The health effects of soy and its constituent isoflavones are one of the most thoroughly researched fields in nutritional science. Over 2000 soy-related papers are published annually and more than one half are related to isoflavones.¹ Overall, soy foods are considered a heart-healthy source of protein because of their high content of polyunsaturated fats, fiber, vitamins, and minerals and low content of saturated fat.² The data related to specific health benefits of isoflavones—such as supporting bone health, decreasing menopausal hot flashes, improving cognitive function, and reducing the risk of certain types of cancers—are inconsistent. Several factors have been identified that contributed significantly to inconsistent results from early studies. These factors were rarely accounted for during the first 20 years of soy research (Box 124-1) and include individual variability of the human response to soy, differences in the isoflavone composition of different soy preparations, and the lack of analytic methods to assess soy constituents in foods, supplements, and biological fluids and tissues.³ The design and evaluation of future soy-food and isoflavone research will be guided by these realizations, and will produce higher-quality data to help answer remaining questions about the health benefits and safety considerations of consuming soy and its bioactive constituents.

BOX 124-1 Historic Overview of Soy and Isoflavone Research

Traditional Use

• Eleventh century BC: evidence of soy being consumed as a traditional food in China

• 1760s: Soy’s first recorded use in the United States

Evidence of Bioactivity

• 1940s: Breeding problems identified in Australian sheep grazing on red clover

• 1950s: Soy studied as a growth promoter in animal feed

• 1960s: Isoflavones established as phytoestrogens

Clinical Research Initiated

• 1970s: Tofu popularized as a health food in the United States

• 1990s: U.S. National Cancer Institute initiates a multimillion-dollar research program evaluating the anticancer effects of soy

Potential Benefits Explored

• 1990s: Research evaluates soy for hypocholesterolemic and mixed estrogen receptor properties, including potential benefits for postmenopausal hot flashes, bone health, and influence on hormone-sensitive cancers

• 1996: First isoflavone supplement becomes available

• 1999: FDA approves health claim for soy protein and coronary heart disease

New Evidence Emerges

• 2001: Researchers begin to explore impact of soy consumption on cognitive function

• 2002: Hypothesis introduced that individuals who possess equol-producing intestinal bacteria are more likely to benefit from soy-food consumption

Guidelines for Next Generation of Soy Research Established

• 2010: NIH identifies methodologic issues that confound soy and isoflavone study results and interpretation and publishes guidelines for the next generation of soy protein–isoflavone research

Data from Messina M. A brief historical overview of the past two decades of soy and isoflavone research. J Nutr 2010;140:1350S-1354S and Barnes S. The biochemistry, chemistry and physiology of the isoflavones in soybeans and their food products. Lymphat Res Biol 2010;8:89-98.

Chemical Composition of Soy

Interest in the constituents of soybeans, particularly soy protein and isoflavones, has catapulted soy to the status of a promising nutrient. Isoflavones remain in soy protein and other soy foods unless the preparation is extracted with alcohol.⁴ Commonly consumed soy foods—including soy milk, protein powders, and soy-based infant formula—are considered rich sources of dietary isoflavones.⁵ These are believed to be the most biologically active compounds in soy, but this has not been determined with certainty.

The principal isoflavones and most researched soy constituents in soy are genistein (4’,5,7-trihydroxyisoflavone) and daidzein (4’,7-dihydroxyisoflavone) (Figure 124-1). Glycetein is another isoflavone found in soy, but relatively little is published on its biological activity. In addition to isoflavones, soybeans contain lignans, coumestans, saponins, plant sterols, phytates, and protease inhibitors. The chemical composition, including the isoflavone content, of different soy-food preparations are variable and dependent on soybean strain, growing conditions, harvest time, and processing method (Table 124-1).

FIGURE 124-1 Structures of genistein and daidzein.

