(Journal of Nutrition. 1999;129:758-767.)
© 1999 The American Society for Nutritional Sciences
Supplement
Dietary Isoflavones: Biological Effects and Relevance to Human Health1
Kenneth D. R. Setchell2 and
Aedin Cassidy*
Clinical Mass Spectrometry, Children's Hospital Medical Center, Cincinnati, Ohio 45229 and
* School of Biological Sciences, University of Surrey GU2 5XH
 |
ABSTRACT
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Substantial evidence indicates that diets high in plant-based foods may
explain the epidemiologic variance of many hormone-dependent diseases
that are a major cause of mortality and morbidity in Western
populations. There is now an increased awareness that plants contain
many phytoprotectants. Lignans and isoflavones represent two of the
main classes of phytoestrogens of current interest in clinical
nutrition. Although ubiquitous in their occurrence in the plant
kingdom, these bioactive nonnutrients are found in particularly high
concentrations in flaxseeds and soybeans and have been found to have a
wide range of hormonal and nonhormonal activities that serve to provide
plausible mechanisms for the potential health benefits of diets rich in
phytoestrogens. Data from animal and in vitro studies provide
convincing evidence for the potential of phytoestrogens in influencing
hormone-dependent states; although the clinical application of diets
rich in these estrogen mimics is in its infancy, data from preliminary
studies suggest beneficial effects of importance to health. This review
focuses on the more recent studies pertinent to this field and
includes, where appropriate, the landmark and historical literature
that has led to the exponential increase in interest in phytoestrogens
from a clinical nutrition perspective.
KEY WORDS: • phytoestrogens • isoflavones • genistein • hormones • cancer • cardiovascular disease • bone
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INTRODUCTION
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Isoflavones are naturally occurring plant chemicals belonging to the
"phytoestrogen" class; they are currently heralded as offering
potential alternative therapies for a range of hormone-dependent
conditions, including cancer, menopausal symptoms, cardiovascular
disease and osteoporosis. Recent epidemiologic evidence and
experimental data from animal studies that have been reviewed recently
(Anderson and Garner 1997
, Cassidy 1996
, Knight and Eden 1996
, Messina et al. 1994
, Murkies et al. 1998
, Setchell 1995 and 1998
) are highly
suggestive of beneficial effects of isoflavones on human health, but
the clinical data supportive of such effects are either not available,
or are awaiting the design and execution of appropriate large-scale
clinical studies. Nevertheless, data from limited small pilot studies
are promising, and this has spurred the current interest in this area.
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Biological actions.
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The isoflavones are strikingly similar in chemical structure to
mammalian estrogens (Setchell and Adlercreutz 1988
). The phenolic ring
is a key structural element of most compounds that bind to estrogen
receptors (Leclerq and Heuson 1979
). When the structures of the
isoflavone metabolite equol and estradiol are overlaid, they can be
virtually superimposed; the distance between the hydroxyl groups at
each end of both molecules is virtually identical (Fig. 1
).On the basis of structure alone, it is not surprising that isoflavones
bind to estrogen receptors
(ER)3
; however, their actions are more those of partial estrogen agonists and
antagonists, a concept that is difficult to fully understand, but that
continues to fascinate steroid biochemists and endocrinologists (Jordon
1990
, Mendelson 1996
). To complicate matters further, estrogens can
have nonclassical actions distinct from their classical genomic actions
(Brann et al. 1995
); these include effects on plasma membranes and on
cell signaling pathways (Kim et al. 1998
). What this implies from a
clinical perspective is that at certain concentrations, which may
depend on many factors including receptor numbers, occupancy and
competing estrogen concentration, rather than acting as estrogen mimics
and initiating estrogen-like actions, they may antagonize and inhibit
estrogen action. These effects will also be tissue specific. This
phenomenon is a well-known characteristic of steroid action, and is one
that has driven pharmacologists to search for new molecules with
selective estrogen action. The recently approved drug, Raloxifen, a
selective estrogen receptor modulator is an example (Dodge et al. 1997
).
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Figure 1. Comparison of the structure of the isoflavone metabolite equol with
that of estradiol showing the striking similarity in planar spatial
arrangement of the two molecules.
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Structure-activity relationships may provide clues to the molecular
basis for this agonism and antagonism (Brzozowski et al. 1997
), and the
absence of a specific lipophilic region in phytoestrogens may affect
binding to the ER (Cunningham et al. 1997
). Differences in the ability
of isoflavones to bind to ER, induce estrogen regulated end products
and activate cell proliferation in estrogen-sensitive human breast
cancer cell lines may help to explain this difference in biological
activity. However, predicting the effects of isoflavones in vivo is
more difficult because the route of administration, chemical form of
the phytoestrogen, its metabolism, bioavailability, half-life, timing
and level of exposure, intrinsic estrogenic state and nonhormonal
secondary mediated actions of isoflavones also have to be considered in
the design of clinical studies investigating their effects.
