The Journal of Nutrition Supplement: Equol, Soy, and Menopause

A Brief Historical Overview of the Past Two Decades of Soy and Isoflavone Research1,2

Mark Messina*

Department of Nutrition, School of Public Health, Loma Linda University, Loma Linda, CA 92350; and Nutrition Matters, Inc., Port Townsend, WA 98368

Abstract

During the past 20 years, a remarkable amount of research into the health effects of soy consumption has been

conducted, which in large part can be attributed to the presence of isoflavones in the soybean. Isoflavones first came to Downloaded from the attention of the scientific community in the 1940s because of fertility problems observed in sheep grazing on a type of isoflavone-rich clover. In the 1950s, as a result of their estrogenic effects in rodents, isoflavones were studied as possible growth promoters for use by the animal feed industry, although shortly thereafter, it was shown that isoflavones could also function as antiestrogens. Despite this early work, it was not until the 1990s, largely because of research sponsored

by the U.S. National Cancer Institute, that the role of soyfoods in disease prevention began to receive widespread jn.nutrition.org attention. Subsequently, isoflavones and soyfoods were being studied for their ability to alleviate hot flashes and inhibit bone loss in postmenopausal women. In 1995, soy protein attracted worldwide attention for its ability to lower cholesterol. At this same time, isoflavones began to be widely discussed as potential alternatives to conventional hormone therapy. In

2002, it was hypothesized that individuals possessing the intestinal bacteria capable of converting the soybean isoflavone at American Society for Nutrition on June 18, 2010 daidzein into the isoflavan equol were more likely to benefit from soy intake. More recently, in vitro and animal research has raised questions about the safety of isoflavone exposure for certain subsets of the population, although the human data are largely inconsistent with these concerns. J. Nutr. 140: 1350S–1354S, 2010.

Asian populations have consumed foods made from soybeans to soy at this time because it was perceived as being a source of for centuries, whereas in the West, certain subpopulations, high-quality protein low in saturated fat that was more namely Seventh-day Adventists and vegetarians, have used efficiently produced than animal sources of protein. soyfoods for ~100 years, although the quintessential soyfood A dramatic increase in soyfood consumption during the last tofu was first introduced on a large scale to the general U.S. decade of the 20th century occurred because of the belief among population beginning only in the early 1970s. Health-conscious many consumers that soyfoods might offer health benefits and ecologically minded consumers were particularly attracted independent of their nutrient content. This increased interest is best viewed in the context of the general recognition underway 1 Published in a supplement to The Journal of Nutrition. Presented at the “Equol, at this time that plants contain large numbers of potentially Soy, and Menopause Research Leadership Conference,” held in Washington, beneficial nonnutritive biologically active components com- DC, June 16, 2009. The Supplement Coordinator for this supplement is Kara monly referred to as phytochemicals. This knowledge led to the Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The The concept of functional foods [initially referred to as designer supplement is the responsibility of the guest editors to whom the Editor of 3 Journal of Nutrition has delegated supervision of both technical conformity to foods by the National Cancer Institute (NCI) ] and to soy being the published regulations of The Journal of Nutrition and general oversight of the one of the first foods widely acknowledged to fall into this scientific merit of each article. Publication costs for this supplement were category. Like all foods, the soybean contains a number of defrayed in part by the payment of page charges. This publication must therefore biologically active components, many of which are being be hereby marked “advertisement" in accordance with 18 USC section 1734 solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. actively investigated including, e.g., saponins and lunasin, but Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement unquestionably it is the isoflavones that are responsible for so Coordinator disclosure: Kara Lewis is currently under contract with and receives much of the scientific interest in this legume. compensation from the supplement sponsor. She was also compensated for Isoflavones, which have been known to exist in plants attending and organizing the Equol, Soy, and Menopause Research Leadership . Conference and for organizing, writing, editing, or reviewing, and collection of for 100 years, have a relatively limited distribution in nature supplemental manuscripts. The opinions expressed in this publication are those such that among commonly consumed foods by humans, they of the authors and are not attributable to the sponsors or the publisher, Editor, or are found in physiologically relevant amounts only in soybeans Editorial Board of The Journal of Nutrition. and foods derived from this legume (1), although a variety of 2 Author disclosure: M. Messina regularly consults for companies that manu- plants such as red clover (2) are also rich sources. Consequently, facture and/or sell soyfoods and/or isoflavone supplements and is the Executive Director of the Soy Nutrition Institute, an organization funded by the soy industry and the United Soybean Board. 3 Abbreviations used: ER, estrogen receptor; NCI. National Cancer Institute; * To whom correspondence should be addressed. E-mail: [email protected]. ODS, Office of Dietary Supplements.

1350S ã 2010 American Society for Nutrition. First published online May 19, 2010; doi:10.3945/jn.109.118315. isoflavone intake among older adults in Japan and Chinese reduce risk of hormone-sensitive cancers as a result was already cities such as Shanghai is ~40 mg/d (3), whereas in Europe and part of the scientific literature in the 1960s (23–26). the United States it is likely no more than 3 mg/d (4–10). In 1990, there was sufficient preliminary evidence for the Although soy protein has also been the subject of considerable NCI to sponsor a workshop on the role of soy in reducing cancer investigation, especially in regard to its hypocholesterolemic risk (34). The findings from this meeting led the NCI to initiate effects (11), recent scientific interest in soy largely parallels the a multi-million dollar research program evaluating the antican- interest in isoflavones. Of the ~2000 soy-related papers cur- cer effects of soyfoods. This declaration of interest, which was rently published annually, more than one-half are related to largely based on the proposed chemopreventive effects of isoflavones. isoflavones, greatly increased interest in both soy and isoflavones Isoflavones, like many phytochemicals of interest to nutri- in a wide range of areas. One of these areas was the alleviation of tionists, are phytoalexins, substances formed by the host tissue menopausal symptoms. in response to physiological stimuli, infectious agents, or their In 1992, Adlercreutz et al. (35) were the first to suggest that products, which accumulate to levels that inhibit the growth of soyfoods, because they contain isoflavones, might at least microorganisms (12). Isoflavones possess properties (i.e. anti- partially account for why Asians and Japanese women in fungal, antimicrobial, and antioxidant) that enhance the survival particular were less likely to report experiencing menopause- of the soybean (12). For this reason, soybean isoflavone related hot flashes. The first trial to examine this hypothesis was concentrations increase greatly in times of stress, such as when published in 1995 (36); since then, .50 trials evaluating the moisture is limited, and are influenced by the environmental efficacy of isoflavone-containing products have been conducted conditions under which the soybean is grown (13,14). (see references for reviews) (37,38). Downloaded from In many respects, the biological effects of isoflavones first Upon reflection, it is now apparent that in the relatively short came to the attention of the scientific community in the 1940s recent history of isoflavone and soy research, 1995 was a because of breeding problems experienced by female sheep in seminal year. In that year, Anderson et al. (11) published a meta- Western Australia grazing on a type of clover rich in isoflavones analysis that attracted widespread attention to the hypocholes- (15–17). Three decades later, Setchell et al. (18) established that terolemic effects of soy protein, although Italian investigators

isoflavone-rich soy, which was part of the standard diet of had demonstrated dramatic reductions in cholesterol in hyper- jn.nutrition.org cheetahs in North American zoos, was a factor in the decline of cholesterolemic participants in response to soy protein as early fertility in these animals. It is easy therefore to understand why as the late 1970s (39,40). In many respects, the meta-analysis nutritionists, if they thought of isoflavones at all at this time, indirectly led to the approval by the U.S. FDA of a health claim viewed them largely as antinutrients. Interestingly, in the 1950s, for soy protein and coronary heart disease 4 years later (41). isoflavones were being studied by the animal feed industry as Interestingly, Anderson et al. (11) suggested that isoflavones at American Society for Nutrition on June 18, 2010 possible growth promoters because of their reported estrogenic might account for 60–70% of the cholesterol-lowering effects of effects in rodents (19–22). By the 1960s, the determination of soy protein; as a result of this suggestion, considerable investi- the relative binding affinities of isoflavones for estrogen gation of the hypocholesterolemic properties of isoflavones was receptor (ER) alpha helped firmly establish these soybean undertaken (42). Although there is only weak support for the constituents as phytoestrogens (23,24). cholesterol-lowering effects of isoflavones, and the cholesterol- For the most part, there was little interest in isoflavones lowering potency of soy protein is less than initially thought within the nutrition community throughout the 1980s. One (43,44), the effects of isoflavones on a variety of coronary heart notable exception is the now-classic work by the pioneering disease risk factors have been extensively evaluated (45). For isoflavone researcher, Kenneth D. R. Setchell, who showed that example, in 2001, Walker et al. (46) were the first to show in response to soy consumption, isoflavone excretion increased that the isoflavone genistein markedly increased nitric oxide- dramatically and that only a minority (;25% of Westerners) of dependent dilation in forearm vasculature. participants possessed the intestinal bacteria capable of con- In the same year in which Anderson et al. (11) published their verting the soybean isoflavone daidzein into the isoflavan equol meta-analysis, a Wake Forest University research group, ex- (25,26). Sixteen years later, Setchell et al. (27) proposed that tremely active in the soy field, helped popularize the notion that equol was an especially beneficial compound and that those isoflavones possessed mixed ER agonist/antagonist properties individuals who possessed equol-producing intestinal bacteria and were a possible alternative to conventional hormone were more likely to benefit from soyfood consumption than therapy (47). Soon thereafter, investigators began referring to those who did not. This hypothesis is currently a very active area isoflavones as natural selective ER modulators (48), a classifi- of research. cation that gained support from the identification of the second The view toward isoflavones as only being phytoestrogens ER, ERb (49), and the finding that isoflavones preferentially required modification as a result of Akiyama et al. (28) bind to ERb compared with ERa (50,51). Later work demon- serendipitously discovering in 1987 that genistein, the primary strated that isoflavones also preferentially transactivate ERb [for soybean isoflavone, was an inhibitor of protein tyrosine kinase, a review, see Reiter et al. (52)]. an enzyme frequently overexpressed in cancer cells (29). Since A final landmark development in 1995 was the publication of then, genistein has been extensively studied for its ability to animal research by Lamartiniere et al. (53–55) showing that affect a diverse array of intracellular signaling cascades (30,31) isoflavone exposure early in life reduces breast cancer risk during that control cell growth (30). As a result of the protein tyrosine adulthood. In addition to the animal studies, this hypothesis has kinase finding, it became clear that isoflavones were complex considerable epidemiologic support (56–59) and is consistent molecules that could no longer be viewed simply as phytoes- with an emerging school of thought that emphasizes the trogens and that soyfoods might account for the low incidence important role early life events have in the etiology of breast of breast cancer in Japan, a notion supported by animal (32) cancer (60–62). and epidemiologic (33) research published in the early 1990s. Not surprisingly, given the recognized role of estrogen, there As already noted, although largely overlooked, the ability of has been considerable interest in the potential skeletal effects of isoflavonoids to function as antiestrogens and thus possibly isoflavones, although in 1996, the investigators responsible for