TABLE 124-1 Percentage and Ratio of Principle Isoflavones in Common Soy Products

Soy Product	Isoflavone Content	Genistein:Daidzein
Whole soybeans	0.1%-0.2%	1:1
Soy flour (defatted)	0.07%-0.3%	1:1
Soy germ	5%	1:4
Synthetic genistein	100%	1:0
Soy protein isolate	0.01%-0.3%	1:1
Tofu	0.02%-0.07%	1:0.7
Soy milk	0.001%-0.02%	1:1
Miso (dry)	0.02%-0.09%	1:0.9

Data from Empie MW. Compositional specificity in soy isoflavone supplements. soy/protein/isoflavone research: challenges in designing and evaluating intervention studies. NIH Workshop; Bethesda, Maryland: July 28-29, 2009.

Isoflavones

Classification

Flavonoids are a subgroup of a larger group of plant constituents, the polyphenols. Flavonoids are further differentiated into isoflavonoids, of which isoflavones are a subcategory (Figure 124-2). Isoflavonoids are not as ubiquitous in nature as some of the other flavonoids, such as flavones and flavonols. Approximately 600 isoflavonoids have been identified and further divided into subclasses according to the oxidation level of the central pyran ring. Isoflavones are the most abundant of the subclasses of isoflavonoids.

FIGURE 124-2 Polyphenol classification.

Absorption, Metabolism, and Excretion

The primary human exposure to isoflavones comes from the diet. Evidence suggests that the absorption and metabolism of isoflavones vary considerably among individuals.⁶ Factors that influence the bioavailability and possibly the bioactivity of isoflavones include absorption, metabolism by intestinal bacteria, liver metabolism, and enterohepatic circulation.

The native isoflavones in soy and unfermented soy foods—genistein and daidzein—occur conjugated to sugar moieties as glycosides and are not readily absorbed. After ingestion, isoflavone glycosides are hydrolyzed by intestinal brush border and bacterial glucosidases and released as the absorbable aglycone isoflavones genistein and daidzein. After absorption, the bioactive isoflavones are attached to glucuronic acid and made more water-soluble. It is predominantly the glucuronidated metabolites of isoflavones that circulate in the blood and are excreted in urine.⁷ In adults, genistein and daidzein can be further metabolized by bacteria to specific metabolites: equol, O-desmethylangolensis, dihydrogenistein, and p-ethylphenol. Emerging evidence suggests that the biological activity of isoflavones may be dependent, at least in part, on the bacterial conversion of isoflavones to corresponding metabolites such as equol, which is highly variable among individuals. It has been reported that 20% to 30% of Westerners have the intestinal bacteria necessary to metabolize daidzein to equol, whereas in Asian countries the frequency of equol producers is 50% to 60%.⁸ The hypothesis that individuals who possess the necessary bacteria to produce equol are more likely to achieve health benefits from soy-food consumption than those who do not is currently being investigated with great interest. Isoflavones attain maximal plasma levels 4 to 8 hours after ingestion and are excreted from the body within 24 hours, mainly in urine and to a lesser extent in feces.⁷

Isoflavone aglycones are thought to be more absorbable than their glucoside counterparts because aglycones have greater hydrophobicity and lower molecular weights. However, the relative bioavailability of isoflavone aglycones versus glucosides is debated. One study examined plasma and urinary concentrations of isoflavones and daidzein metabolites in healthy volunteers after ingesting soy milk, glucosidase-treated soy milk, and fermented soy milk. The researchers concluded that in humans, isoflavone aglycones, which are higher in fermented soy foods, were absorbed faster and in greater amounts than glucosides.⁹ Another study conducted on healthy male subjects (aged 20 to 40 years) found that urinary excretion of genistein and daidzein was greater after consumption of 112 g of tempeh, a fermented soy product, than after 125 g of unfermented soy pieces.¹⁰ These and other findings¹¹ seem to indicate that fermentation of soy products increases the bioavailability of the isoflavones. Other studies have reported conflicting results. One study reported the greater bioavailability of glycoside isoflavones, as measured from the area under the curve of plasma appearance and disappearance,⁷ whereas other studies have reported no significant difference in absorption between aglycone and glycoside isoflavones.^12,¹³