The recent discovery of a second estrogen receptor further complicates
our understanding of the mechanism of action of isoflavones (Kuiper et al. 1998
). Kuiper et al. (1996)
cloned a novel member of the nuclear
receptor family, named ERß to distinguish it from the "classical"
ER
subtype, and the two receptors may play different roles in gene
regulation (Paech et al. 1997
). It is conceivable that there will be
further estrogen receptors discovered in the future, given that there
are a large number of so-called orphan receptors (Willy and Mangelsdorf 1998
) that have been identified and are awaiting the recognition of
specific ligands and function. The tissue distribution (Fig. 2
)and relative ligand binding affinities of the ERß and ER differ,
and this finding may help to explain the selective action of estrogens
in different tissues. It is fascinating that ERß is found in brain,
bone, bladder and vascular epithelia (Kuiper et al. 1997
, Paech et al. 1997
, Tetsuka et al. 1997
), tissues that are responsive to classical
hormone replacement therapy (HRT). Furthermore, the relative molar
binding affinities of different estrogenic compounds reveal that
phytoestrogens and some environmental xenoestrogens have significantly
higher affinities for ERß than ER (Kuiper et al. 1997
), suggesting
that this new receptor may be important to the action of nonsteroidal
estrogens.
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Figure 2. Simplified diagram illustrating the anatomical distribution of the
newly described estrogen receptor, ERß to sites that are specific
targets where classical estrogen replacement therapy is beneficial.
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Sources of isoflavones.
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Unlike the ubiquitously occurring lignans, another class of
phytoestrogens considered important to human health (Setchell et al. 1980
), isoflavones are found almost exclusively in legumes. The
soybean, in particular, provides the most abundant source of
isoflavones, and therefore most soy foods will provide a significant
dietary source of these bioactive nonnutrients (Coward et al. 1993
,
Murphy 1982
, Reinli and Block 1996
).
Isoflavones occur predominantly as glycosides in plants and
consequently are highly polar (water-soluble) compounds (Walz 1931
).
Comprehensive analyses of the isoflavone content of numerous soy foods
have been reported and generally indicate that most contain 0.1–3.0
mg/g of total isoflavone (Coward et al. 1993
, Murphy 1982
). Soy germ
products derived from the hypocotyledon provide one of the most
concentrated (>20 mg/g) sources of isoflavones. Numerous commercial
soy supplements, many of which are made from concentrated extracts of
the soybean, are now available, circumventing the need for nutrition as
a source of isoflavones. The clinical effects of isoflavone
supplements, however, have yet to be fully evaluated, and there are
obvious concerns regarding the potentially adverse effects that could
result from megadosing with these bioactive compounds, a practice all
too common in the supplement area (Setchell et al. 1997
).
Although a high proportion of foods contain soy products, these are
mostly soy oils and soy lecithin; these soy products are devoid of
isoflavones, and the average daily dietary intake of isoflavones in
Western populations is typically negligible (<1 mg/d). Isoflavones
migrate with the protein fraction of the soybean during its processing,
and because soy protein is rarely a normal component of the average
Western diet, this accounts for the low daily intake. The rapidly
changing eating trends in Japan or China now make it difficult to make
an accurate determination of the intake of isoflavones in these
countries in which soy is traditionally a staple. Recent estimates
indicate intakes of 20–50 mg/d (Nagata et al. 1998
), but this may vary
between urban and rural areas, and with generational and other
lifestyle factors.
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Absorption and metabolism.
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The chemical form in which isoflavones occur is an important
consideration because it may influence the biological activity, the
bioavailability, and therefore the physiologic effects of these dietary
constituents. We showed almost two decades ago that intestinal
microflora play a key role in the metabolism and bioavailability of
both lignans and isoflavones (Borriello et al. 1985
, Setchell et al. 1984
). After ingestion, soybean isoflavones are hydrolyzed by
intestinal glucosidases, which release the aglycones, daidzein,
genistein and glycitein (Fig. 3
).These may be absorbed or further metabolized to many specific
metabolites including, equol and p-ethylphenol (Axelson et al. 1984
, Bannwart et al. 1984
, Joannou et al. 1995
, Kelly et al. 1993
). The extent of this metabolism appears to be highly variable
among individuals and is influenced by other components of the diet
(Setchell et al. 1984
). A high carbohydrate milieu, which causes
increased intestinal fermentation, results in more extensive
biotransformation of phytoestrogens, with greatly increased formation
of equol (Fig. 4
),a mammalian isoflavone metabolite of daidzein (Cassidy 1991
). This
metabolic pathway may be clinically relevant to the efficacy of soybean
isoflavones because the estrogenic potency of equol is an order of
magnitude higher than its precursor, daidzein (Shutt and Cox 1972
). The
importance of the microflora in the metabolic handling of
phytoestrogens is illustrated from observations that antibiotic
administration blocks metabolism, germfree animals do not excrete the
metabolites and infants fed soy infant formulas in the first 4 mo of
life, when gut microflora are underdeveloped, cannot form appreciable
amounts of equol (Axelson and Setchell 1981
, Cruz et al. 1994
, Setchell et al. 1997 and 1998
).
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Figure 3. Schematic showing the major biotransformations in the metabolism of
isoflavones in humans and animals.
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Figure 4. In vitro metabolism of daidzein in a colonic model of fermentation of
human fecal flora showing the influence of a high carbohydrate milieu
on the rate of conversion of daidzein to the intestinal bacterially
derived metabolite equol.
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We have determined the plasma half-life of daidzein and genistein,
measured from their plasma appearance and disappearance curves, to be
7.9 h in adults; peak concentrations occur 6–8 h after
administration of the pure compounds (Setchell 1998
). Consequently,
adherence to a soy-containing diet will ultimately lead to high
steady-state plasma concentrations. Knowledge of the pharmacokinetics
of the isoflavones in soy foods (or supplements) is essential in making
recommendations regarding longer-term efficacy in clinical studies.