Overview of soy and isoflavone research 1351S publishing the first animal study to demonstrate skeletal benefits Finally, there is recognition of the need to more precisely suggested it was the ability of genistein to inhibit tyrosine identify those factors contributing to the inconsistent clinical protein kinase activity that was responsible for this effect (63). data such as interindividual differences in isoflavone metabo- That year, the first rodent study was published showing lism, the health status (at-risk compared with normal risk, isoflavone-rich soy protein improved bone mineral density (64) healthy compared with unhealthy) and metabolic profile (i.e. and 2 years later the first clinical study showing this was the case receptor polymorphisms) of study participants and especially, in postmenopausal women appeared in the literature (65). Since differences in the chemical composition of intervention pro- then, .30 trials have examined the effects of isoflavone- ducts. Establishing those variables that play such a role may go containing products on bone mineral density in postmenopausal a long way toward achieving a more precise understanding of women (see references for reviews of the literature) (66,67). The the health effects of soyfood and isoflavone consumption. ability to conduct clinical trials, especially those longer in duration, was greatly aided by the development of isoflavone Acknowledgment supplements, which first became available in 1996. The sole author had responsibility for all parts of the manuscript. Along with research of the possible skeletal benefits of isoflavones for postmenopausal women, there has been interest in understanding the impact of soy on cognitive function. The first clinical trial in this area that reported a benefit was Literature Cited published in 2001 (68). One year before this publication an prospective epidemiologic study, whose primary endpoint was 1. Franke AA, Custer LJ, Wang W, Shi CY. HPLC analysis of isoflavonoids and other phenolic agents from foods and from human fluids. Proc Soc Downloaded from heart disease in men, found an association between tofu intake Exp Biol Med. 1998;217:263–73. and the development of cognitive impairment in older age (69). 2. Wang SW, Chen Y, Joseph T, Hu M. Variable isoflavone content of red However, other epidemiologic data do not concur with this clover products affects intestinal disposition of biochanin A, formono- observation (70). For a review of the clinical trials, the reader is netin, genistein, and daidzein. J Altern Complement Med. 2008;14: referred to the reference (71). 287–97. 3. Messina M, Nagata C, Wu AH. Estimated Asian adult soy protein and

Not surprisingly, the federal government has funded much of jn.nutrition.org the isoflavone research in the United States, but their involve- isoflavone intakes. Nutr Cancer. 2006;55:1–12. ment in this field is not limited to funding. For example, in 1999, 4. Horn-Ross PL, John EM, Canchola AJ, Stewart SL, Lee MM. Phytoestrogen intake and endometrial cancer risk. J Natl Cancer Inst. the USDA in conjunction with Iowa State University created an 2003;95:1158–64. online database of the isoflavone content of foods (72). Also in 5. Goodman-Gruen D, Kritz-Silverstein D. Usual dietary isoflavone intake 1999, The Office of Dietary Supplements (ODS), Office of is associated with cardiovascular disease risk factors in postmenopausal at American Society for Nutrition on June 18, 2010 Research on Women’s Health, and National Institute on Aging women. J Nutr. 2001;131:1202–6. sponsored a workshop to evaluate the effects of phytoestrogens 6. U.S. FDA. 2004Q–0151: Qualified Health Claim (QHC): soy protein on diseases affecting older men and women (73). In 2005, the and cancer. 2004 [cited 2009 Aug 12]. Available from: http://www. ODS sponsored a comprehensive review of the soy-related regulations.gov/search/Regs/home.html#searchResults?Ne=11+8+8053+ clinical literature, which was conducted by the Agency for 8098+8074+8066+8084+1&Ntt=2004Q–0151&Ntk=All&Ntx=mode+ matchall&N=0. Healthcare Research and Quality at Tufts University (74). And in 7. de Kleijn MJ, van der Schouw YT, Wilson PW, Adlercreutz H, Mazur W, July of 2009, the ODS convened a workshop aimed at providing Grobbee DE, Jacques PF. Intake of dietary phytoestrogens is low in guidance for future clinical research involving soy (75). postmenopausal women in the United States: the Framingham study(1– It would be remiss not to mention that although there 4). J Nutr. 2001;131:1826–32. continues to be considerable enthusiasm for the potential health 8. van Erp-Baart MA, Brants HA, Kiely M, Mulligan A, Turrini A, benefits of soyfoods and isoflavones, concerns about the safety Sermoneta C, Kilkkinen A, Valsta LM. Isoflavone intake in four of isoflavones, based largely on their estrogen-like properties, different European countries: the VENUS approach. Br J Nutr. 2003;89 Suppl 1:S25–30. have occurred in parallel. In fact, isoflavones are not just 9. van der Schouw YT, Kreijkamp-Kaspers S, Peeters PH, Keinan-Boker L, classified as phytoestrogens and mixed estrogen agonists/antag- Rimm EB, Grobbee DE. Prospective study on usual dietary phytoes- onists but also as endocrine disruptors (76–78). Evaluations of trogen intake and cardiovascular disease risk in Western women. isoflavone safety have been undertaken by governmental and Circulation. 2005;111:465–71. quasi-governmental agencies in several European countries as 10. Boker LK, van der Schouw YT, de Kleijn MJ, Jacques PF, Grobbee DE, well as in Japan and Israel and at the time of this writing, the Peeters PH. Intake of dietary phytoestrogens by Dutch women. J Nutr. European Food Safety Authority is currently conducting an 2002;132:1319–28. evaluation. Most notable among the concerns, which in all cases 11. Anderson JW, Johnstone BM, Cook-Newell ME. Meta-analysis of the are based almost exclusively on in vitro and animal research (the effects of soy protein intake on serum lipids. N Engl J Med. 1995; 333:276–82. human research, including both clinical and epidemiologic data, 12. Dakora FD, Phillips DA. Diverse functions of isoflavonoids in legumes are supportive of safety) is that isoflavone-containing products transcend anti-microbial definitions of phytoalexins. Physiol Molecular pose a risk to estrogen-sensitive breast cancer patients and Plant Pathol. 1996;49:1–20. women at high-risk of developing this disease (79) and that 13. Eldridge AC, Kwolek WF. Soybean isoflavones: effect of environment isoflavone exposure via the consumption of soy infant formula and variety on composition. J Agric Food Chem. 1983;31:394–6. may harm the long-term development of infants (80). The latter 14. Wang HJ, Murphy PA. Isoflavone composition of American and issue was reviewed by the National Toxicology Program, Center Japanese soybeans in Iowa: effects of variety, crop year, and location. for the Evaluation of Risks to Human Reproduction in 2006 J Agric Food Chem. 1994;42:1674–7. (81,82), and 2009 (83). At the most recent meeting the expert 15. Bennetts HW, Underwood EJ, Shier FL. A specific breeding problem of sheep on subterranean clover pastures in Western Australia. Aust J Agric panel concluded that there was minimal concern about the Res. 1946;22:131–8. safety of soy infant formula (83). For a discussion of the breast 16. Bradbury RB, White DR. Estrogen and related substances in plants. In: cancer (79,84) and infant formula issues (80,85,86), the reader is Harris RS, Marrian GF, Thimann KV, editors. Vitamins and hormones. referred to the references. New York: Academic Press; 1954. pp. 207–30.

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Overview of soy and isoflavone research 1353S 66. Liu J, Ho SC, Su YX, Chen WQ, Zhang CX, Chen YM. Effect of long- 76. Safe S. Endocrine disruptors and human health: is there a problem. term inervention of soy isoflavones on bone mineral density in women: a Toxicology. 2004;205:3–10. meta-analysis of randomized control trials. Bone. 2009;44:948–53. 77. Paganetto G, Campi F, Varani K, Piffanelli A, Giovannini G, Borea PA. 67. Ma DF, Qin LQ, Wang PY, Katoh R. Soy isoflavone intake increases Endocrine-disrupting agents on healthy human tissues. Pharmacol bone mineral density in the spine of menopausal women: meta-analysis Toxicol. 2000;86:24–9. of randomized controlled trials. Clin Nutr. 2008;27:57–64. 78. Wuttke W, Jarry H, Becker T, Schultens A, Christoffel V, Gorkow C, 68. File SE, Jarrett N, Fluck E, Duffy R, Casey K, Wiseman H. Eating soya Seidlova-Wuttke D. Phytoestrogens: endocrine disrupters or replace- improves human memory. Psychopharmacology (Berl). 2001;157:430–6. ment for hormone replacement therapy? Maturitas. 2003;44 Suppl 1: 69. White LR, Petrovitch H, Ross GW, Masaki K, Hardman J, Nelson J, S9–20. Davis D, Markesbery W. Brain aging and midlife tofu consumption. 79. Helferich WG, Andrade JE, Hoagland MS. Phytoestrogens and breast J Am Coll Nutr. 2000;19:242–55. cancer: a complex story. Inflammopharmacology. 2008;16:219–26. 70. Woo J, Cheung CK, Ho SC, Mak YT, Swaminathan R. Protein 80. Chen A, Rogan WJ. Isoflavones in soy infant formula: a review of nutritional status in elderly Chinese in Hong Kong. Eur J Clin Nutr. evidence for endocrine and other activity in infants. Annu Rev Nutr. 1988;42:903–9. 2004;24:33–54. 71. Zhao L, Brinton RD. WHI and WHIMS follow-up and human studies of 81. Rozman KK, Bhatia J, Calafat AM, Chambers C, Culty M, Etzel RA, soy isoflavones on cognition. Expert Rev Neurother. 2007;7:1549–64. Flaws JA, Hansen DK, Hoyer PB, et al. NTP-CERHR expert panel 72. USDA, Iowa State University. USDA-Iowa State University Database on report on the reproductive and developmental toxicity of genistein. the Isoflavone Content of Foods, release 1.3–2002. USDA. 2002 [cited Birth Defects Res B Dev Reprod Toxicol. 2006;77:485–638. 2009 Aug 13]. Available from: http://www.nal.usda.gov/fnic/foodcomp/ 82. Rozman KK, Bhatia J, Calafat AM, Chambers C, Culty M, Etzel RA, Data/isoflav/isoflav.html. Flaws JA, Hansen DK, Hoyer PB, et al. NTP-CERHR expert panel report on the reproductive and developmental toxicity of soy formula.

