Isoflavones and coronary heart disease

1 Introduction

The increase in coronary heart disease (CHD) incidence associated with decreased ovarian function at the menopause (McGrath et al., 1998; Bittner, 2002) is in part attributable to a less favourable blood lipid profile and arterial dysfunction. Replacement of the natural hormones by exogenous oestrogen and progesterone, in the form of hormone replacement therapy (HRT), has been consistently shown to decrease plasma concentrations of low-density lipoprotein (LDL)-cholesterol and increase concentrations of the beneficial high-density lipoprotein (HDL)-cholesterol (Erberich et al., 2002).

As a result, HRT has been widely advocated as an effective means of delaying the progression of atherosclerosis in postmenopausal women. However, recent findings from long-term controlled intervention studies have proved disappointing, with no benefit, or increased incidence, of CHD reported in a number of well-controlled trials (Grady et al., 2002; Skouby, 2002; Kuller, 2003). This lack of efficacy has been attributed to the fact that synthetic oestrogens are associated with increased likelihood of blood clot formation and a rise in circulating concentrations of triglycerides and the inflammatory marker C-reactive protein (CRP), which are independent risk factors for CHD (Manson et al., 2003). Of concern is evidence from recent large-scale studies that demonstrate, conclusively, that HRT is associated with increased incidence of endometrial and breast cancer (Beral et al., 1999). Lack of efficacy of HRT with respect to CHD progression, and clear evidence of increased risk of hormone-dependent cancers, has led to interest in the search for alternative therapies to counteract this loss of natural oestrogens at the menopause.

The oestrogenicity of isoflavones was first documented over 50 years ago, when isoflavones present in the diet of sheep were found to be responsible for the permanent infertility induced in these animals. Subsequent epidemiological evidence in humans suggested that high soy consumption, the main dietary source of isoflavones, was cardioprotective, in part attributed to the ability of the isoflavones in soy to act as oestrogen mimics. Demonstration of the ability of soy products to bring about a beneficial change in the blood lipoprotein profile led the US Food and Drug Administration (FDA, 1999) to approve a claim that `25g of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease’. It is currently uncertain whether soy isoflavones contribute to the cholesterol-lowering effects that are reported in controlled trials of soy and soy products. Recent studies have shown small hydrolysed soy peptides that may enter the circulation and up-regulate LDL receptors in the liver could be an important mechanism for the hypocholesterolaemic effects of soy and indicate the active cholesterol-lowering component to be the protein rather than isoflavones (Lovati et al., 2000).

Furthermore, in vitro and animal data are emerging that suggest that isoflavones may be cardioprotective by mechanisms independent of blood lipids, but the underlying mechanisms are only partly understood. The recent discovery of a second oestrogen receptor (ERO) (Gustaffson, 1999), which binds isoflavones with much higher affinity than ERa, and advances in analytical techniques allowing the measurement of a wide range of isoflavone metabolites in biological fluids, is greatly contributing to this knowledge.

2 Chemical structure of isoflavones

Isoflavones are non-nutrient plant components, which belong to the phytoestrogen family (Kurzer and Xu, 1997). Phytoestrogens are diphenolic compounds. Ring A and Ring B are separated by a heterocyclic pyrone ring and have a similar chemical structure to mammalian oestrogens. Comparison of the chemical structure of 170-estradiol and equol, a gut metabolite of daidzein (a major food isoflavone), indicates that the two compounds are almost super-imposable.

The first evidence that isoflavones have oestrogenic properties was documented in the 1940s when it was established that the infertility of Australian sheep was attributable to the high concentration of the precursor isoflavones, formononetin and biochanin A, found in clover-rich pastures (Bennett et al., 1946). Although oestrogenicity assays reported low oestrogenic potency for dietary isoflavones (100±1000 times less than 17,3-estradiol, with genistein being the most potent) (Miksicek, 1993), the fact that their high circulating levels could exceed endogenous estradiol concentrations by up to 10 000-fold (Adlercreutz et al., 1993) was thought to explain their physiological effects.

Isoflavone R1 R2 R3 R4

R5

Genistein OH H OH OH

H

Daidzein H H OH OH

H

Glycitein H OCH3 OH OH

H

Formononetin H H OH OCH3

H

Biochanin A OH H OH OCH3

H

Genistin OH H O-G OH

H

Daidzin H H O-G OH

H

Glycitin H OCH3 O-G OH

H

Chemical structure of isoflavones found in plants.