Clearly, maintenance of steady-state plasma concentrations should be
achieved; on the basis of our data for clearance rates, this is best
accomplished by divided doses of the soy food or supplements, rather
than by single daily intakes. The pharmacokinetic behavior of
phytoestrogens contrasts with that of the environmental xenoestrogens
which, because of their very long half-lives, bioaccumulate and persist
in fat tissues for years. It is this difference that may explain in
part the potential dangers of synthetic xenoestrogens as endocrine
disruptors (Safe 1995
).
Plasma concentrations of 50–800 ng/mL are achieved for daidzein,
genistein and equol in adults consuming modest quantities of soy foods
containing ~50 mg/d of total isoflavones. These values are similar to
the plasma concentrations of Japanese consuming their traditional diet
(Adlercreutz et al. 1993b
). In infants fed soy formulas and ingesting
similar daily intakes, plasma concentrations are even higher (Setchell et al. 1997
). Overall, when soy is consumed on a regular basis, plasma
isoflavone levels far exceed normal plasma estradiol concentrations,
which in men and women generally range between 40 and 80 pg/mL. It was
this early observation that led us to hypothesize that with such
disproportional levels one could anticipate hormonal effects from
phytoestrogens (Setchell et al. 1984
); these were subsequently
established in premenopausal women adhering to a diet of soy protein
(Cassidy et al. 1994 and 1995
). This comes as no surprise because there
are well-documented examples, mostly deleterious, of hormonal effects
in several animal species resulting from ingesting phytoestrogens
(Bennetts et al. 1946
, Setchell et al. 1987a
).
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Clinical effects of isoflavones.
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Further research is required to characterize the pharmacokinetics of
isoflavones. The optimal dose of isoflavone required to have clinical
effects remains to be established for most of the hormone-dependent
conditions under investigation. Results from our previous human studies
in healthy premenopausal women indicate that 50 mg/d of aglycones is
sufficient to have significant endocrine effects (Cassidy et al. 1994
),
whereas half this dose appears biologically inactive (Cassidy et al. 1995
). Dose and duration of intake will likely be the major factors
that influence the clinical and biological outcome of a
phytoestrogen-rich diet. Rigorous compliance to dietary regimens of soy
foods containing isoflavones remains a major challenge in clinical and
nutritional studies. Improvements in food technology are required, and
genetic modifications of the soybean and other plants to enhance
isoflavone production seem inevitable.
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Premenopausal women.
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Controlled intervention studies in premenopausal women provide direct
evidence to suggest that diets containing phytoestrogens can produce
estrogenic effects (Cassidy et al. 1994 and 1995
). A daily intake of
textured vegetable protein (TVP) containing 45 mg of isoflavones
modified characteristics of the menstrual cycle of healthy
premenopausal women by prolonging its length, specifically the length
of the follicular phase and suppressing the magnitude of the normal
midcycle surge in follicle-stimulating hormone (FSH) and luteinizing
hormone (LH). This effect did not occur with an isoflavone-free soy
protein, thus providing evidence that dietary soy protein–containing
phytoestrogens have an endocrine-modulating effect and that this occurs
at the level of the hypothalamic-pituitary-gonadal axis. Similar
effects on menstrual cycle length from dietary phytoestrogens have been
reported by others; however, in one study it was a lengthening of the
luteal phase that was reported (Phipps et al. 1993
). This is difficult
to explain because changes in menstrual cycle length are confined
almost exclusively to the follicular phase; luteal phase length is
extremely constant and difficult to modify (Ferrin et al. 1993
).
Menstrual cycle length, as epidemiologic data show, is one of the risk
factors for breast cancer; the reasons for this association are unclear
(Wu et al. 1996a
). Mean cycle length in Western countries, in which
breast cancer risk is high, is 28–29 d, whereas the average length of
the menstrual cycle is 32 d in Japanese women, for whom breast
cancer risk is four to five times lower (Henderson et al. 1985
).
Interestingly, plasma circulating estrogen concentrations in Asian
women are 20–30% lower than those in Western women (Bernstein et al. 1990
, Key and Pike 1988
, Key et al. 1990
; Shimizu et al. 1990
, Wang et al. 1991
), and the combined effect of longer menstrual cycles with
lower estrogen concentrations translates to an overall lower integrated
lifetime exposure to estrogens. Whether this may be a factor in
explaining the lower risk of breast cancer in Asian women is
speculative. Critical studies of the effects of phytoestrogens on
estrogen status and metabolism in premenopausal women remain to be
undertaken, but the facts that dietary isoflavones can modulate
endocrine status and influence ovarian cyclicity appear to be favorable
with regard to potential breast cancer prevention. In this regard, it
is of interest that the commonly used chemotherapeutic antiestrogen,
tamoxifen, which also has similar effects on the endocrine regulation
of the menstrual cycle, is in use as a pharmacologic means of
preventing breast cancer in women at high risk for this disease (Jordan et al. 1987
, Jordan 1997
, Powles 1997
).
Phytoestrogen concentrations in the urine and plasma of Japanese women
consuming a traditional diet are high, as are those of vegetarians
(Adlercreutz et al. 1991 and 1993a
); the incidence of breast,
endometrial and ovarian cancers in these groups is low. Almost 15 years
ago, it was shown that breast cancer patients excreted lower amounts of
phytoestrogens compared with similarly aged subjects who did not have
breast cancer (Adlercreutz et al. 1982
). More recently, a case-control
study confirmed the idea that phytoestrogen-rich diets may offer
protective benefits, by demonstrating an inverse relationship between
the odds-adjusted risk for breast cancer and urinary phytoestrogen
excretion (Ingram et al. 1997
). This latter finding was surprising
given that the total phytoestrogen intake of the subjects in this
study, which can be roughly estimated from the urinary phytoestrogen
excretion, was clearly negligible (Messina et al. 1997
).