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1354S Supplement The Journal of Nutrition Supplement: Equol, Soy, and Menopause

Equol: History, Chemistry, and Formation1,2

KennethD.R.Setchell3*andCarloClerici4

3Pathology and Laboratory Medicine, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and 4Clinica di Gastroenterologia ed Epatologia, Universita` degli Studi di Perugia, Italy University of Perugia, Perugia 06156, Italy

Abstract

Equol, first isolated from equine urine in 1932 and identified 50 years later in human urine as a metabolite of the soy isoflavones, daidzin and daidzein, is produced by intestinal bacteria in some, but not all, adults. This observation led to the term equol-producers to define those adults that could make equol in response to consuming soy isoflavones and the hypothesis that the health benefits of soy-based diets may be greater in equol-producers than in equol nonproducers. By Downloaded from virtue of a chiral center, equol occurs as a diastereoisomer and intestinal bacteria are enantiospecific in synthesizing exclusively the S-(-)equol enantiomer, an enantiomer that has selective affinity for the estrogen receptor-b. Both enantiomers are of interest from a clinical and pharmacological perspective and are currently being developed as nutraceutical and pharmacological agents. The wide range of biological activities these enantiomers possess warrants their investigation for the treatment of a number of hormone-related conditions involving estrogen-dependent and jn.nutrition.org androgen-related conditions. The following review describes the history, chemistry, and factors governing the intestinal bacterial formation of equol. J. Nutr. 140: 1355S–1362S, 2010. at American Society for Nutrition on June 18, 2010

Introduction the autumn months, the amounts of equol declined and by It is now 57 years since the first report appeared describing a new winter it was impossible to isolate it from urine. The authors phenolic compound in an estrogenic fraction of pregnant mare concluded that, “so far as can be determined, no dietary factor urine (1). It was suggested that the compound be given the name was the cause of this (seasonal) variation…” (2). It later became equol, after the equine source of the material. Efforts to obtain apparent that this was not the case when in SWAustralia reports large-scale quantities of the compound led to the recognition emerged of a catastrophic “failure to breed” associated with that it was also present in appreciable amounts in the urine of uterine abnormalities and endometriosis in sheep grazing on stallions and nonpregnant mares and the conclusion that it was Trifolium subterranium clover (3). Reductions in sperm counts not associated with the presence of high estrogen states. During and motility were also documented in ewes (4). This clover disease, as it was so-called, was found to be the result of extremely high circulating concentrations of equol, formed by 1 Published in a supplement to The Journal of Nutrition. Presented at the “Equol, rumenal bacteria from the ingestion of large amounts of the Soy, and Menopause Research Leadership Conference”, held in Washington, methoxylated isoflavone, formononetin, abundant in several DC, June 16, 2009. The supplement coordinator for this supplement is Kara Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The indigent species of clover (5–7). Equol was even found as a supplement is the responsibility of the guest editors to whom the Editor of The component in urinary calculi of sheep and cattle (8). Equol has Journal of Nutrition has delegated supervision of both technical conformity to since been reported to be present in the urine and/or plasma of the published regulations of The Journal of Nutrition and general oversight of the many other animal species, including cows (9), hens (10–14), scientific merit of each article. Publication costs for this supplement were monkeys (15,16), chimpanzees (17,18), dogs (19), mice (20), defrayed in part by the payment of page charges. This publication must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 rats (20–22), and pigs (16,23), but there are marked differences solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. in the extent of metabolism of isoflavones into equol by these Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement species. Rodents, e.g., very efficiently convert daidzin/daidzein Coordinator disclosure: Kara Lewis is currently under contract with and receives to equol (24), whereas pigs and humans have been reported to compensation from the supplement sponsor. She was also compensated for attending and organizing the Equol, Soy, and Menopause Research Leadership do this less efficiently (16,24). In the decades leading to 1970, a Conference and for organizing, writing, editing, or reviewing, and collection of great deal of work was performed defining the metabolism of supplemental manuscripts. The opinions expressed in this publication are those isoflavones and biological actions of equol (6,25–28). While its of the authors and are not attributable to the sponsors or the publisher, Editor, or estrogenic effects were well documented based upon field Editorial Board of The Journal of Nutrition. 2 observations and classical bioassays, it was not until after the Author disclosures K. D. R. Setchell was supported by funding from the NIH 5 (grant nos. R01AT-003313 and R01AT-002190) and has intellectual property on discovery of the first estrogen receptor (ER) in the mid-1960s equol enantiomers, including patents licensed by Cincinnati Children’s Hospital (29) that the relative affinity of equol for the ER could be Medical Center, Cincinnati, OH, to industry and is a consultant to Otsuka Pharmaceuticals Company, Tokyo, Japan. C. Clerici, no conflict of interest. * To whom correspondence should be addressed. E-mail: kenneth.setchell@ 5 Abbreviations used: BBM, brush border membrane; ER, estrogen receptor; cchmc.org. ISP, isolated soy proteins; RMB, relative molar binding.

ã 2010 American Society for Nutrition. 1355S First published online June 2, 2010; doi:10.3945/jn.109.119776. quantified. When the relative molar binding affinities of a number of phytoestrogens for sheep uterine ER were compared, equol was found to have much higher affinity than its precursor daidzein in competing with radioactive estradiol for binding to the cytosolic receptor (27), supporting the theory that it may be advantageous to be able to convert daidzein to equol (24). There was little interest in equol for several decades until the chance discovery in 1980 of high concentrations of an unknown estrogen-like compound in rat urine (30) accompanying the mammalian lignans, enterolactone and enterodiol (31–33). At the time, it was referred to as compound 386/192, a notation for the molecular ion and base peak in the mass spectrum of its trimethylsilyl ether derivative. In common with most endoge- nous steroid hormones, including estrogens, it was conjugated predominantly to glucuronic acid and a lesser extent to sulfuric acid (34). Its presence in such high concentrations in rat urine fortuitously afforded a means of isolating sufficient quantities for structural elucidation studies by infrared spectroscopy, FIGURE 1 Cumulative number of publications on equol by year NMR, and GC-MS (35) and the subsequent confirmation that since its first identification in human urine. Downloaded from it was identical in chemical structure to the equol first isolated from pregnant mares urine in 1932 by Marrian et al. (1,2). This nonsteroidal estrogens. It has a molecular composition of confirmation was made possible because one of us (K.D.R.S.) C15H1403 and a molecular weight of 242.27 Daltons. The was gifted from the curator of the UK Medical Research heterocyclic structure contains 2 reactive hydroxyls and 1 rela- Council’s Steroid Reference Collection (the late Professor D.N. tively inert and unreactive oxygen in the central furan ring.

Kirk) the original 4.0 mg sample of equol isolated from pregnant Physicochemically, it is nonpolar and relatively insoluble in jn.nutrition.org mares urine by Marrian et al in 1932. Equol was also found to solution, something that should be considered when conducting occur as a minor constituent in the urine of many adults. The in vitro experiments, particularly at high concentrations. It is link between equol and soy came about after a series of studies in also extremely acid-labile and can readily be destroyed (.60%) which different plant-based foods were fed to rats maintained on in the general work-up of samples, particularly if acidic a purified diet. The introduction of soy protein led to a huge hydrolytic steps are used (39). Despite having 2 phenolic rings, at American Society for Nutrition on June 18, 2010 increase in the urinary excretion of equol and following this it exhibits poor UV absorption characteristics, meaning that observation, the soy isoflavone daidzin was isolated and shown HPLC with UV detection is unsuitable for its measurement in to be a precursor to equol (35). It was also found that the most biological fluids. As a result of a chiral carbon at position introduction of soy protein to the diet led to an increased C-3 of the molecule, equol exists in 2 enantiomeric forms, R-(+) excretion of equol in some but not all adults (36), whereas in equol and S-(-)equol, and the latter is the natural diastereoiso- vitro incubation of cultured fecal flora from equol-producing mer produced by intestinal bacteria in the intestine of humans individuals with either daidzein or soy protein resulted in the and rats (40). This makes it distinct from its precursor formation of equol (36). The finding of high concentrations of isoflavone, daidzein, and the 2 other major isoflavones of soy, equol in the urine of adults consuming soy foods prompted the genistein and glycitein (41). Equol can be readily synthesized hypothesis that this nonsteroidal estrogen may be beneficial in from daidzein by catalytic hydrogenation, but this yields the (6) the prevention and treatment of many hormone-dependent equol form (42) and it is the form that has been commercially conditions (36). available and mostly utilized in studies of its biological potency Progress in research studies of equol was hampered by the and properties. Indeed, unless otherwise stated, it can be lack of sufficient amounts of the compound for biological and assumed that all previously reported experiments used (6)equol clinical testing and by the divergent interest and focus on and not the individual enantiomers. It cannot always be assumed genistein, the other soy-derived isoflavone that was shown to be that the racemate will behave in an identical manner to that of an potent inhibitor of tyrosine protein kinases (37) and a the individual enantiomers and this was recently shown to be compound that was readily available in bulk. Almost 20 y after the case for its pharmacokinetics (43). It is probable that the finding of equol in human urine, it was proposed that the biological effects may be underestimated when testing with the efficiency with which adults convert daidzein to equol when racemate and this may particularly hold true for binding consuming diets containing soy foods could enhance the clinical affinities to receptors. The racemic mixture can be readily effectiveness of soy-based diets—the so called equol-hypothesis separated by chiral chromatography and the earliest studies used (24)—and this has driven a resurgence of interest in equol, as is this approach to isolate sufficient amounts of each enantiomer to evident from the almost exponential increase since 1980 in the determine the estrogen binding affinities (40,44) and the number of publications cited by a PUBMED search of this as a pharmacokinetics (43). More recently, methods for the selective keyword (Fig. 1). At the recent 8th International Soy Symposium synthesis of S-(-)equol (45,46) and R-(+)equol (45) have been held in Tokyo, Japan, for the first time an entire session was described. Methods for the synthesis of [13C]labeled isoflavone devoted to equol (38). This overview will focus on some of the analogs (47–50) have been described that can represent suitable key areas related to this unique molecule and is not intended to starting points for the preparation of stable-labeled [13C]equol be a comprehensive review of the topic. for use as tracers in metabolic studies or for internal standards in stable-isotope dilution mass spectrometric assays (43). The Chemistry synthesis of S-(-)equol and R-(+)equol by chiral chemistry (45) Equol [7-hydroxy-3-(49-hydroxyphenyl)-chroman], an isofla- now affords the large-scale production of enantiomeric pure van, belongs to the general class of compounds referred to as compounds for use in clinical and animal studies. Finally, the