The discovery and sequencing of the ER (oestrogen receptor ) gene in 1996 offered a further explanation for divergent actions of synthetic, endogenous and plant sources of oestrogens (Kuiper et al., 1996). In contrast to the ER, which is present in high concentrations in the ovaries and testes, ER is predominantly expressed in non-gonadal tissues including the blood vessels and bones. This receptor shows a much higher binding affinity for isoflavones compared with the -receptor. Genistein demonstrates a binding affinity for ER and ER of 4 and 87 per cent of oestrogen binding, whereas daidzein, the other major isoflavone, displays affinities of 0.1 and 0.5% respectively (Kuiper et al., 1998). In addition X-ray crystallography data suggested that, despite the structural similarities to oestrogens, the ligand binding sites of natural oestrogens and isoflavones may be reversed, with the B ring hydroxyl group of isoflavones interacting with the ER (oestrogen receptor) in contrast to the A ring hydroxyl of 17-estradiol (Setchell, 1998, 2001). This specificity of isoflavones for ER, and the orientation of isoflavones with the

ER, has led to the recent classification of isoflavones as selective oestrogen receptor modulators (SERMs) rather than phytoestrogens or oestrogen mimics. The specific tissue distribution of the two oestrogen receptor subtypes (ER and ER) and interaction of isoflavones, particularly with the ER, will undoubtedly impact on their functional effects on cells and tissues. This may explain the suggested selective benefits of isoflavones on cardiovascular and bone health, without the associated negative effects in certain tissues such as the breast. However, as yet, such selective benefit is indicated largely from epidemiological evidence and more information from intervention trials and cell studies is required to substantiate these beneficial effects.

3 Dietary sources, bioavailability and metabolism of isoflavones

3.1 Dietary sources of isoflavones

Isoflavones are found naturally in soybeans and to a much lesser extent in legumes. Unprocessed soybeans contain 1±5 mg of isoflavones/g of dry weight (Wang and Murphy, 1994). In certain Asian countries such as parts of China and Japan, where soy is a traditional staple, intakes of isoflavones of 20±50 mg per day are common (Nagata et al., 1998). In Westernised countries, where soy is not a commonly consumed food except in a small minority of the population, typical isoflavones intakes are less than 1 mg/day. Clover and Chinese vine are also rich in isoflavones, and although these plants do not form part of the human food chain, they serve as an important source of isoflavones for commercially available supplements.

Soybeans contain three main isoflavones, present in one of four chemical forms. The free isoflavone aglycones are genistein, daidzein and glycitein. Isoflavones predominantly occur in plants as the water-soluble -glucosides (complexed to glucose) genistin, daidzin, glycitin, or as acetyl--glucosides or malonyl--glucosides (Song et al., 1998). Formononetin and biochanin A are the precursors of daidzein and genistein respectively.

On harvesting the soybean the processing techniques used are important determinants of the total isoflavone content and chemical form in the consumed food (Kurzer and Xu, 1997; Coward et al., 1998). Soy flour and texturised vegetable protein (TVP) generally contain about one-third of the isoflavone content (1±1.5 mg/g dry weight) of the original soybean. Because of harsh processing techniques, soy concentrate is a relatively poor isoflavone source. Secondary soy products such as tempeh burgers and tofu contain less than 20 per cent of the isoflavone content found in whole soybeans, because of the addition of large quantities of non-soybean ingredients to the products.

Processing is also known to influence the chemical form of the isoflavones (Coward et al., 1998). Unprocessed soy foods contain mainly 6′ O­malonyldaidzin and O-malonylgenistin but the heating process used during the production of TVP converts these forms into the more stable -glucosides.

Non-fermented foods (e.g. tofu) are generally rich in 3-glucosides, whereas fermented soy (e.g. tempeh) is rich in aglycones, owing to enzymatic hydrolysis during fermentation.

The isoflavone form present in isoflavone supplements is highly variable and dependent on the source of the isoflavones and the manufacturing techniques. In addition a recent publication by Setchell and co-workers (2001) indicates that there are often considerable discrepancies between the true composition of supplements and the claims made on the label.

3.2 Isoflavone metabolism in the gut lumen

Isoflavones are thought to undergo extensive metabolism in the intestinal tract prior to absorption. Important factors that regulate and therefore determine isoflavone metabolism, bioavailability and subsequent biological activities are the presence and activity of intestinal microflora, the chemical forms in which isoflavones are ingested, and the presence of certain components in the diet. Adequate information exists for the metabolism of dietary isoflavones with limited information also available concerning their post-absorption metabolism (pharmacokinetics). However, information about the metabolism of the plethora of isoflavone extracts present in commercial supplements is distinctly lacking.