Interest in phytoestrogens as natural anticancer agents was stimulated
from animal studies using the classical animal model of chemically
induced breast cancer. In this model, soy protein–containing
isoflavones were found to reduce tumor formation significantly in a
dose-dependent manner (Barnes et al. 1990
). This effect was completely
abolished with soy protein that was devoid of isoflavones. These animal
studies are supported by numerous in vitro studies that have shown that
daidzein and genistein can inhibit cell growth (Adlercreutz 1995
,
Barnes 1995
). The effects, however, may not be due entirely to their
hormonal actions (Barnes and Peterson 1995
). Soon after these animal
studies were reported, genistein was shown to be a specific and quite
potent inhibitor of many tyrosine kinases that are involved in the
regulation of cell growth (Akiyama et al. 1987
). More recently,
genistein has been found to augment transforming growth factor-ß, an
essential growth factor that inhibits the cell cycle (Kim et al. 1998
)
and therefore progression of cell growth. Genistein can also influence
key transcription factors that are involved in the expression of stress
response–related genes involved in programmed cell death (Zhou and Lee 1998
). Like flavonoids, the isoflavones possess antioxidant activity,
which offers further potential protective actions for phytoestrogens
(Ruiz-Larrea et al. 1997
, Wei et al. 1995
). The antiproliferative
effects of phytoestrogens on breast cancer cells in culture (reviewed
in Adlercreutz 1995
, Barnes 1995
) together with reported antiangiogenic
effects, albeit at supraphysiologic concentrations (Fotsis et al. 1993
,
Jaggers et al. 1996
), provide further stimulus for the interest in
these compounds as potential anticancer agents. There is, however, a
danger in trying to simplify the explanations for the many positive
effects observed for phytoestrogens in cancer studies to a single
mechanism of action. It is more likely that the beneficial effects of
these compounds, particularly in vivo, are the result of multiple
actions that are of both a primary and secondary nature.
Despite the euphoria surrounding the potential value of phytoestrogens
in cancer prevention, supporting clinical data are lacking at present.
Prospective large-scale clinical studies will be required to address
this issue because the epidemiologic data (reviewed in Messina et al. 1994
) are not convincing. This may be, in part, because these
epidemiologic studies did not initially question the role of
phytoestrogens and their relationship with breast cancer. Six recently
reported case-control studies examined soy intake and breast cancer
risk (Hirayama 1986
, Hirohata et al. 1985
, Lee et al. 1991
, Nomura et al. 1978
, Wu et al. 1996b
, Yuan et al. 1995
); a significant reduction
in risk was found in three of the studies. It is conceivable that the
low incidence of hormone-dependent disease in populations consuming soy
as a staple may be more a function of lifetime exposure to
phytoestrogens, particularly from an early age (Setchell et al. 1997
),
and that this may program adaptive responses, thereby lowering
susceptibility to cancer later in life (Colditz and Frazier 1995
).
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Postmenopausal women.
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Epidemiologic data and clinical experience indicate that estrogen
therapy after the menopause offers protection from cardiovascular
disease, reduces the extent of osteoporosis, improves cognitive
function and relieves menopausal symptoms associated with acute ovarian
estrogen loss (Col et al. 1997
). Given the poor compliance with
conventional HRT that is driven by a fear of possible increased risk of
developing breast cancer, and because of side effects (Breckwoldt et al. 1995
, Zumoff 1993
), alternative sources of exogenous estrogen are
constantly being sought. Diet has been claimed to offer potential
relief from some of the symptomology of the menopause, with vegetarians
reporting fewer symptoms, although much of the evidence is anecdotal.
Hypothetically, soy isoflavones have the potential to provide an
exogenous source of estrogen, and the lower incidences of osteoporosis,
breast cancer and menopausal symptoms for women in countries consuming
soy as a staple have been attributed in part to the intake of
isoflavones (Adlercreutz et al. 1992
).
The incidence of hot flushes ranges from 70–80% in menopausal women
in Europe, to 57% in Malaysia and 18% in China (Sturdee 1997
). A
number of clinical trials of soy foods have been conducted in
postmenopausal women aimed at evaluating the effects on hot flushes and
vaginal cytology. Results and conclusions have been variable but
promising with regard to an estrogenic effect; however, a strong
placebo effect has been observed (Albertazzi et al. 1998
, Baird et al. 1995
, Brzezinski et al. 1997
, Murkies et al. 1995
). With a
phytoestrogen-rich diet, there was a 40–55% reduction in hot flushes
over a 12-wk period in three of these studies, whereas the control
diets resulted in a 20–35% reduction in hot flushes. Effects on
vaginal epithelia have been reported to range from increases in
maturation index to no significant effects (Baird et al. 1995
, Wilcox et al. 1990
). This histological endpoint, however, is highly operator
dependent for interpretation. Difficulties in comparing these studies
relate to the inconsistencies in the design of the trials and the
duration and type of diet used, particularly because the optimum intake
of isoflavones required to be effective is yet to be established. In
only one of these studies was the level of isoflavone measured in serum
(Brzezinski et al. 1997
), which may be an important endpoint to monitor
in view of the high interindividual variability in metabolism of
isoflavones. Further studies must address the issue of dose response;
however, given the difficulty of compliance to soy diets, it is
probable that this could best be done using supplements. At best, the
results from these initial clinical studies are promising and
indicative of an estrogenic effect.