1356S Supplement production of S-(-)equol from daidzein-rich soy germ by a istered [13C]daidzein and [13C]genistein tracers show conclu- specific equol-producing bacterium, Lactococcus garvieae (51), sively that in humans, S-(-)equol is formed from daidzein and offers an alternative biological means to producing specifically not genistein (78). Although the major degradative pathway for S-(-)equol (52). With these breakthroughs, it is now possible to genistein leads to p-ethylphenol, a phenol first found in goat study in some detail the effects of equol in animals and humans urine (79) and 4-hydroxyphenyl-2-propionic acid in rats (80), and such studies will likely shed further light on the relevance of recently it was shown that an anaerobic bacterium from mouse this metabolite to the clinical outcomes. intestine could produce 5-hydroxy-equol from genistein by analogous reactions to those that yield equol from daidzein Role of intestinal bacteria in the formation of S-(-)Equol (66,74). All of these conversions are time dependent and slow, The early evidence in support an intestinal bacterial origin for and in humans it takes 12–36 h for the appearance of [13C]equol equol in humans and animals was as follows: 1) germ-free in plasma after oral administration of [13C]daidzein (78), which animals fed a soy diet do not excrete equol in urine (30); 2) S-(-) is consistent with a colonic origin for its formation. equol is not excreted in the urine or found in the plasma of either Biotransformations that take place after oral administration newborn infants that lack a developed microflora (53) or in of soy isoflavones are summarized in Figure 2. The production of infants up to the age of 4-mo fed exclusively soy infant formula S-(-)equol from daidzin requires 3 key steps. Daidzin first from early life (54,55); 3) incubation of soy, or daidzein with undergoes hydrolysis to split the glucoside moiety and effect human fecal flora from adults that produce equol, leads to the release of the bioavailable aglycon, daidzein. This step is crucial formation of S-(-)equol (36,40); 4) some antibiotics will knock- to all soy isoflavones, because the conjugated forms (glucosides) out the production of equol (18,56,57). do not cross the enterocyte and are consequently not bioavail- Downloaded from Even though it was known for decades that intestinal bacteria able (81). Hydrolysis is very efficient and begins in the proximal were responsible for the production of S-(-)equol, it is only in intestine by the action of brush border membrane b-glucosidases recent years that specific bacteria capable of converting daidzin/ (82). Bacterial b-glucosidases are also capable of performing this daidzein to S-(-)equol have been isolated and identified (Table 1). hydrolysis and many of the common bacteria that reside in the Interestingly, whereas it was reported that 1.59% of injected intestinal tract do this (62,63,83). The efficiency of this initial 14 14

[ C]genistein was recovered as [ C]equol in domestic fowl hydrolytic step by brush border membrane glucosidases is also jn.nutrition.org (77), metabolic and pharmacokinetic studies in humans admin- exemplified by the very high plasma concentrations of daidzein

TABLE 1 List of intestinal bacteria that cultured in vitro have been found to biotransform isoflavones to S-(-)equol

or related intermediates at American Society for Nutrition on June 18, 2010

Reference Bacterium strain Source Reaction1

Maruo et al., 2008 (58) Adlercreutzia equolifaciens Human Daidzein / Equol Minamida et al., 2006 (59) Asaccharobacter celatus AHU1763 Rat Daidzein / Equol Minamida et al, 2008 (60) Asaccharobacter celatus gen,nov.sp. nov strain do03 Rat Daidzein / Equol Ueno and Uchiyama, 2002 (61) Bacteroides ovatus Human Daidzein / Equol Tsangalis et al., 2002 (62) Bifidobacterium Human Daidzein / Equol Tsangalis et al., 2002 (62) Bifidobacterium animalis Pure culture Daidzein / Equol Raimondi et al., 2009 (63) Bifidobacterium sp (22 strains) Human Daidzin / Daidzein Hur et al., 2000 (64) Clostridium sp HGH6 Bovine Daidzin / Dihydrodaidzein Tamura et al., 2007 (65) Clostridium-like bacterium Human Daidzin / Dihydrodaidzein Matthies et al., 2008 (66) Coriobacteriaceae sp MT1B9 Mouse Daidzein / Equol Matthies et al., 2008 (66) Coriobacteriaceae sp MT1B9 Mouse Genistein / 5-Hydroxy-equol Wang et al., 2005 (67) Eggerthella sp Julong 732 Human Dihydrodaidzein / Equol Kim et al., 2009 (68) Eggerthella sp Julong 732 Human Dihydrodaidzein / Equol Yokoyama and Suzuki, 2008 (69) Eggerthella sp YY7918 Human Daidzein / Equol Decroos et al., 2005 (70) Enterococcus faecium Human Daidzein / Equol Tamura et al., 2007 (65) Escherichia coli (HGH21 and HGH6) Human Daidzin / Daidzein Yu et al., 2008 (71) Eubacterium sp D1 and D2 Pig Daidzein / Equol Decroos et al., 2005 (70) Finegoldia magna Human Daidzein / Equol Decroos et al., 2005 (70) Lactobacillus mucosae Human Daidzein / Equol Wang et al., 2006 (72) Lactobacillus sp Niu-O16 Human Daidzein / Equol Ishimi et al., 2008 (73) Lactococcus garvieae (Lc 20-92) Human Daidzein / Equol Ueno and Uchiyama, 2002 (61) Ruminococcus productus Human Daidzein / Equol Matthies et al., 2009 (74) Slackia sp HE8 Human Daidzein / Equol Matthies et al., 2009 (74) Slackia sp HE9 Human Genistein / 5-Hydroxy-equol Jin et al., 2009 (75) Slackia equolifaciens (Strain DZE) Human Daidzein / Equol Jin et al., 2008 (76) Strain PUE Human Purarin / Daidzein Ueno and Uchiyama, 2002 (61) Streptococcus intermedius Human Daidzein / Equol Decroos et al., 2005 (70) Veillonella sp Human Daidzein / Equol Decroos et al., 2005 (70) Mixture of Lactobacillus mucosae EP12, Enterococcus faecium EP11, Human Daidzein / Equol Finegoldia magna EP13, and Veillonella sp

1 In most cases the definitive structure of the equol product was not determined, but it can be assumed that in all cases the product is the S-(-)equol enantiomer.

Equol, part 1: history 1357S of equol in urine (94,95). In the future, it is likely that molecular techniques in bacteriology may help to more accurately define equol-producer status.

Frequency of equol-producers All animal species tested produce equol in response to con- sumption of isoflavones whether from soy protein or clover (16,23,24). Rodents in particular have very high plasma S-(-) equol concentrations, in part because most commercial rodent diets are formulated with soy protein (20,96). For this reason, careful consideration is needed when using rodents, particularly if the primary endpoints being examined are in any way influenced by estrogens, either directly or indirectly, through upstream signaling pathways with estrogen response elements FIGURE 2 Principal metabolic biotransformations of the soy iso- (20,97–99). Frequently, there is a lack of awareness on the part flavones daidzein and genistein. The structure for daidzein is shown and of investigators of the type and composition of the diets being genistein has the identical structure but with an additional hydroxyl used by their animal facilities or by the vendors providing group at position C-5 of the A-ring, highlighted by the wavy arrow. rodents for research studies. A high batch-to-batch variability in the isoflavone content of commercial rodent unpurified diet Downloaded from makes it impossible to control for the background level of these and genistein in infants fed soy infant formula (54,55) that have phytoestrogens (97). immature and undeveloped gut microflora (53). Daidzein is When the association between soy isoflavones and equol was reduced to S-(-)equol through the intermediate dihydrodaidzein first made, it was noted that not all healthy adults produced then converted by deoxygenation to yield S-(-)equol. Daidzein is equol when challenged with soy protein (36). The first reported