Isoflavones are present in food mostly in a conjugated form. After ingestion, isoflavone conjugates are hydrolysed by intestinal and bacterial 3-glucosidases, thereby releasing the aglycones, genistein, daidzein and glycitin (Setchell et al., 2002; Hur et al., 2002). In addition to aglycones derived directly from the diet, these compounds can also be derived from dietary precursors. The precursors biochanin A and formononetin are transformed into genistein and daidzein in the gut lumen.

The aglycones may be absorbed directly from the small intestine by passive diffusion mechanisms or undergo further biotransformation to a range of metabolites, by specific enzymes produced by a limited range of as-yet largely unknown bacterial species in the colon. Genistein is transformed to dihydrogeni­stein, which is further metabolised to 6′-hydroxy-O-desmethylangolensin (DMA), whereas daidzein is transformed to dihydrodaidzein, which is further metabolised to both equol (70 per cent) and O-desmethylangolensin (O-DMA) (5±20 per cent) (Setchell and Adlercreutz, 1988; Joannou et al., 1995). As the oestrogenic potency of equol is 10-fold higher than its precursor daidzein, the transformation of daidzein to equol is considered to be a clinically relevant step in the therapeutic potential of soy isoflavones (Shutt and Cox, 1972; Cassidy et al., 2000).

It has been widely acknowledged that the activity of intestinal microflora is an important factor in the metabolism of isoflavones (Lampe et al., 1998; Rowland et al., 2000, Cassidy et al., 2000). The administration of antibiotics to laboratory animals blocks isoflavone metabolite production in the resultant germ-free animal. In addition in newborn infants, whose gut microflora is underdeveloped, no equol is detectable in the urine or plasma when soy formulas are fed. Furthermore it is now well recognised that adults differ in their ability to produce equol. A number of investigators have concluded that only 30-40 per cent of individuals produce equol following daidzein ingestion (Lampe et al., 1998). This lack of ability to synthesise equol has been attributed to an absence of specific bacterial enzymes in the large gut. Therefore, there is an increased interest in identifying the microorganisms that are responsible for isoflavone metabolism and furthermore for introducing specific dietary components such as fibre that may stimulate their growth (Bingham et al., 2003). Three strains of bacteria that are capable of metabolising daidzein to equol have been identified, namely Bacteroides ovatus, Ruminococcus productus and Streptococcus intermedius (Ueno et al., 2002). However, it is likely that other strains are involved.

Of total isoflavone ingested, approximately 20-50 per cent is absorbed when dietary intakes are less than 0.5 mg/kg body weight. Isoflavone intakes greater than 0.5 mg/kg body weight can reduce absorption efficiency (Setchell et al., 2001). This information, in combination with the fact that isoflavones are rapidly excreted in the urine (usually within 24 hours), suggests that consuming modest levels on a regular basis rather than large amounts intermittently can best achieve the maximum benefit from isoflavone consumption.

Rates of absorption and absorption efficiency are dependent on the chemical form in which the isoflavone is consumed and on the matrix of the food itself. Aglycones are generally absorbed more rapidly than the 0-glycoside equivalent (Izumi et al., 2000; Setchell et al., 2001). However, the bioavailability of aglycones is thought to be less than the complexed isoflavones, as aglycones are more likely to be degraded in the gut lumen (Kelly et al., 1993; Joannou et al., 1995).

3.3 Post-absorption isoflavone metabolism and pharmacokinetics

In order for recommendations to be made regarding efficacious and safe long­term isoflavone intake, it is important to understand the post-absorptive metabolism of isoflavones and their metabolites. Once absorbed, isoflavones are mainly metabolised in the gut and liver where they are converted into either a glucuronidated (attached to glucose) or sulphated (attached to sulphate) form. There are two conjugation sites on genistein and on daidzein and each of these sites can be sulphated or glucuronidated (Shelnutt et al., 2002). These conjugates are more easily transported in the blood and excreted in bile or urine than the parent aglycones due to their greater water solubility.