Isoflavone Content of Soy Products

A 2009 National Institutes of Health (NIH) workshop on designing, implementing, and reporting clinical studies of soy interventions has identified product composition and integrity as key challenges to accurately interpreting soy clinical studies.³ The type and proportion of isoflavones will vary depending on plant genetics, growing and harvesting conditions, storage before and after processing, plant parts used, and processing and extraction method. The typical isoflavone content and genistein:daidzein ratios from different soy foods are listed in Table 124-1. Differences in the isoflavone content of soy products may account for different biological effects and consequently contribute to the heterogeneous results observed among clinical and epidemiologic studies of soy foods.

Soy foods are the primary dietary source of isoflavones and the only foods that provide isoflavones in physiologically relevant amounts. Other plants, such as red clover (Trifolium pratense) and kudzu (Pueraria lobata), contain isoflavones, but these are generally not consumed via the diet. Traditional Asian diets provide a different isoflavone profile when compared with Western diets. Traditionally, Asians consumed minimally processed and fermented soy foods. Fermented soy foods account for approximately 30% of total soy-food consumption among Asians,¹⁴ which correlates with a higher consumption of isoflavone aglycones. Forms of soy consumed in Asia include boiled soybeans, miso soup with tofu, natto, and soy milk.¹⁵ Americans consume much less soy overall but also more processed soy foods, such as soy flour, textured vegetable protein, and isolated soy protein. Soy foods, including protein powders and soy-based infant formula, are considered rich sources of dietary isoflavones and contain 1 to 4.2 mg isoflavones per gram.⁵ Approximately 25% of formula-fed infants consume soy formula, and these infants have isoflavone concentrations that are significantly higher on a body-weight basis than do adults with high soy intake.¹⁶ The impact of early exposure to higher levels of isoflavones remains unclear. However, Asian populations whose isoflavone exposure begins earlier in life and continues throughout life have been the source of epidemiologic data demonstrating a relationship between soy consumption and health benefits.

Individuals from Western cultures consume approximately 1 to 2 mg of isoflavones daily,¹⁷ whereas total mean intake of isoflavones among Asian populations ranges from 25 to 50 mg daily.³ In 1999, the U.S. Food and Drug Administration (FDA) approved a food-labeling health claim for soy protein in the prevention of coronary heart disease,¹⁸ which subsequently led to a significant increase in retail soy-food consumption. Sales of U.S. soy foods increased from $2 billion in 1999 to $4.3 billion in 2005, and the number of soy-food products increased from hundreds to thousands.⁵ Soy is also increasingly being used in food processing. Soy flour and soy protein isolates are added to processed meats, meat substitutes, breads, and other processed foods, making these items additional hidden sources of dietary isoflavones. For a comprehensive list of foods and their isoflavones content, the reader is referred to extensive published lists.^19,²⁰ Overall, the combined effect of increased availability of frank soy-food products and hidden isoflavones in processed foods suggests an overall net increase in isoflavone intake among Western populations.

Dietary supplements are an additional source of isoflavones that contribute to an individual’s total isoflavone intake. Soy, red clover, and kudzu root are the primary materials used to manufacture isoflavone supplements. These three materials provide differing amounts and types of isoflavones. For example, red clover contains formononetin and biochanin A, which are methoxylated forms of genistein and daidzein. Kudzu contains high levels of daidzein and puerarin, another methoxylated derivative of daidzein (Table 124-2).