also metabolized to O-desmethylangolensin as a result of cleavage dietary intervention study of the 6 healthy adults fed 40 g of jn.nutrition.org across the hetercyclic ring (84), but this metabolite appears to be textured vegetable protein for 7 consecutive days found only 4/6 of little interest in that it has no known biological activity. excreted equol in urine; the term equol-producers was thus Bacteria are enantioselective in metabolizing daidzein to coined. Many other studies of larger sample sizes have provided exclusively S-(-)equol and not R-(+)equol (40,67,72). It is not a consensus that only 25–30% of the adult population of completely clear whether the conversion of daidzein to S-(-) Western countries produce S-(-)equol when fed soy foods at American Society for Nutrition on June 18, 2010 equol is performed by a single bacterium or whether there are containing isoflavones (94,100,101). This is significantly lower distinctly different bacteria that execute these reactions, or both. than the reported 50–60% frequency of equol-producers in The large variability in the levels of dihydrodaidzein and S-(-) adults from Japan, Korea, or China (102–105) or in Western equol in human urine (85) would indicate that there is more than adult vegetarians (95). The reasons for these differences are a single bacterium responsible for producing S-(-)equol. Fur- unclear but are important to understand if the hypothesis that thermore, the finding that certain antibiotics selectively inhibit the ability to produce equol when consuming soy foods (equol the formation of equol but not dihydrodaidzein when human hypothesis) is advantageous in terms of enhancing health feces from equol-producers are incubated with daidzein also benefits can be clearly demonstrated (24,106). Thus far, the supports this contention (56). The list of intestinal bacteria that data are inconclusive, but this is partly because, to our can produce equol in culture is ever increasing (Table 1); a knowledge, none of the clinical studies have preselected partic- number of strains have been isolated that perform only ipants on the basis of equol-producer status but rather have conversion of daidzin/daidzein to dihydrodaidzein whereas retrospectively subanalyzed data from equol-producers, and others appear to be able to completely convert daidzein to most are underpowered. Significant differences in gene expres- equol (Table 1). One in particular, Lactococcus garvieae, that is sion between equol-producers and equol nonproducers have found in some Italian cheeses (51) and has been isolated from been demonstrated in postmenopausal women exposed to an human feces (61,73) was shown to efficiently convert daidzin to isoflavone supplement, with the most notably significant alter- S-(-)equol and has been used to produce the first natural S-(-) ations in expression of a number of estrogen-responsive genes equol–containing nutraceutical (52,86). Whether demonstrating (107). formation of S-(-)equol in culture can be considered represen- It would appear that equol-producer status is a relatively tative of intraluminal colonic formation of S-(-)equol remains stable phenomenon, as evidenced from repeat testing over uncertain. It may, e.g. under specific conditions be possible to prolonged periods of time in adults (24,89,95,108). There have demonstrate conversion of daidzein to equol in vitro, yet such been many studies looking at associations between equol- conditions may not necessarily be reflective of the intraluminal producer status and dietary components, including fat and milleu in the human intestinal tract. For example, culturing carbohydrate composition (93,101), PUFA (109), dairy intakes human fecal flora from equol-producers under conditions that (110), lactose (111), green tea consumption (112), seaweed enhance fermentation, such as a high nonstarch polysaccharide (113), and soy food intake (103,105,110), but no clear conclu- medium, enhances conversion of daidzein to equol (87), as do sions can be made. Prolonged soy food consumption appears not hydrogen gas, butyrate, and proprionate (59,70). Despite the to be a factor driving equol formation (114). One possible ability to manipulate equol production in vitro, human dietary explanation for the differences in the frequency of equol- intervention studies using prebiotics or probiotics have had little producers among populations, we speculate, may be related to impact on its formation (88–92). Therefore, some caution is the type of soy foods consumed (115). There are marked required in relying on in vitro fecal cultures from adults as a differences in the isoflavone composition of Western and Asian means of confirming equol-producer status (93). Such ap- soy foods (Fig. 3) (41). Asians consume a high proportion of proaches should always be coupled with specific measurement isoflavone aglycons, because fermented soy foods account for

1358S Supplement approach is to feed a soy isoflavone-containing food, such as soymilk, or an isoflavone supplement, such as soy germ, that is preferentially high in daidzein (128) for 3 consecutive days to attain a steady state (94,95,108,129). Urine is then collected and daidzein and S-(-)equol measured. We recently opted to define an equol-producer based on the ratio of equol:daidzein (95), because this reflects the product/precursor relationship and overcomes the drawbacks of using absolute urinary (or plasma) equol concentrations (36,94,101,130), which are subject to considerable variation due to inter-individual differences in isoflavone pharmacokinetics (78,102,119,120,131–133). Defin- ing an equol-producer by the equol:daidzein ratio also circum- vents the need for accurately timed 24-h urine collections.

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bacterium capable of daidzein and genistein conversion. Appl 96. Thigpen JE, Setchell KDR, Ahlmark KB, Locklear J, Spahr T, at American Society for Nutrition on June 18, 2010 Environ Microbiol. 2009;75:1740–4. Caviness GF, Goelz MF, Haseman JK, Newbold RR, et al. 75. Jin JS, Kitahara M, Sakamoto M, Hattori M, Benno Y. Slackia Phytoestrogen content of purified, open- and closed-formula labora- equolifaciens sp. nov., a human intestinal bacterium capable of tory animal diets. Lab Anim Sci. 1999;49:530–6. producing equol. Int J Syst Evol Microbiol. Epub 2009 Sep 4. 97. Thigpen JE, Setchell KDR, Padilla-Banks E, Haseman JK, Saunders 76. Jin JS, Nishihata T, Kakiuchi N, Hattori M. Biotransformation of C- HE, Caviness GF, Kissling GE, Grant MG, Forsythe DB. Variations in glucosylisoflavone puerarin to estrogenic (3S)-equol in co-culture of phytoestrogen content between different mill dates of the same diet two human intestinal bacteria. Biol Pharm Bull. 2008;31:1621–5. produces significant differences in the time of vaginal opening in CD- 77. Cayen MN, Carter AL, Common RH. The conversion of genistein to 1 mice and F344 rats but not in CD Sprague-Dawley rats. Environ equol in the fowl. Biochim Biophys Acta. 1964;86:56–64. Health Perspect. 2007;115:1717–26. 78. Setchell KDR, Faughnan MS, Avades T, Zimmer-Nechemias L, 98. Thigpen JE, Haseman JK, Saunders HE, Setchell KDR, Grant MG, Brown NM, Wolfe BE, Brashear WT, Desai P, Oldfield MF, et al. Forsythe DB. Dietary phytoestrogens accelerate the time of vaginal Comparing the pharmacokinetics of daidzein and genistein with the opening in immature CD-1 mice. Comp Med. 2003;53:607–15. 13 use of C-labeled tracers in premenopausal women. Am J Clin Nutr. 99. Thigpen JE, Setchell KDR, Saunders HE, Haseman JK, Grant MG, 2003;77:411–9. Forsythe DB. Selecting the appropriate rodent diet for endocrine 79. Grant JK. p-Ethylphenylsulphuric acid in goat urine. Biochem J. disruptor research and testing studies. ILAR J. 2004;45:401–16. 1948;43:523–4. 100. Atkinson C, Frankenfeld CL, Lampe JW. Gut bacterial metabolism of 80. Coldham NG, Howells LC, Santi A, Montesissa C, Langlais C, King the soy isoflavone daidzein: exploring the relevance to human health. LJ, Macpherson DD, Sauer MJ. Biotransformation of genistein in the Exp Biol Med (Maywood). 2005;230:155–70. rat: elucidation of metabolite structure by product ion mass 101. Rowland IR, Wiseman H, Sanders TA, Adlercreutz H, Bowey EA. fragmentology. J Steroid Biochem Mol Biol. 1999;70:169–84. Interindividual variation in metabolism of soy isoflavones and 81. Setchell KDR, Brown NM, Zimmer-Nechemias L, Brashear WT, lignans: influence of habitual diet on equol production by the gut Wolfe BE, Kirschner AS, Heubi JE. Evidence for lack of absorption of microflora. Nutr Cancer. 2000;36:27–32. soy isoflavone glycosides in humans, supporting the crucial role of 102. Watanabe S, Yamaguchi M, Sobue T, Takahashi T, Miura T, Arai Y, intestinal metabolism for bioavailability. Am J Clin Nutr. 2002;76: Mazur W, Wahala K, Adlercreutz H. Pharmacokinetics of soybean 447–53. isoflavones in plasma, urine and feces of men after ingestion of 60 g 82. Day AJ, DuPont MS, Ridley S, Rhodes M, Rhodes MJ, Morgan MR, baked soybean powder (kinako). J Nutr. 1998;128:1710–5. Williamson G. Deglycosylation of flavonoid and isoflavonoid glyco- 103. Arai Y, Uehara M, Sato Y, Kimira M, Eboshida A, Adlercreutz H, sides by human small intestine and liver beta-glucosidase activity. Watanabe S. Comparison of isoflavones among dietary intake, FEBS Lett. 1998;436:71–5. plasma concentration and urinary excretion for accurate estimation 83. Steer TE, Johnson IT, Gee JM, Gibson GR. Metabolism of the of phytoestrogen intake. J Epidemiol. 2000;10:127–35. soybean isoflavone glycoside genistin in vitro by human gut bacteria 104. Akaza H, Miyanaga N, Takashima N, Naito S, Hirao Y, Tsukamoto and the effect of prebiotics. Br J Nutr. 2003;90:635–42. T, Fujioka T, Mori M, Kim WJ, et al. Comparisons of percent equol 84. Bannwart C, Adlercreutz H, Fotsis T, Wahala K, Hase T, Brunow G. producers between prostate cancer patients and controls: case- Identification of O-desmethylangolensin, a metabolite of daidzein controlled studies of isoflavones in Japanese, Korean and American and of matairesinol, one likely plant precursor of the animal lignan residents. Jpn J Clin Oncol. 2004;34:86–9. enterolactone in human urine. Finn Chem Lett. 1984;120–5. 105. Song KB, Atkinson C, Frankenfeld CL, Jokela T, Wahala K, Thomas 85. Kelly GE, Joannou GE, Reeder AY, Nelson C, Waring MA. The WK, Lampe JW. Prevalence of daidzein-metabolizing phenotypes variable metabolic response to dietary isoflavones in humans. Proc differs between Caucasian and Korean American women and girls. Soc Exp Biol Med. 1995;208:40–3. J Nutr. 2006;136:1347–51.