Effects of soy diets on estrogen-sensitive biochemical markers have
been demonstrated in postmenopausal women. Wilcox et al. (1990)
showed
a modest suppression in FSH in a group of postmenopausal women after 6
wk of phytoestrogen-containing diets; in a separate study, a decrease
in plasma LH and a stimulation of plasma sex hormone binding globulin
(SHBG) and HDL cholesterol concentrations was observed (Gavaler et al. 1991
). Serum concentrations of SHBG, an important transport protein of
estrogen, were also raised significantly over a 12-wk period in
postmenopausal women consuming a phytoestrogen-rich diet comprising soy
foods and flaxseed (Brzezinski et al. 1997
). These findings contrast
with the lack of effect of phytoestrogens on SHBG levels in
premenopausal women (Cassidy et al. 1994 and 1995
), or men (Shultz et al. 1991
). In free-living trials in postmenopausal women, 60 g/d soy
given over a 4-wk period led to a suppression of LH levels, but there
was no significant effect on serum SHBG or estradiol concentration
(Cassidy et al. 1998
). Collectively, these studies suggest that
phytoestrogens are able to act as weak estrogens, particularly in the
presence of the low endogenous estrogen status of the postmenopausal
woman.
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Cardiovascular health.
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Mortality from cardiovascular disease is similar in men and women, and
heart disease is the major cause of death in postmenopausal women.
Estrogen deficiency is associated with significant alterations in
lipoprotein metabolism, with serum cholesterol concentrations
increasing markedly in the postmenopausal years. Preventing or reducing
the increase in serum cholesterol is associated with reducing risk for
cardiovascular disease. The cardioprotective effects of estrogen
replacement therapy are well established (Stampfer et al. 1991
) and are
mediated via effects on lipid metabolism (O'Brien and Nguyen 1997
),
which include a lowering of LDL cholesterol (LDLc) and increases in the
levels of HDL cholesterol (HDLc), and by direct actions on the blood
vessel wall (Sarrel 1990
). The hypocholesterolemic effect of soy
protein has been recognized for more than 30 years (reviewed in Carroll 1982
). Animal studies show that substituting soy protein for dietary
animal protein reduces serum total and LDLc concentrations (Sirtori et al. 1993
). Studies in rhesus monkeys show that soy has favorable
effects on plasma lipid and lipoprotein concentrations (Anthony et al. 1996 and 1997
). Similar responses are seen with acute loss of estrogen
as in the ovariectomized rat model (Arjmandi et al. 1997
).
Although the clinical response to soy protein in terms of its effect on
plasma lipids has been variable and highly dependent upon the initial
serum cholesterol level, a meta-analysis of 38 clinical studies
concluded that the mean reduction in serum total cholesterol was 9.3%,
whereas LDLc decreased an average of 12.9% with soy protein (Anderson et al. 1995a
). The effect on HDLc was marginal; a small increase was
observed. The average intake of soy protein was 47 g, and not
surprisingly, the cholesterol-lowering effect was greatest in those
subjects with the highest entry level serum cholesterol. In some cases
soy protein may be as effective as the statins, the HMG CoA reductase
inhibitors, at lowering elevated serum cholesterol, and the FDA is
currently reviewing a food health claim for soy protein in lowering
serum lipids. A mean 9.6% reduction in total serum cholesterol was
observed in healthy premenopausal women with normal cholesterol levels
when 60 g of soy protein was given daily over a 1-mo period
(Cassidy et al. 1994 and 1995
), whereas increases of 14% in HDLc
occurred over a 4-wk period of soy intake by postmenopausal women in
similarly designed studies (Cassidy et al. 1998
). Ingestion of 60 g/d
of a soy protein isolate by normocholesterolemic men resulted in no
significant changes to plasma lipids even though plasma isoflavone
concentrations were raised (Gooderham et al. 1996
).
The exact mechanism of the hypocholesterolemic effect of soy protein
remains elusive and is almost certain to be multifactorial. Soy protein
has no cholesterol; it has the effect of increasing fecal bile acid
excretion and altering bile acid synthesis rates, one of the primary
mechanisms responsible for the regulation of cholesterol homeostasis;
hepatic cholesterol secretion is also increased (Duane 1997
). Soy
isoflavones may also exert their effects by upregulating LDL-receptor
activity; recent evidence from animal studies using a LDL-receptor
knock-out C57BL/6 mouse model, established that in the absence of the
LDL receptor, the effect on plasma lipids and on atherosclerosis seen
in wild-type animals by soy isoflavone administration was abolished
(Kirk et al 1998
). Some time ago, it was suggested that isoflavones,
because of their estrogenic activity, might play a role in modulating
lipoprotein metabolism (Setchell 1985
); substantial support for this
theory has been confirmed in studies from Clarkson's group (Anthony et al. 1996 and 1997
, Anthony and Clarkson 1998
). Crouse et al. (1998)
studied 156 patients, both men and women, with moderately elevated
serum total and LDLc who were randomized to receive 25 g of a soy
protein beverage containing differing amounts of isoflavones in the
range 0–58 mg/d. Only the soy drinks containing isoflavones lowered
serum total cholesterol and LDLc; the reduction was ~10% over a 9-wk
period. Like earlier studies (reviewed in Anderson et al. 1995a
), the
largest effect was observed in those patients with the highest serum
cholesterol levels at entry. A linear dose-response relationship was
observed between isoflavone content and cholesterol reduction; the
hypocholesterolemic effect was lost when the isoflavones were removed
from the soy protein by alcohol extraction (Crouse et al. 1998
). This
study suggests that the active component of soy may be the isoflavones.