The plasma concentration of individual isoflavones has been investigated in a limited number of studies. Higher plasma genistein concentrations are consistently observed when equal amounts of genistein and daidzein are consumed (King, 1998); this is because genistein is retained in the body to a greater extent owing to its higher lipid solubility, and daidzein has a wider body

tissue distribution. In a study investigating the effects of consuming a soy protein isolate beverage powder (60 g/day for 28 days) in 20 male subjects, plasma genistein and daidzein concentrations reached 0.9 µM and 0.5 pM, respectively (Gooderham et al., 1996). Barnes (1995) reported the maximal physiologically achievable plasma isoflavone concentration to be 18.5 ^ In a study evaluating the pharmacokinetics and safety of purified unconjugated soy isoflavone preparations fed at a concentration of up to 16 mg/kg bodyweight in postmenopausal women, half-lives for free genistein, daidzein, and glycitein in the circulation averaged 3.8, 7.7 and 3.4 h, respectively (Bloedon et al., 2002). The studies conducted to date have largely focused on the metabolism of the free parent aglycones, which represent a relatively small proportion of the total isoflavones present in plasma. Information on the metabolism of the sulphate and glucuronide conjugates are needed in order to get an accurate assessment of efficacy and safety (Shelnutt et al., 2002). Also pharmacokinetic data have been accumulated using supplements as the isoflavone source, with little data available on isoflavone metabolism following soy ingestion.

4 The effect of isoflavones on coronary heart disease (CHD)

Isoflavones are suggested to have beneficial effects on a number of oestrogen-related conditions such as menopausal symptoms, osteoporosis, cancer and cardiovascular disease. Over the past two decades it is the potential cardioprotective benefits that have received most attention.

Cardiovascular disease is the primary cause of death in Western countries. The lower rates of cardiovascular disease in areas of South East Asia such as Japan compared with Western countries has been suggested to be partly attributable to the greater consumption of soy foods in these countries (Beaglehole, 1990; Adlercreutz et al., 1992; Adlercreutz and Mazur, 1997). However, this observation is by no means conclusive as other lifestyle factors, such as lower dietary intakes of saturated fat or higher intakes of fresh vegetables and/or fish, may be in part responsible. In addition, isoflavones may not be the only constituent of soy responsible for the protective effects. Despite these uncertainties, this putative link has stimulated extensive research in the area of isoflavones and cardiovascular health. Proposed mechanisms of action include a beneficial effect on blood lipid metabolism, LDL oxidation, endothelial function and platelet aggregation.

4.1 Mechanisms of action: lipid metabolism

Raised total and LDL-cholesterol levels, low-HDL cholesterol and raised fasting triglyceride concentrations in the blood are known risk factors for the development of cardiovascular disease. It has been recognised for many years that reductions in circulating total cholesterol, LDL-cholesterol and triglycerides, and increases in HDL-cholesterol can result from soy protein consumption. This has been shown in animals (Huff et al., 1977; Anthony et al., 1996, 1998; Balmir et al., 1996; Wagner et al., 1997; Clarkson et al., 2001), and in many human intervention and cross-sectional studies (Nagata et al., 1998). In a meta-analysis of 38 placebo-controlled trials soy protein consumption was associated with an average 9 per cent decrease in total cholesterol concentrations in 34 of the studies (Anderson et al., 1995). Furthermore, 26 out of 31 studies reported an average 13 per cent decrease in LDL-cholesterol, and 22 out of 30 studies reported an average 10 per cent decrease in plasma triglyceride levels. Significantly, this meta-analysis showed that reductions in plasma cholesterol were most likely to be seen in subjects with raised cholesterol concentrations. Since 1995, numerous further studies have been reported on the lipid-lowering effects of soy protein and soy isoflavones.

The possible mechanisms for the hypocholesterolaemic effects of soy are not yet clear, although soy isoflavones have been proposed as a putative cholesterol-lowering soy constituent. However, while the majority of soy protein studies have shown lipid-lowering effects, the small number of studies that have supplemented with isoflavones alone have to date produced negative results (e.g. Hodgson et al., 1998; Simons et al., 2000; Dewell et al., 2002). This does not rule out a possible hypocholesterolaemic effect of isoflavones. Larger changes in lipid profiles have been observed following supplementation with intact soy protein compared to soy protein where the isoflavone content has been removed via ethanol extraction in monkeys (Anthony et al., 1996, 1998) and postmenopausal women (Gardner et al., 2001). Concern does, however, exist regarding the impact of the harsh ethanol extraction conditions on the composition of the resultant isoflavone-free product. It is also possible that other constituents of soy may have cholesterol-lowering properties or that isoflavones may act in conjunction with soy components such as saponins, fibre or specific amino acids (Erdman, 2000). It has been suggested that isoflavones may directly increase bile excretion (Lissin and Cooke, 2000), which would reduce the hepatic cholesterol pool and increase the receptor-mediated uptake of LDL from the circulation. Saponins also increase bile excretion, and there may be a greater hypocholesterolaemic effect through isoflavones and saponins acting together. In vitro studies have shown that specific subunits of 7S soy globulins increase LDL uptake and degradation by upregulation of LDL receptors in HepG2 cells (Lovati et al., 1992, 2000; Manzoni et al., 2003). Soy fibre may decrease intestinal cholesterol absorption. In addition, isoflavones may also reduce cholesterol levels by improving LDL receptor activity (Kirk et al., 1998). Almost all isoflavone intervention studies to date have administered the treatment and placebo as a capsule. If isoflavones are active as part of a food,