Equol, part 1: history 1361S 106. Lampe JW. Is equol the key to the efficacy of soy foods? Am J Clin 120. Zubik L, Meydani M. Bioavailability of soybean isoflavones from Nutr. 2009;89:S1664–7. aglycone and glucoside forms in American women. Am J Clin Nutr. 107. Niculescu MD, Pop EA, Fischer LM, Zeisel SH. Dietary isoflavones 2003;77:1459–65. differentially induce gene expression changes in lymphocytes from 121. Setchell KDR, Zhao X, Shoaf SE, Ragland K. The pharmacokinetics postmenopausal women who form equol as compared with those of S-(-)equol administered as SE5-OH tablets to healthy postmeno- who do not. J Nutr Biochem. 2007;18:380–90. pausal women. J Nutr. 2009;139:2037–43. 108. Frankenfeld CL, Atkinson C, Thomas WK, Goode EL, Gonzalez A, 122. Setchell KDR, Cole SJ. Variations in isoflavone levels in soy foods Jokela T, Wahala K, Schwartz SM, Li SS, Lampe JW. Familial and soy protein isolates and issues related to isoflavone databases and correlations, segregation analysis, and nongenetic correlates of soy food labeling. J Agric Food Chem. 2003;51:4146–55. isoflavone-metabolizing phenotypes. Exp Biol Med (Maywood). 123. Valentin-Blasini L, Sadowski MA, Walden D, Caltabiano L, 2004;229:902–13. Needham LL, Barr DB. Urinary phytoestrogen concentrations in 109. Bolca S, Possemiers S, Herregat A, Huybrechts I, Heyerick A, De the U.S. population (1999–2000). J Expo Anal Environ Epidemiol. Vriese S, Verbruggen M, Depypere H, De Keukeleire D, et al. 2005;15:509–23. Microbial and dietary factors are associated with the equol producer 124. Peeters PH, Slimani N, van der Schouw YT, Grace PB, Navarro C, phenotype in healthy postmenopausal women. J Nutr. 2007;137: Tjonneland A, Olsen A, Clavel-Chapelon F, Touillaud M, et al. 2242–6. Variations in plasma phytoestrogen concentrations in European 110. Nagata C, Ueno T, Uchiyama S, Nagao Y, Yamamoto S, Shibuya C, adults. J Nutr. 2007;137:1294–300. Kashiki Y, Shimizu H. Dietary and lifestyle correlates of urinary 125. Ward H, Chapelais G, Kuhnle GG, Luben R, Khaw KT, Bingham S. excretion status of equol in Japanese women. Nutr Cancer. Breast cancer risk in relation to urinary and serum biomarkers of 2008;60:49–54. phytoestrogen exposure in the European Prospective into Cancer- 111. Tamura A, Shiomi T, Hachiya S, Shigematsu N, Hara H. Low Norfolk cohort study. Breast Cancer Res. 2008;10:R32. activities of intestinal lactase suppress the early phase absorption of 126. Travis RC, Spencer EA, Allen NE, Appleby PN, Roddam AW, Downloaded from soy isoflavones in Japanese adults. Clin Nutr. 2008;27:248–53. Overvad K, Johnsen NF, Olsen A, Kaaks R, et al. Plasma phyto- 112. Miyanaga N, Akaza H, Takashima N, Nagata Y, Sonoda T, Mori M, oestrogens and prostate cancer in the European Prospective Investi- Naito S, Hirao Y, Tsukamoto T, et al. Higher consumption of green gation into Cancer and Nutrition. Br J Cancer. 2009;100:1817–23. tea may enhance equol production. Asian Pac J Cancer Prev. 127. Goodman MT, Shvetsov YB, Wilkens LR, Franke AA, Le Marchand 2003;4:297–301. L, Kakazu KK, Nomura AM, Henderson BE, Kolonel LN. Urinary 113. Teas J, Hurley TG, Hebert JR, Franke AA, Sepkovic DW, Kurzer MS. phytoestrogen excretion and postmenopausal breast cancer risk:

Dietary seaweed modifies estrogen and phytoestrogen metabolism in the multiethnic cohort study. Cancer Prev Res (Phila Pa). 2009;2: jn.nutrition.org healthy postmenopausal women. J Nutr. 2009;139:939–44. 887–94. 114. Vedrine N, Mathey J, Morand C, Brandolini M, Davicco MJ, Guy L, 128. Murphy PA, Barua K, Hauck CC. Solvent extraction selection in the Remesy C, Coxam V, Manach C. One-month exposure to soy determination of isoflavones in soy foods. J Chromatogr B Analyt isoflavones did not induce the ability to produce equol in postmen- Technol Biomed Life Sci. 2002;777:129–38. opausal women. Eur J Clin Nutr. 2006;60:1039–45. 129. Atkinson C, Newton KM, Aiello Bowles EJ, Lehman CD, Stanczyk at American Society for Nutrition on June 18, 2010 115. Clerici C, Setchell KDR, Battezzati PM, Pirro M, Giuliano V, Asciutti FZ, Westerlind KC, Li L, Lampe JW. Daidzein-metabolizing pheno- S, Castellani D, Nardi E, Sabatino G, et al. Pasta naturally enriched types in relation to mammographic breast density among premeno- with isoflavone aglycons from soy germ reduces serum lipids and pausal women in the United States. Breast Cancer Res Treat. 2009; improves markers of cardiovascular risk. J Nutr. 2007;137:2270–8. 116:587–94. 116. Wakai K, Egami I, Kato K, Kawamura T, Tamakoshi A, Lin Y, 130. Zhao JH, Sun SJ, Arao Y, Oguma E, Yamada K, Horiguchi H, Nakayama T, Wada M, Ohno Y. Dietary intake and sources of Kayama F. Identification of equol producers in a Japanese population isoflavones among Japanese. Nutr Cancer. 1999;33:139–45. by high-performance liquid chromatography with coulometric array 117. Somekawa Y, Chiguchi M, Ishibashi T, Aso T. Soy intake related to for determining serum isoflavones. Phytomedicine. 2006;13:304–9. menopausal symptoms, serum lipids, and bone mineral density in 131. King RA, Bursill DB. Plasma and urinary kinetics of the isoflavones postmenopausal Japanese women. Obstet Gynecol. 2001;97:109–15. daidzein and genistein after a single soy meal in humans. Am J Clin 118. Izumi T, Piskula MK, Osawa S, Obata A, Tobe K, Saito M, Kataoka Nutr. 1998;67:867–72. S, Kubota Y, Kikuchi M. Soy isoflavone aglycones are absorbed faster 132. Shelnutt SR, Cimino CO, Wiggins PA, Badger TM. Urinary pharma- and in higher amounts than their glucosides in humans. J Nutr. cokinetics of the glucuronide and sulfate conjugates of genistein and 2000;130:1695–9. daidzein. Cancer Epidemiol Biomarkers Prev. 2000;9:413–9. 119. Setchell KDR, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, 133. Bloedon LT, Jeffcoat AR, Lopaczynski W, Schell MJ, Black TM, Dix Brashear WT, Kirschner AS, Cassidy A, Heubi JE. Bioavailability of KJ, Thomas BF, Albright C, Busby MG, et al. Safety and pharma- pure isoflavones in healthy humans and analysis of commercial soy cokinetics of purified soy isoflavones: single-dose administration to isoflavone supplements. J Nutr. 2001;131:S1362–75. postmenopausal women. Am J Clin Nutr. 2002;76:1126–37.

1362S Supplement The Journal of Nutrition Supplement: Equol, Soy, and Menopause

Equol: Pharmacokinetics and Biological Actions1,2

KennethD.R.Setchell3*andCarloClerici4

3Pathology and Laboratory Medicine, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and 4Clinica di Gastroenterologia ed Epatologia, Universita` degli Studi di Perugia, Italy University of Perugia, Perugia 06156, Italy

Abstract

Equol [7-hydroxy-3-(49-hydroxyphenyl)-chroman], an isoflavan produced by intestinal bacteria in response to soy isoflavone S intake in some but not all humans, exhibits a wide range of biological properties. It exists as the diastereoisomers -(-) Downloaded from equol and R-(+)equol. Intestinal bacteria produce exclusively S-(-)equol, which has selective affinity for estrogen receptor (ER)-b. The evidence is conflicting on whether there is an advantage to producing S-(-)equol in response to soy isoflavone intakes, but the ability to now synthesize these diastereoisomers opens the way for future clinical trials to directly examine their potential in a number of hormone-dependent conditions. In this review, the plasma and urinary pharmacokinetics of S R

