[CrossRef][Medline]

Chang YC and Nair MG (1995) Metabolism of daidzein and genistein by intestinal bacteria. J Nat Prod 58: 1892–1896.[CrossRef][Medline]

Correia MA (1995) Rat and human liver cytochromes P450, in Cytochrome P450: Structure, Mechanism and Biochemistry (Ortiz de Montellano PR, ed) pp 607–630. Plenum Press, New York.

Frankenfeld CL, McTiernan A, Aiello EJ, Thomas WK, LaCroix K, Schramm J, Schwartz SM, Holt VL, and Lampe JW (2004) Mammographic density in relation to daidzein-metabolizing phenotypes in overweight, postmenopausal women. Cancer Epidemiol Biomarkers Prev 13: 1156–1162.[Abstract/Free Full Text]

Heinonen S, Wahala K, and 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 2742: 211–219.

Heinonen SM, Hoikkala A, Wahala K, and Adlercreutz H (2003) Metabolism of the soy isoflavones daidzein, genistein and glycitein in human subjects. Identification of new metabolites having an intact isoflavonoid skeleton. J Steroid Biochem Mol Biol 87: 285–299.[CrossRef][Medline]

Joannou GE, Kelly GE, Reeder AY, Waring M, and Nelson C (1995) A urinary profile study of dietary phytoestrogens. The identification and mode of metabolism of new isoflavonoids. J Steroid Biochem Mol Biol 54: 167–184.[CrossRef][Medline]

Kelly GE, Nelson C, Waring MA, Joannou GE, and Reeder AY (1993) Metabolites of dietary (soya) isoflavones in human urine. Clin Chim Acta 223: 9–22.[CrossRef][Medline]

Kostelac D, Rechkemmer G, and Briviba K (2003) Phytoestrogens modulate binding response of estrogen receptors alpha and beta to the estrogen response element. J Agric Food Chem 51: 7632–7635.[CrossRef][Medline]

Kulling SE, Honig DM, and Metzler M (2001) Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo. J Agric Food Chem 49: 3024–3033.[Medline]

Kulling SE, Honig DM, Simat TJ, and Metzler M (2000) Oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein. J Agric Food Chem 48: 4963–4972.[Medline]

Lake BG (1987) Preparation and characterization of microsomal fractions for studies on xenobiotic metabolism, in Biochemical Toxicology (Snell K and Mullock B eds), pp 138–215. IRL Press, Oxford.

Lamberton J, Suares H, and Watson K (1978) Catalytic hydrogenation of isoflavones. The preparation of (+/–)-equol and related isoflavans. Aust J Chem 31: 455–457.

Lund TD, Munson DJ, Haldy ME, Setchell KD, Lephart ED, and Handa RJ (2004) Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback. Biol Reprod 70: 1188–1195.[Abstract/Free Full Text]

Marrian GF and Haselwood GAD (1932) Equol, a new active phenol isolated from ketohydroxyestrin fraction of mare's urine. Biochem J 26: 1226–1232.

Morito K, Hirose T, Kinjo J, Hirakawa T, Okawa M, Nohara T, Ogawa S, Inoue S, Muramatsu M, and Masamune Y (2001) Interaction of phytoestrogens with estrogen receptors alpha and beta. Biol Pharm Bull 24: 351–356.[CrossRef][Medline]

Muthyala RS, Ju YH, Sheng S, Williams LD, Doerge DR, Katzenellenbogen BS, Helferich WG, and Katzenellenbogen JA (2004) Equol, a natural estrogenic metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta. Bioorg Med Chem 12: 1559–1567.[CrossRef][Medline]

Omura T and Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J Biol Chem 61: 461–467.

Rimbach G, De Pascual-Teresa S, Ewins BA, Matsugo S, Uchida Y, Minihane AM, Turner R, VafeiAdou K, and Weinberg PD (2003) Antioxidant and free radical scavenging activity of isoflavone metabolites. Xenobiotica 33: 913–925.[Medline]

Rowland I, Faughnan M, Hoey L, Wahala K, Williamson G, and Cassidy A (2003) Bioavailability of phyto-oestrogens. Br J Nutr 89: S45–S58.

Rowland IR, Wiseman H, Sanders TA, Adlercreutz H, and Bowey EA (2000) Interindividual variation in metabolism of soy isoflavones and lignans: influence of habitual diet on equol production by the gut microflora. Nutr Cancer 36: 27–32.[CrossRef][Medline]

Setchell KD, Brown NM, and Lydeking-Olsen E (2002) The clinical importance of the metabolite equol—a clue to the effectiveness of soy and its isoflavones. J Nutr 132: 3577–3584.[Abstract/Free Full Text]

Setchell KD, Clerici C, Lephart ED, Cole SJ, Heenan C, Castellani D, Wolfe BE, Nechemias-Zimmer L, Brown NM, Lund TD, et al. (2005) S-equol, a potent ligand for estrogen receptor beta, is the exclusive enantiomeric form of the soy isoflavone metabolite produced by human intestinal bacterial flora. Am J Clin Nutr 81: 1072–1079.[Abstract/Free Full Text]

Shutt DA and Braden AWH (1968) The significance of equol in relation to the oestrogenic responses in sheep ingesting clover with a high formononetin content. Aust J Agric Res 19: 545–555.[CrossRef]

Turner R, Baron T, Wolffram S, Minihane AM, Cassidy A, Rimbach G, and Weinberg PD (2004) Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein. Free Radic Res 382: 209–216.[CrossRef]

Wahala K, Koskimies JK, Mesilaakso M, Salakka AK, Leino TK, and Adlercreutz H (1997) The synthesis, structure and anticancer activity of cis- and trans-4',7-dihydroxyisoflavan-4-ols. J Org Chem 62: 7690–7693.[CrossRef]

Wessely F and Prillinger F (1939) Die Konstitution des Equols. Chem Universitätslaborat Wien 72: 629–633.

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