then more studies are needed to investigate the absorption, biokinetics and lipid-lowering effects of isoflavones incorporated into various foods.

4.2 Antioxidant function

The oxidation of LDL in the arterial wall leads to its uptake by macrophages and to the formation of lipid-filled foam cells, a key feature of atherosclerosis. Soy protein and isoflavone treatment have been shown to reduce lipid peroxidation in monkeys and rabbits (Wagner et al., 1997; Yamakoshi et al., 2000). Many authors have reported on the antioxidant effect of soy protein supplementation in humans, mainly represented as a delay in copper-mediated LDL oxidation ex vivo (Tikkanen et al., 1998; Ashton et al., 2000; Wiseman et al., 2000; Steinberg et al., 2003) but also by other methods commonly used to assess lipid peroxidation/antioxidant status (Wiseman et al., 2000; Scheiber et al., 2001; Bazzoli et al., 2002; Jenkins et al., 2002; Fritz et al., 2003). Again, however, the evidence for the effects of isoflavones alone is not strongly supported by the intervention studies conducted up until now. Samman and colleagues (1999) found no change in copper-mediated LDL oxidation in premenopausal women following two months of isoflavone supplementation, while Hodgson et al. (1999b) reported no effect of supplementation on urinary F2-isoprostane concentrations (a marker of whole body lipid peroxidation).

In vitro studies have indicated that equol and genistein are more potent antioxidants than daidzein, genistin, biochanin A and formononetin, with equol being more potent than genistein (Wei et al., 1993, 1995; Kapiotis et al., 1997; Ruiz-Larrea et al., 1997; Arora et al., 1998; Mitchell et al., 1998). It is thought that isoflavone structure is a major determinant of antioxidant potency, with the hydroxyl groups at the C4 and C5 positions of the molecule being integrally involved. The cellular processes responsible for the antioxidant activity of isoflavones are not clear. Scavenging of free radicals is a potential mechanism. However, a number of isoflavones showed no significant scavenging effects on a range of radicals in a recent study (Guo et al., 2002). Other possible mechanisms include the inhibition of hydrogen peroxide production (a source of the destructive hydroxyl radical) and stimulation of antioxidant enzymes such as catalase (Wei et al., 1995).

An increase in endothelial cell glutathione concentrations was also observed following exposure of the cells to physiologically achievable concentrations of genistein and daidzein, which would increase the antioxidative effects of these isoflavones (Guo et al., 2002). Kerry and colleagues (Kerry and Abbey, 1998) noted that genistein inhibited LDL oxidation, but that the hydrophilic isoflavone was poorly incorporated into the LDL particle. It was consequently shown (Meng et al., 1999) that esterification of isoflavones increased incorporation into LDL with a number of isoflavone-fatty acid esters inhibiting LDL oxidation. In vivo, lipophilic isoflavones may therefore be more important with respect to LDL oxidation than the native hydrophilic form of the compounds (Kaamanen et al., 2003).

As is the case for the pharmacokinetic data, most in vitro studies have investigated the effects of the aglycones, genistein and daidzein, for which there is a relatively low tissue exposure. Rimbach et al. (2003) compared the free radical-scavenging properties of the metabolites equol, 8-hydroxydaidzein, O­desmethylangiolensin and 1,3,5-trihydroxybenzene in comparison to their parent aglycones, genistein and daidzein. 8-Hydroxydaidzein was the most potent scavenger of hydroxyl and superoxide anion radical, and the isoflavone metabolites exhibited higher antioxidant activity than the parent compounds, indicating that the metabolism of isoflavones affects their free radical scavenging and antioxidant properties.