However, it should be realized that alcohol will extract many other
components from soy protein, and therefore it is difficult to make
definitive conclusions regarding the exact component responsible for
the cholesterol-lowering effect, particularly because animal studies in
which isoflavones were added to casein failed to demonstrate a similar
cholesterol-lowering effect to that of soy with comparable levels of
isoflavones. Nevertheless, these studies are supported by previous data
from monkeys, which similarly suggested that the isoflavone component
of soy protein accounts for a proportion of the hypocholesterolemic
effect (Anthony et al. 1997
). The magnitude of the cholesterol-lowering
effect of soy is similar to that observed for other plant-based foods
such as oat bran or garlic and is unlikely to be of major clinical
value as an exclusive means of treating patients with genetically based
hypercholesterolemia. However, the real potential of soy and
phytoestrogens appears to be in preventing the rise in serum
cholesterol, which is diet-induced in almost 40% of the adult
population, and therefore in lowering the overall risk for
cardiovascular disease. Interestingly, in a health checkup study of
1242 men and 3596 women living in Japan, a strong inverse relationship
(P < 0.0001) between serum cholesterol and daily
intake of soy products was found, with an average intake of soy protein
calculated from dietary questionnaires of 8.0 g for men and
6.88 g for women (Nagata et al. 1998
). This implies that the
intake of total isoflavones is likely to be 
25 mg on the basis of the
expected composition of isoflavones in the types of soy foods consumed.
In addition to effects on lipids, there may be other benefits from
phytoestrogens that are of relevance to reducing risk for
cardiovascular disease. The antioxidant properties, albeit weaker for
isoflavones than for flavonoids, which appear to have cardioprotective
effects, may contribute to reducing oxidation of lipids (Kapiotis et al. 1997
). Studies by Ruiz-Larrea et al. (1997)
showed that genistein
enhances resistance of LDLc to oxidation in vitro, and it is the most
potent antioxidant of the isoflavones in soy protein. Atherosclerosis,
a secondary response to hypercholesterolemia and dyslipidemia, severely
impairs coronary vascular reactivity. Honore et al. (1997)
have shown
in athersclerotic rhesus monkeys that isoflavones, like steroidal
estrogens, can enhance coronary vascular reactivity by increasing blood
vessel dilation, thereby improving blood flow. The mechanism of this
effect on the endothelium, which is presumed to be not merely by
effects on plasma lipids, is unclear. In vitro studies confirm that
genistein inhibits the process of coagulation, a key promoter of plaque
formation, and that this effect may be mediated through its inhibition
of growth factors such as platelet-derived growth factor and effects on
thrombin formation (Sargeant et al. 1993
, Wilcox and Blumenthal 1995
).
These effects may be the result of genistein's potent inhibition of
tyrosine kinase (Akiyama et al. 1987
) because this enzyme is central to
thrombin formation and inflammation in general. Animal studies have
shown a reduction in the extent of atherosclerosis in the internal
carotid artery of postmenopausal monkeys given phytoestrogens; this
effect was similar to that observed when premarin was used (Anthony and Clarkson 1998
).
|
Osteoporosis.
|
|---|
Ipriflavone is a synthetic isoflavone lacking estrogenic activity but
strikingly similar in chemical structure to daidzein and genistein; one
of its metabolites is daidzein (Yoshida et al. 1985
). When given at
doses of 600 mg/d, Ipriflavone prevents bone loss and increases bone
formation (Cheng et al. 1994
); this drug is approved as an alternative
to HRT for preventing bone loss in estrogen-deficient states (Brandi 1993
). The possibility that phytoestrogens may offer a natural
alternative to conventional HRT for the prevention of bone loss due to
estrogen deficiency associated with loss of ovarian function during the
menopause has led to a number of animal and clinical investigations of
the effects of either soy protein or genistein on bone remodeling.
Animal studies have used the ovariectomized rat as a model; although
this is not an ideal model for postmenopausal bone loss in women, the
findings are encouraging with regard to demonstrable protective effects
from phytoestrogens. At low doses, genistein was found to exert modest
effects on bone retention in the highly stressed oophorectomized
lactating rat model. Using a dose of 1 mg/d, genistein had an effect
similar to that of Premarin (5 g/d) in maintaining trabecular bone
tissue, but at higher doses (3.2 and 10 mg/d), this effect was lost
(Anderson et al. 1995b
). The bone-conserving effects of soy
protein–containing isoflavones were also confirmed in separate studies
using the ovariectomized adult rat; bone density was shown to be
greater in ovariectomized animals fed a soybean protein compared with
animals fed a casein-based diet (Arjmandi et al. 1996
). Coumesterol, a
coumestan, and the mycotoxin, zearalanol, two related phytoestrogens
that are rarely consumed in the human diet, were both shown to have
positive effects in conserving bone in the ovariectomized rat model
when given intramuscularly, although the observed effect was weaker
than that of estradiol (Draper et al. 1997
). Interestingly, these
investigators could not demonstrate any effect from an orally
administered isoflavone extract of clover (131.25 mg/wk), which
contained mainly formononetin and biochanin A, but only 9% of the
content was genistein. Nonestrogenic derivatives of coumestrol also
have similar effects (Tsutsumi et al. 1995
).