TABLE 124-2 Isoflavone Composition of Various Commercially Available Isoflavone Dietary Supplements

Other Soy Constituents

Soy is a source of a variety of bioactive compounds that include protein, lignans, coumestans, saponins, plant sterols, phytates, protease inhibitors, and isoflavones. There are over 100 different phytochemicals present in soy, and much is unknown about the potential interactions that occur among components within soy foods or supplement formulations. The types and amounts of isoflavones and other soy constituents will vary based on the initial source, processing, and storage conditions. Although isoflavones are believed to be the most biologically active compounds in soy, the presence or absence of other potentially active constituents may provide synergistic, additive, or other effects on the physiologic impact of a particular soy product. Common and potentially misleading assumptions in the interpretation of research on the biological actions of soy are that the effects of soy-food consumption reflect the activity of one or a small number of its components and, conversely, that the activity of a purified soy component reflects the effects of eating soy foods.

For a summary of soy constituents and their functions, see Table 124-3; for a detailed review, see Kang et al.²¹

TABLE 124-3 Soy Constituents and Their Functions

Constituent	Function
Protease inhibitors (Bowman-Birk inhibitor, Kunitz trypsin inhibitor)	Inhibit oncogene expression Inhibit chemically induced carcinogenesis Implicated in pancreatic hypertrophy (animal studies)
Lignans (enterolactone, enterodiol)	Phytoestrogen effects (agonistic/antagonistic) Antitumor Antiviral
Phytosterols, beta-sitosterol	Binds cholesterol in the gut
Coumestans (coumestrol)	Phytoestrogen effects (agonistic/antagonistic)
Saponins	Antioxidant Bind cholesterol and bile acids in the gut Antiviral (human immunodeficiency virus)
Phytates	Antioxidant Chelate metal ions (e.g., iron) Enhance natural killer cell activity
Isoflavones (genistein, daidzein, and their metabolites)	Phytoestrogen effects (agonistic/antagonistic) Antimutagenic Antioxidant Antiinflammatory Antiproliferative Antihypertensive Angiogenesis inhibition

Protease Inhibitors

Researchers have looked with interest at protease inhibitors (PIs) and their potential anticancer and antiinflammatory effects. Soybeans contain several possibly active PIs, including Bowman-Birk inhibitor (BBI), lunasin, and Kunitz trypsin inhibitor (KTI). BBI is particularly effective in suppressing carcinogenesis, and concentrated BBI, known as BBIC, was approved by the FDA as an Investigational New Drug in 1998.²²

Researchers have questioned the concept that PIs contribute significantly to the anticancer effects of soy. This is partly because raw and cooked soy products are equally effective in reducing cancer incidence even though heating destroys virtually all PI activity.²³ Another point to consider is that PIs are peptides, and ingested PIs (such as BBIC) may not survive digestion and reach target tissues intact. Data suggest that lunasin may be the actual bioactive cancer-preventive agent in BBIC, and that BBI simply protects lunasin when soybeans and other PI foods are eaten by humans.²⁴

Saponins

Saponins are found in many plants, including soybeans. They appear to have anticancer properties by virtue of their antioxidant and antimutagenic properties.³⁰ They are also known to reduce plasma and LDL cholesterol levels by affecting intestinal cholesterol absorption.³¹ It has been suggested that saponins found in soybeans have important synergistic functions and may contribute to the LDL-lowering effect of soy protein.³²

Phytates

Although phytic acid (inositol hexaphosphate) has been implicated in blocking the absorption of minerals, the phytate content of plants, including soy, seems to be responsible for some of the anticancer properties of vegetable-based foods. Phytic acid is a highly charged antioxidant, capable of scavenging hydroxyl radicals and chelating metal ions such as the prooxidant iron. Graf and Eaton ³³ reported the iron-chelating ability of phytate to be more important than the activity of fiber in the dietary prevention of colon cancer. Vucenik et al³⁴ reported antitumor effects of phytic acid both in vitro and in animal models.