In conclusion, soy protein has significant antioxidant effects in vivo but this has not been shown after isoflavone supplementation. The evidence from in vitro studies is strongly supportive of an antioxidant role for isoflavones, but caution is needed in interpreting these results as pharmacological doses were employed in the majority of cases.

4.3 Blood pressure and endothelial function

Hypertension is a classical risk factor for cardiovascular disease, and is a result of reduced blood vessel flexibility and increased resistance to blood flow due to narrowing of the vessel. This causes endothelial injury and dysfunction, thereby potentiating the development of atherosclerosis and increasing the risk of CHD and stroke.

A number of human studies have shown a decrease in blood pressure after soy protein supplementation (Washburn et al., 1999; Vigna et al., 2000; Teede et al., 2001; Jenkins et al., 2002; Rivas et al., 2002), but again this has not been the case when encapsulated isoflavones were administered (Hodgson et al., 1999a; Han et al., 2002). Vascular and endothelial function, as measured by systemic arterial compliance, pulse wave velocity and flow-mediated vasodilatation, have also been reported to improve after soy protein supplementation (Teede et al., 2001; Yildirir et al., 2001; Steinberg et al., 2003). In contrast to the negative findings for cholesterol and LDL oxidation, isoflavone supplementation has been shown to improve vascular function (Nestel et al., 1997, 1999; Squadrito et al., 2003), although there are conflicting results (Hale et al., 2002). The human data is supported by the animal data (Honore et al., 1997; Williams and Clarkson, 1998; Karamsetty et al., 2001; Nevala et al., 2002).

The most likely mechanism by which isoflavones may improve endothelial function is via the oestrogen receptors, ERa and ERO (Kuiper et al., 1997; Register and Adams, 1998). ERa and ERO both mediate non-genomic endothelial derived nitric oxide synthase (eNOS) activation, the key enzyme responsible for the production of nitric oxide, a major vasodilator (Chen et al., 1999; Chambliss et al., 2002). Furthermore, increased nitric oxide production was observed following isoflavone supplementation in animals (Squadrito et al., 2000; Catania et al., 2002) and in human volunteers (Squadrito et al., 2003).

The evidence for the effect of isoflavones on vascular reactivity and dilatation is convincing, and may prove to be of greater physiological importance in cardiovascular disease than the lipid-lowering effects.

4.4 Platelet aggregation

Thrombus (large blood clot) formation in a vessel already narrowed by atherosclerosis can cause a complete occlusion, leading to tissue damage in the affected coronary tissue. Platelet aggregation is a key feature of thrombus formation. Studies in rats (Peluso et al., 2000) and monkeys (Williams and Clarkson, 1998; Kondo et al., 2002) have shown lower rates of platelet aggregation following consumption of soy protein or isoflavones; and both genistein and daidzein have been shown to inhibit in vitro platelet aggregation (Nakashima et al., 1991; Gottstein et al., 2003). However, soy protein supplementation had no effect on collagen-induced platelet aggregation in normocholesterolaemic men (Gooderham et al., 1996), and therefore the importance of isoflavones in reducing platelet aggregation in vivo is not yet clear.

The mechanisms by which isoflavones affect platelet aggregation have not yet been established, but a number of mediators in the aggregatory process may be involved, e.g. cyclooxygenase (via protein tyrosine kinase), cyclic-3′,5’­ adenosine monophosphate (cAMP), lipoxygenase or hydrogen peroxide, a second messenger in platelet activation (Beretz et al., 1982; Landolfi et al., 1984; Pignatelli et al., 1998). Although demonstration of efficacy has been observed in animal and in vitro aggregation studies and the underlying mechanisms are partly understood, data on the impact of isoflavones on platelet aggregation in humans are lacking

4.5 Cell adhesion molecules and inflammatory cytokines

The production of inflammatory cytokines and cell adhesion molecules (CAMs) by the arterial endothelium are key cellular events involved in the development of atherosclerosis. Activation of the endothelium results in the release of vascular cytokines such as interleukin-1 (IL-10) and tumour necrosis factor alpha (TNF-a). These cytokines induce the expression of CAMs such as intracellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1), which, together with activated monocyte chemoattractant protein-1 (MCP-1), recruit monocytes through the vascular wall, where they are involved in foam cell formation.