It would appear that the action of nonsteroidal phytoestrogens on bone
remodeling differs from that of classical estrogens (Blair et al. 1996
). The recent identification of the ERß in bone (Kuiper et al. 1998
), coupled with its ligand specificity toward phytoestrogens
(Kuiper et al. 1997
), may be of relevance in explaining the effects of
isoflavones in this tissue. Alternatively, these effects need not be
restricted to direct hormonal actions. Growth factors and cytokines
play a role in regulating osteoclast activity (Manolagas and Jilka 1995
), and several of these are tyrosine kinase–mediated pathways that
could conceivably be influenced by genistein. More recently it has been
suggested that genistein might stimulate the production of TGFß in
osteoclasts (Kim et al 1998
).
Limited clinical studies of phytoestrogens have thus far been
performed, but preliminary data suggest that the above effects seen in
animals are similarly observed in humans. Demonstrating effects on bone
density requires long-term studies, and compliance to soy foods is a
major problem that must be addressed in the design of human studies.
Potter et al. (1998)
found significant increases in bone mineral
content in postmenopausal women consuming isolated soy protein over a
6-mo period compared with a diet containing casein. In a short-term
study, Pansini et al. (1997)
examined biochemical markers of bone
turnover in 17 postmenopausal women and showed a 10 and 24% reduction
in the urinary excretion of D-pyrodinoline
(P < 0.05) and N-telopeptide
(P < 0.001), respectively, after 3 mo of a diet of
60 g of isolated soy protein. The incidence of
osteoporosis-related fractures is lower in Asia than in most Western
communities (Ho 1996
, Tobias et al. 1994
), but it is difficult to
discern whether this difference can be accounted for by the intake of
isoflavones from soy foods, particularly because there are many other
factors that can account for these epidemiologic findings.
Nevertheless, the potential clinical or nutritional use of
phytoestrogens as an alternative to HRT for preventing or limiting bone
loss in the peri- and postmenopausal years would be of major
significance in terms of women's health.
|
Men.
|
|---|
To date, there have been very few reported studies examining the
effects of phytoestrogens specifically in men. There appear to be no
clear gender differences in the bioavailability or metabolism of
isoflavones or lignans, and therefore many of the potential benefits
attributed to dietary phytoestrogens are probably applicable to men.
The hypocholesterolemic effects of soy protein have been demonstrated
in many studies of hypercholesterolemic adult men (see, for example,
Potter et al. 1993
), and these have been discussed above. Two dietary
intervention studies searched for evidence of hormonal effects in men,
and the results suggested only minimal effects. A study of middle-aged
men fed 60 g/d of TVP found no significant hormonal modifications
(Cassidy et al. 1998
), whereas 60 g/d of a soy isolate beverage had no
significant effects on serum cholesterol levels or platelet aggregation
(Gooderham et al. 1996
). These findings contrast with the significant
hormonal effects observed in women from similar intakes of soy foods;
the reasons for this gender difference in responsiveness are unclear.
Prostatic cancer is responsive to estrogen therapy, and it is known
that phytoestrogen intake is higher in countries in which the incidence
rates of prostatic cancer and other conditions linked to estrogen
exposure (hypospadia, testicular cancers) are low. Analysis of the
plasma and prostatic fluid from Asian men (Morton et al. 1997
), who
have a low risk for prostate cancer relative to European men, found
high concentrations of the isoflavones, equol and daidzein; genistein
was not measured. This type of data has consequently led to speculation
that dietary phytoestrogens may play a role in the reduced risk for
prostatic cancer that is evident from some epidemiologic studies. In
the absence of solid data from clinical studies, circumstantial
evidence in support of this contention comes only from invitro and animal studies.
In vitro, the isoflavone, genistein inhibits steroid 5-reductase and
17ß-hydroxysteroid dehydrogenase in genital skin fibroblasts (Evans et al. 1995
, Makela et al. 1995a
); these two enzymes are involved in
the synthesis of androgens and estrogens. Genistein and biochanin A
have both been shown to inhibit the growth of prostatic cancer cells in
vitro, irrespective of whether these were androgen-dependent or
-independent cell lines (Peterson and Barnes 1991 and 1993
).
The severity and incidence of prostatitis in the lateral lobe of the
prostate in a rat model of prostatitis were reduced when the animals
were fed a soy-rich diet (Sharma et al. 1992
). Inflammation may play a
key role in tumor formation by mechanisms involving cytokine release,
free radical formation and subsequent DNA damage, and a reduction in
the extent of inflammation may in general be considered to be
beneficial in reducing cancer risk. In subsequent studies, Pollard and Luckert (1997)
showed in a rat model that an isoflavone-rich diet
reduced the incidence of prostate cancer and prolonged the disease-free
period by 27%. However, these effects were observed only if the soy
was fed before the induction of cancer. Mice treated neonatally with
diethylstilbestrol showed hyperplastic and dysplastic changes in the
prostate, which were partially prevented by the addition of soy
isoflavones (Makela et al. 1995b
). However in adult male animals, no
estrogenic or antiestrogenic effects of soy were apparent (Makela et al. 1995c
).
Studies are necessary and will undoubtedly be carried out to establish
whether phytoestrogens exert consistent biological effects in men. This
will be particularly important given the current concerns,
controversial as they are, concerning the relationship between the
declining sperm count and environmental estrogen exposure (Sharpe and Skakkebaek 1993
).
|
Infants.
|
|---|
Few studies have examined the effects of phytoestrogens in infants, yet
this has been a particularly controversial area (Essex 1996
, Irvine et al. 1995
, Robertson 1995
). More than a decade ago, soy infant formulas
were first shown to contain significantly high levels (40 µg/mL) of
the isoflavones daidzein and genistein (Setchell et al. 1987b
). Similar
concentrations were confirmed in later studies of five commercially
available soy infant formulas (Setchell et al. 1997
), and exposure
levels for 4-mo-old infants fed these formulas exclusively were
calculated to be in the range of 6–11mg/(kg body weight · d).