-(-)equol and -(+)equol are reviewed and summarized, and some of the more recent evidence supporting potential jn.nutrition.org biological effects of S-(-)equol is considered. J. Nutr. 140: 1363S–1368S, 2010. at American Society for Nutrition on June 18, 2010 Introduction (3) that the relative affinity of equol for both receptors could be In Part 1 of this overview of equol (1), the history, chemistry, and quantified (4–6). The results from these studies places the factors that influence equol production were reviewed. Part 2 natural soy isoflavone metabolite, S-(-)equol, into a category of a separately reviews the pharmacokinetics and the biological selective ER modulator and consequently prompts many ques- properties of equol that have led to the current interest in this tions as to whether it could confer some specific benefits in unique isoflavone metabolite. hormone-related conditions. The hormonal effects of equol are well documented from The ability of both S-(-)equol and its diastereoisomer, R-(+) early observations using estrogen bioassays. It was not until after equol, to antagonize the in vivo actions of dihydrotestosterone (7) the discovery of the first estrogen receptor (ER)-a (2) and the further makes equol a unique molecule with potential for discovery that a second ER (ERb) was present in specific tissues the treatment or prevention of androgen-mediated conditions. For these reasons equol is currently attracting considerable interest as a potential pharmaceutical or nutraceutical agent. 1 The Journal of Nutrition Published in a supplement to . Presented at the “Equol, The following will review its pharmacology and biological effects. Soy, and Menopause Research Leadership Conference”, held in Washington, DC, June 16, 2009. The supplement coordinator for this supplement is Kara Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The Pharmacokinetics of equol supplement is the responsibility of the guest editors to whom the Editor of The To our knowledge, data from the first pharmacokinetic study of Journal of Nutrition has delegated supervision of both technical conformity to The Journal of Nutrition equol was described in a single healthy adult female adminis- the published regulations of and general oversight of the 6 scientific merit of each article. Publication costs for this supplement were tered 25 mg of ( )equol given as a single oral bolus dose (8). The defrayed in part by the payment of page charges. This publication must therefore plasma (6)equol concentration appearance/disappearance curve be hereby marked "advertisement" in accordance with 18 USC section 1734 suggested that equol differed in its pharmacokinetic behavior solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. from the soy isoflavones daidzein and genistein. Most notably it Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement Coordinator disclosure: Kara Lewis is currently under contract with and receives had a much higher apparent bioavailability and slower clearance compensation from the supplement sponsor. She was also compensated for rate (8). This was confirmed in later studies when the plasma attending and organizing the Equol, Soy, and Menopause Research Leadership pharmacokinetics of S-(-)equol and R-(+)equol were compared Conference and for organizing, writing, editing, or reviewing, and collection of in 3 healthy adults (6). More recently, using [13C]labeled tracers, supplemental manuscripts. The opinions expressed in this publication are those the plasma and urinary pharmacokinetics of enantiomeric pure of the authors and are not attributable to the sponsors or the publisher, Editor, or Editorial Board of The Journal of Nutrition. S-(-)equol and R-(+)equol were determined in 12 healthy adults 2 Author disclosures: K. D. R. Setchell was supported by funding from the NIH (6 males, 6 females) (9). Both enantiomers were rapidly (grant nos. R01AT-003313 and R01AT-002190) and has intellectual property on absorbed and reached peak plasma concentrations after 2–3 h equol enantiomers, including patents licensed by Cincinnati Children’s Hospital when taken with a meal. In an evaluation of the pharmacoki- Medical Center, Cincinnati, OH to industry and is a consultant to Otsuka Pharmaceuticals Company, Tokyo, Japan. C. Clerici, no conflict of interest. netics of a new S-(-)equol–containing supplement (SE5-OH) * To whom correspondence should be addressed. E-mail: kenneth.setchell@ given to 12 healthy postmenopausal women, the average peak cchmc.org. plasma concentration was observed after 1–2 h when taken

ã 2010 American Society for Nutrition. 1363S First published online June 2, 2010; doi:10.3945/jn.109.119784. without a meal (10). The differences in the absorption rates of Biological properties of equol S-(-)equol between these 2 studies is explained by a meal-effect The diastereoisomers of equol share many similarities yet altering gastric emptying time and slowing the initial absorption some significant differences in biological properties. The more rate. Such differences will influence peak plasma concentrations, planar-looking S-(-)equol enantiomer is strikingly similar in as was evident from the much higher dose-adjusted Cmax values conformational structure to estradiol and, not surprisingly, this attained with the S-(-)equol supplement given without a meal enantiomer binds to the ER (Fig. 1). When first isolated from compared with the pure compounds given with a meal (10). equine urine in 1932, long before the discovery of the first ER Therefore, in practice, the maximal effect of S-(-)equol is more (2), it was reported to have no estrogenic activity when injected likely to occur if it is administered before a meal. Independent of into ovariectomized mice in doses up to 0.086 mg (25); however, this difference, the pharmacokinetics of enantiomeric pure S-(-) its uterotrophic activity was later acknowledged. The earliest in equol was similar to that of the S-(-)equol supplement produced vitro study of the binding of S-(-)equol, isolated from sheep by the fermentation of soy germ isoflavones with Lactococcus urine showed it to have a relative molar binding affinity of 0.4 garvieae (10). S-(-)equol has an terminal elimination half-life of compared with estradiol, which was about 4 times the affinity 7–8 h in healthy adults and, therefore, steady-state levels will be of its precursor, daidzein (26). Later, 5 mg of equol, presumed more readily attained by dosing twice daily to minimize peaks to be the racemic form because it was chemically synthesized and troughs in circulating concentrations. Within the constraints by methods at the time that were not enantioselective, when of small sample-sizes, data from all these studies suggested no injected subcutaneously into 3-wk-old female rats increased obvious gender differences in the pharmacokinetics of S-(-) uterine weight to the same extent as 0.005 mg of estradiol (27) equol. Two interesting findings arose from a comparison of the and it was shown to antagonize the binding of estradiol to the Downloaded from pharmacokinetics of the [13C]labeled enantiomers. Racemic (6) ER. Others have reported similar relative binding affinities using [13C]equol showed slower absorption, attained lower peak a selection of different in vitro systems (28–30). It should, plasma concentrations, and had lower systemic bioavailability however, be pointed out that in most cases these early studies compared with S-(-)[13C]equol and R-(+)[13C]equol. Also, the would have examined binding to ERa, because they predated apparent systemic bioavailability of R-(+)[13C]equol was signif- the discovery of ERb (3) and because this is the major ER 13

icantly greater than that of S-(-)[ C]equol (9). subtype localized to the uterus (31). These early data are also jn.nutrition.org S-(-)equol and R-(+)equol undergo little biotransformation possibly underestimated because of the use of racemic mixtures. in humans, save phase II metabolism by conjugation to glu- Several more recent studies have since reported the binding curonic acid and to a minor extent sulfuric acid. S-(-)equol characteristics of the individual enantiomers toward ERa and circulates in plasma and is excreted in urine as predominantly ERb (4–6,32–34). S(-)equol produced from incubation of soy the 7-glucuronide conjugate (11–13). In this respect, its me- at American Society for Nutrition on June 18, 2010 tabolism is similar to that of the soy isoflavones daidzein and genistein (14–18). Conjugation is extremely efficient in humans and takes place on first-pass absorption within the enterocyte and also the liver. Uridine diphosphate-59-glucuronosyltransferase is widely distributed throughout the gastrointestinal tract (19) and it is probable that it is the uridine diphosphate- 59-glucuronosyltransferase 1A10 isoform that catalyzes glu- curonidation, because this one conjugates genistein (15). For equol, its major route of elimination is by renal excretion into urine. The percent fractional elimination in urine after oral administration is extremely high and in some adults it can be close to 100% (9,10), which is far higher than that of daidzein (30–40%) and genistein (7–15%) (20,21). Recoveries averaged 82% when S-(-)equol was given as a supplement and 61.3 6 19.5% for enantiomeric pure S-(-)[13C]equol. The bioavailabil- ity of R-(+)equol was higher than its diastereoisomer S-(-)equol based on the plasma pharmacokinetics and urinary recovery of the [13C] tracers (9). Overall, the very high bioavailability of S- (-)equol would indicate that relatively modest doses (10–30 mg twice a day) would result in high steady-state plasma concen- trations in the range observed for plasma S-(-)equol derived from soy foods. Endogenous estrogens, as with most hormones, circulate predominantly bound to albumin and sex hormone binding globulin (22) and also to a-fetoprotein (23). Less than 5% of estradiol is present in the free (unbound form), which is the fraction that is available for receptor occupancy. For equol it has been reported that 49.7% circulates in the free form, which is significantly higher than daidzein (18.7% free), its precursor (22). Thus, the biological activity of equol should be enhanced by its reduced binding to serum proteins and greater availability for receptor binding. In a dose-dependent manner, equol in vitro inhibits the binding of estradiol and testosterone for serum FIGURE 1 A comparison of the conformation structures of estra- proteins (24). diol, S-(-)equol, and R-(+)equol.

1364S Supplement isoflavones with enteric bacteria when tested in competitive Equol can be broadly classified as a polyphenol and due to the binding assays with human ERa and ERb, and in a gene high number of p-electrons, it has hydrogen/donor properties expression assay, was found to bind more strongly to ERb than and will scavenge free radicals. The in vitro antioxidant property to ERa (4). The preferential binding of S(-)equol to ERb has been of equol, presumed to be racemic equol, is well documented confirmed in multiple studies (5,6,32) and indicates the molecule (47–50) and the antioxidant activities of the individual enanti- shares the characteristics of a selective ER modulator in this omers should be similar. (6)Equol has the highest antioxidant regard. However, S-(-)equol induces transcription either similarly, activity of all the isoflavones that have been tested. To date there or more strongly, with ERa than with ERb (4,5), as does R(+) are no in vivo human data on the extent to which administering equol (5), indicative of both being agonists. So the differential equol may influence lipid peroxidation, an important risk factor effects of 2 almost identical molecules on the ER subtypes is quite for atherosclerosis, but LDL oxidation by cultured monocyte/ striking and shows how the presence of a chiral center in the macrophages was shown to be inhibited by an antioxidant effect molecule confers quite different biological properties. mediated through inhibition of superoxide radical production Given the present interest in S-(-)equol as a possible (51). The effect of equol on inhibition of nitric oxide (NO) pharmaceutical or nutraceutical agent for a number of hormone- production by inducible NO synthase gene expression in murine dependent disorders (9,10,35), the question of whether the ER macrophages was reported as being mediated through upstream agonist action could pose some risk for women with breast signaling pathways, specifically by Akt activation and down- cancer or for those in high risk groups remains to be addressed regulation of nuclear factor-kB activity (52); inducible NO (36–38). Recent studies using animal models of breast cancer synthase is implicated in the development of atherosclerosis. have examined the role of equol on the growth of mammary These findings are perhaps not unexpected, because genistein is Downloaded from tumors (39,40). In one model, S-(-)equol did not stimulate the antiinflammatory by an effect on reducing NO production (53). growth of human ER positive MCF-7 cells transplanted into the Several studies show equol to be a vasorelaxant, inducing athymic mouse (39). This important finding is in striking endothelial and NO-dependent relaxation (54–60), suggesting contrast to the marked stimulatory effect of the soy isoflavone equol may be helpful in reducing risk of cardiovascular disease. genistein reported earlier by the same investigators in this same The isoflavone intermediate dihydrodaidzein and the closely