CAMs are increasingly regarded as important molecular markers of atherosclerosis. The nuclear transcription factor, NFnB, is a mediator in TNF­a-induced expression of cell adhesion molecules. NFnB is activated by an atherogenic diet (Liao et al., 1993) and oxidised LDL (Brand et al., 1997), and activation is inhibited by various antioxidants (Kunsch and Medford, 1999). Therefore it is of great interest that genistein attenuated NF-nB DNA binding and TNF-a release in human monocytes (Shames et al., 1999). Genistein, but not daidzein, inhibited TNF-a-induced NF-KB activation in ex vivo human lymphocytes following consumption of 100 mg isoflavones/day for 3 weeks, as well as in cultured human lymphocytes (Davis et al., 2001). However this small pilot study (n = 6) was not placebo-controlled.

Isoflavones have been found to have no effects on CAM expression in vivo (Blum et al., 2003; Steinberg et al., 2003). Nevertheless, genistein has been shown to inhibit CAM surface expression and monocyte cell adhesion in cultured endothelial cells (McGregor et al., 1994; Weber et al., 1995; May et al., 1996). Therefore it appears that isoflavones have the potential to attenuate this inflammatory response, but whether this can be achieved at physiologically relevant isoflavone concentrations remains to be established.

4.6 Summary of cardioprotective benefits of soy isoflavones

Overall the evidence from human, animal and in vitro studies suggests that soy protein, and perhaps isoflavones, have potential as a dietary means of reducing cardiovascular risk. Evidence for a cholesterol lowering and antioxidant effect of soy is convincing. However, data are not currently available to suggest that isoflavones are the active ingredient. The vasodilatory effect of isoflavones has been repeatedly observed in human trials and it is likely that chronic isoflavone intake could positively increase the elasticity of blood vessels. Although there is an ever-increasing body of in vitro evidence to suggest that isoflavones are anti-inflammatory, improve endothelial cell function and decrease platelet aggregation, the concentrations used in vitro are higher than those that are physiologically achievable in human studies. Efficacy in humans remains to be established.

5 Potential risks of isoflavones

Recently, the safety of soy and its constituent isoflavones has been questioned. Concerns have arisen from animals and in vitro studies, which suggest that isoflavones may be involved in cancer development, thyroid dysfunction and reduced fertility.

5.1 Isoflavones and cancer risk

Although there is substantial epidemiological evidence to suggest that isoflavones may be anti-carcinogenic, data from a limited number of intervention studies have generated concerns regarding the safety of isoflavones. Short-term dietary soy supplementation (45 mg isoflavones/day) induced proliferation in breast tissue of premenopausal women with breast cancer (McMichael-Phillips et al., 1998), raising concern that isoflavones may stimulate oestrogen-dependent tumours in the breast. In a further study, consumption of soy protein isolate (38 mg isoflavones/day) was associated with the appearance of hyperplasic cells and increased secretion of breast fluid (Petrakis et al., 1996). However, data are limited and require further investigation.

Animal studies have shown conflicting results. Some studies have shown that genistein has a suppressive effect on chemically induced tumours (Constantinou et al., 1996; Fritz et al., 1998), whereas other studies showed that genistein could stimulate the growth of mammary implanted tumours (Hilakivi-Clarke et al., 1999). So far there is no direct association between isoflavone consumption and other cancer types.

5.2 Isoflavones and thyroid function

Results from several studies have raised speculation on the effect of isoflavones on thyroid function. Goitrogenic effects in infants fed soy-based infant formula were first reported in the 1960s. The subsequent substitution of soy flour with soy protein isolate and the supplementation of the formula with iodine overcame the goitrogenic effects of soy-based infant formula (Fomon, 1993). Since then there have been no reports of goitre in children fed soy formula.

There have been a few intervention studies investigating the effects of isoflavones on thyroid function. In these studies, supplementation of isoflavones in pre-menopausal and post-menopausal women led to an alteration in the levels of thyroxine, triiodothyronine and thyroid binding globulin (TBG). However, the changes in hormone concentrations were considered to be of too small a magnitude to be clinically important (Duncan et al., 1999a,b; Persky et al., 2002). Unlike human studies, some cell studies posed concerns on isoflavone safety and indicated that isoflavones can interfere with thyroid function, although high levels of isoflavones were generally used. In these studies isoflavones inhibited thyroid peroxidase, a key enzyme in the production of thyroid hormones (Divi and Doerge, 1996, 1997). Some animal studies have also suggested the interference of isoflavones with thyroid function (Ikeda et al., 2000; Balmir et al., 1996).

Because of the potential interactions between isoflavones and thyroid function, chronic high-dose isoflavone consumption may not be advisable in individuals with reduced thyroid function.