This was confirmed in a subsequent study (Huggett et al. 1997
). This is
an order of magnitude higher than the dose of isoflavones consumed by
adults [<1 mg/(kg body weight · d)] with comparable intakes
of isoflavones (50 mg/d) from soy protein foods (Setchell et al. 1997
).
Absorption of isoflavones by the infant was clearly demonstrated from
the appearance of daidzein and genistein in the urine of 4-mo-old
infants fed soy formulas, although there was a high variability in the
concentrations excreted (Cruz et al. 1994
). Equol, a specific bacterial
metabolite of daidzein, was not detected in the urine (Cruz et al. 1994
); in later studies, it was either not detectable or present only
in traces in the serum of 4-mo-old infants fed soy infant formulas
(Setchell et al. 1997
), thus highlighting the requirement for a mature
intestinal flora for this biotransformation. Interestingly, equol was
found in the serum of infants fed cow's milk or human breast milk.
Cow's milk has been shown to contain isoflavones (Bannwart et al. 1988
), and the maternal transfer of isoflavones into human breast milk
has been demonstrated (Franke and Custer 1996
, Setchell et al. 1998
).
Isoflavone concentrations in human breast milk are <10 ng/mL
(mean ± SD, 5.6 ± 4.4 ng/mL); although
these values increase when lactating women consume a soy diet, the
contribution of phytoestrogens from human milk is still trivial
relative to that from soy infant formulas (Franke and Custer 1996
,
Setchell et al. 1998
). Contrary to what has been stated previously
(Slavin 1996
), human breast milk is not a useful source of
phytoestrogens. Concentrations of genistein and daidzein combined in
the plasma from 4-mo-old infants fed soy formulas were found to range
from 654 to 1775 ng/mL (mean 980 ng/mL), which is approximately an
order of magnitude higher than plasma concentrations reported for
adults consuming similar levels of isoflavones from soy foods, and
13,000- to 22,000-fold greater than plasma estradiol concentrations in
infants (40–80 pg/mL). The finding of high plasma concentrations of
daidzein and genistein is probably accounted for by the reduced
intestinal bacterial degradation of the ingested isoflavones, combined
with frequent feeding during the day, which would facilitate a high
steady-state circulating level, especially given the relatively long
half-life of plasma clearance (Setchell et al. 1997
). Although there is
evidence for reduced intestinal biotransformation of isoflavones by
infants (Cruz et al. 1994
, Setchell et al. 1997
), the presence of the
aglycones, genistein and daidzein in plasma indicates that there is
efficient hydrolysis of the glycosidic bond in early life. Over the
first 2 y of life, there is an age-dependent increase in the
activity of ß-glucosidase (Mykkanen et al. 1997
), and at 4 mo of age,
there is clearly sufficient activity to efficiently hydrolyze the
isoflavone glycosides that constitute the major fraction of
phytoestrogens of soy infant formulas (Setchell et al. 1997
).
The safety of soy-based infant formulas has been under scrutiny because
of the presence of phytoestogens and their potential for hormonal
actions at critical times of development. Data are lacking in infants
for biological effects from exposure to phytoestrogens, with the
exception of a study of cholesterol homeostasis showing that the
fractional synthesis rate of cholesterol is inversely related to the
urinary isoflavone concentration in infants fed different formulas
(Cruz et al. 1994
). Despite the use of these feedings for more than 30
years, there is no obvious evidence to support negative effects from
exposure to phytoestrogens. Two studies that used the classical animal
model of chemically induced mammary cancer have examined the effects of
early lifetime exposure to phytoestrogens (Lamartiniere et al. 1995
,
Murrill et al. 1996
). Prepubertal and neonatal rats exposed to
genistein were shown later in life to have increased resistance to
tumor formation, suggesting that early lifetime exposure may confer
protection later in life against the susceptibility to breast cancer.
It is conceivable that it may be the lifetime exposure to a diet rich
in phytoestrogens that explains the epidemiologic data showing a lower
incidence of hormone-dependent diseases in populations consuming high
intakes of soy and other plant-based foods. A strong case could
therefore be made for supplementing infant diets with phytoestrogens
rather than removing these phytoprotectants, as appears to be under
consideration (Janas & Ostrom 1998
).
|
CONCLUSION
|
|---|
Determining the mechanisms involved in explaining the potential
health benefits of a diet high in plant-based foods remains a major
challenge to nutritionists and scientists. Phytoestrogens represent
just one of many important bioactive nonnutrients found in many plants
commonly consumed in the human diet. The myriad of biological
properties that have been associated with phytoestrogens has resulted
in the current euphoria over their potential for the prevention and/or
treatment of many hormone-dependent diseases. Animal and in vitro
studies convincingly argue a case for positive effects from
phytoestrogens in many disease states. However, the clinical data
supporting many of the currently claimed health benefits of
phytoestrogens remain to be established definitively. Nevertheless, the
limited studies thus far performed in humans clearly confirm that diet
can have significant hormonal effects and that these may be of benefit
in the prevention of many of the common diseases seen in Western
populations in which the diet is typically devoid of these bioactive
nonnutrients.
|
FOOTNOTES
|
|---|
2 To whom correspondence should be 
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ABSTRACT
INTRODUCTION