Pharmacology

Soy constituents have been shown to have estrogenic,⁴ antiestrogenic,⁴ antiviral,³⁵ anticarcinogenic,³⁶^–³⁸ bacteriocidal, and antifungal³⁹ effects. Isoflavones have demonstrated selective estrogen-receptor modulating,⁴⁰ antimutagenic,³⁷ antioxidant,^41,⁴² mild antiinflammatory,⁴³ antihypertensive,⁴³ and antiproliferative effects.^38,⁴⁴

Hormonal Effects in Infants

Soy formula is an infant food made from soy protein and other soy components; it is fed to millions of infants worldwide as a supplement or replacement for human milk or cow’s milk. Soy formula has been shown to be comparable with milk-based formulas in supporting infant growth and development.⁴⁵ The potential for estrogenic effects to occur in infants fed with soy formula has raised the possibility of long-term safety considerations.

In the United States and Canada, approximately 25% of formula-fed infants are fed soy-based formulas.⁵ The range of total isoflavone content in soy formula has been reported to be 10 to 47 mg/L.^46,⁴⁷ Genistein is the predominant isoflavone found in soy formula (about 58% to 67%), followed by daidzein (about 29% to 34%) and glycetein (about 5% to 8%).⁴⁸ Infants fed soy formula also have higher blood levels of genistein and daidzein compared with other populations, with relatively high levels among vegan and Asian infants also consuming a traditional diet high in soy foods.⁴⁸ Data from the United States show that the concentrations of total genistein in blood samples from infants fed soy formula were higher than the maximum total genistein concentrations available for any other population. The average blood levels of total genistein in the soy formula-fed infants were approximately 160 times higher than the mean levels of total genistein in omnivorous adults in the United States, and a similar pattern was observed for urinary concentrations of genistein and daidzein.⁴⁹

Adverse effects of phytoestrogens on the development and reproductive capacity of livestock, wildlife, and experimental animals have been reported.⁵⁰ Historically, there have been relatively few studies of children fed soy formula, and data are insufficient to judge if soy feeding results in adverse health consequences. The Arkansas Children’s Nutrition Center is conducting a prospective longitudinal study comparing the growth, development, and health of breastfed children with those of children fed soy- and milk-based formulas from birth through age 6. After the first 5 years of the study, all of the children were found to be growing and developing within normal limits; there were no indications of adverse effects in the soy-fed children.⁴⁵ In 2009, the National Toxicology Program Center for the Evaluation of Risks to Human Reproduction convened an expert panel to evaluate soy formula. This panel reviewed and evaluated all available data on soy formula and concluded that there is minimal concern for adverse developmental effects in infants fed soy infant formula.⁴⁸

Millions of Asians have consumed large quantities of soy foods for hundreds of years without any apparent health risk and seemingly with health benefits. Long-term human studies of children fed soy formula and conclusions from the 2009 National Toxicology Program expert review panel collectively suggest that soy formula supports normal growth and is not associated with any adverse health effects.^45,⁴⁸ Healthy dietary choices during the early phases of life positively influence long-term health. Therefore, additional studies are needed to affirm the safety of soy-based formulas and to assess the potential beneficial effects of consuming phytoestrogens in the form of soy isoflavones early in life.

Hormonal Effects in Adults

Much of the interest in the biological activity of isoflavones relates to their structural similarity to estrogens. In the 1960s it was discovered that isoflavones had a weak binding affinity for estrogen receptor alpha. It was later determined that isoflavones had mixed estrogen receptor agonist-antagonist properties and that isoflavones preferentially bind to estrogen receptor beta,⁵¹ which has led some researchers to classify isoflavones as natural selective estrogen receptor modulators.⁴⁰ The binding of isoflavones to mammalian estrogen receptors is approximately 100 times weaker than the binding affinity of physiologic estrogens. However, this can be offset by higher circulating levels of isoflavones.⁵²

After ingestion, isoflavone glycosides are converted by gut bacteria in the intestines to compounds with molecular weights and structures similar to those of steroid hormones (Figure 124-3). The pattern of isoflavone and lignan excretion in the urine is similar to that seen with endogenous estrogens.⁵³ Hormonal effects of isoflavones have been evaluated in several human clinical trials with equivocal results. Overall, isoflavones appear to have a modest hormonal effect in women, but the clinical implications of these modest hormonal changes remain to be determined.