5.3 Isoflavones, fertility and development

Concerns that phytoestrogens may have adverse effects on mammalian development and fertility were initially posed when animals fed phytoestrogen-rich plants displayed loss of fertility (Bennett et al., 1946; Moersch et al., 1967; Obst and Seamark, 1975). Subsequent animal studies provided evidence that dietary exposure to phytoestrogens can adversely affect reproduction (Adams, 1995; Kallela et al., 1984). The fact that exposure to potent oestrogens in utero can have long-term adverse effects on human development and fertility in males and in female offspring, has raised concerns that exposure to phytoestrogens, including isoflavones, may also give rise to similar effects later in life.

A main concern is the consumption of soy-based milk by infants since chemical compounds in milk are of particular significance for the differentiation and development progress throughout the neonatal period. Furthermore, the concentrations of isoflavones in soy infant formula and the exposure levels for infants fed on soy infant formulas (per kg body weight), are much higher than the dose consumed by Asian adults (Setchell et al., 1997, 1998).

Human studies examining the effect of phytoestrogens on fertility and development are limited and the majority of information is derived from animal studies. One human study examined the effect of soy-based formula feeding on subsequent sexual development and fertility. Apart from small increases in the duration and discomfort of menstruation in young adult females fed soy formula as infants, there was no evidence for adverse clinical effects on sexual development or reproductive health (Strom et al., 2001). An association between consumption of soy infant formula and premature thelarche (breast development before 8 years of age) has also been suggested (Freni-Titulaer et al., 1986). Rodent and primate studies suggest that isoflavone consumption can affect tissue differentiation and reproductive function as well as causing hormonal changes (Awoniyi et al., 1997; Harrison et al., 1999; Lewis et al., 2003). However, the extrapolation of the data from rodent and primate studies to humans is very difficult because there are significant species differences in sexual development and reproductive function, and in the concentration and tissue distribution of oestrogen receptors. Furthermore, many studies used the subcutaneous route of isoflavone administration that bypasses the gut and hepatic metabolism, which can affect phytoestrogen bioactivity. Additionally, many studies do not report doses on a body weight basis. Therefore, it is not meaningful to make direct comparisons of these studies

6 Future trends

Although plausible mechanisms exist that support the case for cardioprotective benefits of isoflavones, the evidence is largely based on isoflavone consumption in the form of soy. Adequately powered human intervention studies that can definitely establish the benefits of either encapsulated isoflavones or isoflavone­fortified foods are needed. This work needs to be supported by kinetic experiments examining the absorption and subsequent metabolism of different isoflavone sources.

Also little information exists on the inter-individual variability in the gut metabolism, absorption, pharmacokinetics and physiological impact of increased isoflavone intake. It is likely that there are subsets of the population that respond to isoflavone supplementation to a greater extent than others. For example, it has been speculated that those individuals who can metabolise daidzein to equol in the colon may be more responsive to isoflavone supplementation relative to non-producers (Setchell et al., 1984). In addition, the effectiveness of isoflavones on cardiovascular risk factors may be modulated by genotype. Polymorphisms in glucuronidation and sulphation enzymes and in oestrogen receptor-a and-,3 genes may impact on responsiveness. Although this would be of interest to public health, this information is currently lacking.

A wide range of cell culture work has already been carried out to establish mechanisms of action of isoflavones. Further work in this area needs to be conducted using the forms of isoflavones present in the plasma, rather than the native aglycones, at physiologically relevant concentrations.

Isoflavone safety remains an issue and needs further investigation. It is unlikely that chronic isoflavone consumption has any negative effect on cancer risk or thyroid function in the majority of individuals, but may be a concern in those with a family history of breast cancer or thyroid dysfunction, and those who already suffer from these conditions. The evidence suggesting that physiologically achievable circulating isoflavone concentrations impact on sexual development or fertility in adults is currently weak, although there is a substantial body of animal data showing adverse effects, albeit at very high doses. The impact of high in utero exposure to isoflavones, as well as soy milk consumption by infants, on sexual development in later life is of particular concern. Consequently, there are unresolved questions regarding the suitability of high isoflavone supplementation during pregnancy or the use of soy based infant formulas.

In conclusion, isoflavones offer a possible alternative to HRT therapy in postmenopausal women. However, further work is needed to fully establish the safety of chronic high intakes and efficacy with respect to cardiovascular risk.

Jean-Paul Marat

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