Metabolism of isoflavones and lignans by the gut microflora: a study in germ-free and human flora associated rats

Abstract

We have investigated the metabolism of isoflavones and lignans in germ-free (GF) rats and rats associated with human faecal bacteria (human flora associated [HFA] rats), in order to provide unequivocal evidence for the role of the gut microflora in the absorption and metabolism of these phytoestrogens. Furthermore, we have investigated whether certain metabolic characteristics (high equol-producing and low equol-producing status) of human intestinal floras can be transferred to GF rats. Germ-free rats fed a soy-isoflavone containing diet excreted large quantities of daidzein and genistein in urine indicating that the gut microflora is not required for the absorption of isoflavones. The isoflavone metabolites equol, O-desmethylangolensin and the lignan enterolactone were not detectable in urine from the GF rats, but were present in HFA rat urine, indicating that they were products of gut microflora activity. Colonization of GF rats with a faecal flora from a human subject with the capacity to convert daidzein to equol, resulted in the rats excreting substantial amounts of the metabolite. In contrast, equol was undetectable in urine of HFA rats associated with a faecal flora from a low equol-producing subject. The results therefore show that the inability of some subjects to produce equol is a consequence of the lack of specific components of the gut microflora.

Introduction

The isoflavones genistein and daidzein are found almost exclusively in soy and soy products, usually in the form of glucosides (genistin and daidzin), and can reach concentrations up to 1 mg/g wet weight. (Coward et al., 1993, Wang & Murphy, 1994, Reinli & Block, 1996, Mazur & Adlercreutz, 1998). The lignans are more widely distributed and are found in cereals, especially rye, oats, a few fruits and in berries as well as in wine and tea and, in much larger quantities, in flaxseed. (Thompson et al., 1991) The major lignans are syringiresinol, pinoresinol, lariciresinol, isolariciresinol, matairesinol and secoisolariciresinol, usually found as glucosides and diglucosides (Axelson et al., 1982, Heinonen et al., 2001).

Both isoflavones and lignans have structural similarities to the human female hormone, 17β-estradiol, and other steroid hormones (Setchell and Adlercreutz, 1988) and can bind to the oestrogen receptor in many tissues and exert weak estrogenic activity (Setchell, 1995a, Setchell, 1995b, Wang & Kurzer, 1998). There is evidence that they have potential health benefits in humans particularly against hormone-dependent diseases such as breast and prostate cancer and osteoporosis.(Adlercreutz & Mazur, 1997, Bingham et al., 1998).

One of the important aspects of isoflavones and lignans is that they are metabolized, in some cases extensively, in the human body (Xu et al., 1995, Heinonen et al., 1999). There is considerable evidence that hydrolysis of the isoflavone glucosides is necessary for their absorption from the gut since the glucosides have not been detected in plasma (Andlauer et al., 2000, Setchell et al., in press). The site or sites of hydrolysis have not been fully elucidated, although it is known that glucosidase enzymes are produced in the small intestine and by members of the gut microflora (Day et al., 1998, Rowland et al., 2003). The aglycones undergo further metabolism, with genistein being converted to p-ethyl phenol and 4-hydroxyphenyl-2-propionic acid, while daidzein can be reduced to the isoflavan equol and O-desmethylangolensin (O-Dma) (Axelson et al., 1984, Kelly et al., 1993, Setchell, 1998, Coldham et al., 1999, Heinonen et al., 1999, Wiseman, 1999). There is considerable interindividual variation in production of certain metabolites, particularly equol and O-Dma. For example, only about 35% of subjects excrete substantial amounts of equol after consuming soy (Lampe et al., 1998; Rowland et al., 2000). The lignans also undergo extensive metabolism, with matairesinol being converted to enterolactone and secoisolariciresinol to enterodiol. Enterodiol may then be converted to enterolactone. These reactions have been shown to occur in in vitro incubations of lignans with human faeces (Borriello et al., 1985). Antibiotic treatment of human subjects decreases the excretion of the metabolites of the lignans and isoflavones, providing some evidence of the importance of the bacterial flora (Adlercreutz et al., 1986, Kilkkinen et al., 2002), but other evidence for the role of the microflora is scanty.

The role of gut bacteria in metabolism and disease is often complicated by the difference in gut flora between animals and man. In order to overcome these problems, human flora associated rats (HFA rats) have been developed (Mallett et al., 1987) whereby germ-free rats are colonized with a complete human faecal microflora. Such rats retain the bacteriological and metabolic characteristics of the human microflora (Rumney & Rowland, 1992, Hirayama et al., 1995).

In the present study we have investigated the metabolism of isoflavones and lignans in germ-free rats and rats associated with human faecal bacteria, in order to provide unequivocal evidence for the role of the gut microflora in the absorption and metabolism of phytoestrogens. Furthermore, we have also investigated whether the metabolic characteristics (high equol-producing and low equol-producing status) of human intestinal floras can be transferred to germ-free rats.