model (41), an observation that led to the issue of whether soy related dehydroequol are also vasodilatory (55,61). No study jn.nutrition.org foods are safe for women with breast cancer. To date, there are has yet examined the vasodilatory actions of S-(-)equol or R-(-) no human data to support this concern, but 2 recent large equol separately and it is too early to know whether either prospective clinical studies of breast cancer survivors suggest enantiomer may be effective in the clinical arena. Studies of soy that soy food consumption is associated with more favorable isoflavones have yielded mixed results with regard to the effects prognosis, in reducing risk of recurrence and improving survival on endothelial function (62–69), but equol-producer status was at American Society for Nutrition on June 18, 2010 (42,43). In a different animal model, S-(-)equol did not stimulate not directly examined. In one recent clinical study of hypercho- the growth of mammary tumors induced by the chemical lesterolemic patients, brachial artery-mediated vasodilatation carcinogen dimethylbenz[a]anthracene, but neither did it prove was significantly greater in equol-producers compared with to be chemopreventive (40). R-(+)equol on the other hand was equol-nonproducers after 4 wk of dietary intervention with a soy found in this same model to be potently chemopreventive (40). isoflavone-containing food that resulted in a high proportion of Combining data from these 2 animal models of breast cancer equol-producers (70). Similar differential effects between equol- suggests that S-(-)equol should not increase risk for breast cancer producers and nonproducers were observed in arterial stiffness and R-(+)equol could be a useful chemopreventive agent. If these in a study of postmenopausal women taking tibolone (71) and animal data can be reliably extrapolated to humans, then the these translated into lower diastolic blood pressure (72). This ability to make equol when consuming soy foods could be was not the case in the former study (69). Because inflammation advantageous in reducing the risk of breast cancer. plays a key role in the onset of cardiovascular disease (73), it is While much of the interest in equol has centered on its possible, given equol’s documented effect on the NO pathway, estrogenic effects, equol enantiomers have a myriad of other that it may act as an antiinflammatory agent. Serum high biological properties with the potential to be of value in many sensitivity C-reactive protein concentration, a surrogate marker clinical areas, including cancer, cardiovascular disease, osteo- of inflammation and cardiovascular risk, was shown in a recent porosis, and menopausal symptoms (8); several of these areas study to be reduced in equol-producers by a soy isoflavone- are discussed below. containing food (70). In a recent study, equol and genistein, but Uniquely, both S-(-)equol and R-(+)equol bind dihydrotes- not daidzein, modulated the inflammatory response in activated tosterone and inhibit the in vivo stimulatory effect of this potent macrophages by inhibition of NO and prostaglandin E2 while androgen on prostate growth (7). Neither S-(-)equol nor R-(+) regulating gene transcription of cytokines and inflammatory equol bind to the androgen receptor, but its selective androgen- markers (74). modulating activity, combined with S-(-)equol having selective In vitro cell culture and animal studies have provided affinity for ERb, suggests that S-(-)equol may have potential in a impressive data on the bone-trophic effects of isoflavones (75), number of androgen-mediated conditions, in particular prostate but recent clinical studies of soy isoflavone supplementation in cancer treatment or prevention. The pharmaceutical industry postmenopausal women have proved disappointing (76–80). has more recently turned its attention toward ERb agonists in None of these trials have prescreened for equol-producer status the search for the next generation of drugs to treat prostate and randomized accordingly and all have used isoflavone cancer (44) and in this regard S-(-)equol may be a potential mixtures of predominantly conjugated rather than aglycon candidate. A small case control of prostate cancer patients from forms. Interestingly, the aglycon genistein given at a dose of 54 South Korea, Japan, and the US found a low frequency of equol- mg/d for 3 y to postmenopausal women was reported to have producers among the patients compared with age-matched impressive effects on bone, with increases in spine and hip bone controls (45), while in a separate Japanese study, the risk for mineral density (81). Studies from Japan show more favorable prostate cancer was reported to show an inverse dose-response responses in measures of bone loss in those women who are relationship with plasma equol concentrations (46). equol-producers (82). Equol in its racemic form has been shown

Equol, part 2: pharmacokinetics 1365S to have modest effects in preventing bone loss in animal models 8. Setchell KDR, Brown NM, Lydeking-Olsen E. The clinical importance of osteoporosis (83–87) but has yet to be used in clinical trials. of the metabolite equol-a clue to the effectiveness of soy and its S-(-)equol is also being studied for its effects on reducing the isoflavones. J Nutr. 2002;132:3577–84. incidence and frequency of menopausal symptoms (88), partic- 9. Setchell KDR, Zhao X, Jha P, Heubi JE, Brown NM. The pharmaco- kinetic behavior of the soy isoflavone metabolite S-(-)equol and its ularly hot flushes. Data from Japan have indicated that the diastereoisomer R-(+)equol in healthy adults determined by using severity of overall menopausal symptoms is significantly lower stable-isotope-labeled tracers. Am J Clin Nutr. 2009;90:1029–37. in women who are equol-producers treated with a soy isoflavone 10. Setchell KDR, Zhao X, Shoaf SE, Ragland K. The pharmacokinetics of supplement (89). S-(-)equol administered as SE5-OH tablets to healthy postmenopausal In conclusion, S-(-)equol is a unique nonsteroidal estrogen women. J Nutr. 2009;139:2037–43. that binds preferentially to ERb and at the same time antago- 11. Axelson M, Setchell KDR. Conjugation of lignans in human urine. FEBS Lett. 1980;122:49–53. nizes the in vivo action of the potent androgen dihydrotestos- 12. Axelson M, Kirk DN, Farrant RD, Cooley G, Lawson AM, Setchell terone. It occurs as 2 distinct diastereoisomers and both have KDR. The identification of the weak oestrogen equol [7-hydroxy- properties that warrant their further investigation for the 3-(49-hydroxyphenyl)chroman] in human urine. Biochem J. 1982;201: prevention and/or treatment of a number of estrogen- and 353–7. androgen-mediated diseases or disorders as was first proposed in 13. Axelson M, Sjovall J, Gustafsson BE, Setchell KDR. Soya: a dietary 1984 (90). The ability to now synthesize bulk quantities of source of the non-steroidal oestrogen equol in man and animals. enantiomeric pure S-(-)equol and R-(+)equol should permit J Endocrinol. 1984;102:49–56. future clinical studies to be conducted that will more clearly 14. Sfakianos J, Coward L, Kirk M, Barnes S. Intestinal uptake and biliary excretion of the isoflavone genistein in rats. J Nutr. 1997;127:1260–8. define the potential benefits of these diastereoisomers. More 15. Doerge DR, Chang HC, Churchwell MI, Holder CL. Analysis of soy Downloaded from importantly, such direct studies of the pure compounds will isoflavone conjugation in vitro and in human blood using liquid enable a better understanding of the extent to which there are chromatography-mass spectrometry. Drug Metab Dispos. 2000;28: advantages to producing equol from soy foods, as has been 298–307. proposed. If the equol hypothesis can be substantiated, then for 16. Shelnutt SR, Cimino CO, Wiggins PA, Ronis MJJ, Badger TM. those adults who are unable to produce equol due to a lack of Pharmacokinetics of the glucuronide and sulfate conjugates of genistein and daidzein in men and women after consumption of a soy beverage.

intestinal equol-producing bacteria or some other factors, one jn.nutrition.org Am J Clin Nutr. 2002;76:588–94. option is to administer these enantiomers in the form of a 17. Shelnutt SR, Cimino CO, Wiggins PA, Badger TM. Urinary pharma- nutraceutical or pharmaceutical. Major clinical studies are likely cokinetics of the glucuronide and sulfate conjugates of genistein and to emerge in the near future that will permit a better understanding daidzein. Cancer Epidemiol Biomarkers Prev. 2000;9:413–9. of the potential value of equol in numerous clinical areas, not just 18. Ronis MJ, Little JM, Barone GW, Chen G, Radominska-Pandya A, those discussed above. Badger TM. Sulfation of the isoflavones genistein and daidzein in at American Society for Nutrition on June 18, 2010 human and rat liver and gastrointestinal tract. J Med Food. 2006;9: 348–55. Note added in proof: Work by Setchell et al (9) has shown that 19. Strassburg CP, Manns MP, Tukey RH. Expression of the UDP- the unconjugated fraction of [2-13C]S-(-)equol accounted for 6 glucuronosyltransferase 1A locus in human colon. Identification and only 0.10 0.05% of the total [2-13C]S-(-)equol in plasma characterization of the novel extrahepatic UGT1A8. J Biol Chem. following oral administration of 20 mg of the [13C]equol tracer 1998;273:8719–26. to 12 healthy adults. 20. Setchell KDR, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, Kirschner AS, Cassidy A, Heubi JE. Bioavailability of Acknowledgments pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr. 2001;131:S1362–75. The manuscript was written and approved by both authors. 21. Setchell KDR, Faughnan MS, Avades T, Zimmer-Nechemias L, Brown NM, Wolfe BE, Brasheer WT, Desai P, Oldfield MF, Botting NP, Cassidy A. Comparing the pharmacokinetics of daidzein and genistein with the use of 13C-labeled tracers in premenopausal Literature Cited women. Am J Clin Nutr. 2003;77:411–9. 1. Setchell KDR, Clerici C. Equol: history, chemistry and formation. 22. Nagel SC, vom Saal FS, Welshons WV. The effective free fraction of J Nutr. 2010;140:1355–62. estradiol and xenoestrogens in human serum measured by whole cell uptake assays: physiology of delivery modifies estrogenic activity. Proc 2. Jensen EV. On the mechanism of estrogen action. Perspect Biol Med. Soc Exp Biol Med. 1998;217:300–9. 1962;6:47–59. 23. Garreau B, Vallette G, Adlercreutz H, Wahala K, Makela T, 3. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Benassayag C, Nunez EA. Phytoestrogens: new ligands for rat and Cloning of a novel receptor expressed in rat prostate and ovary. Proc human a-fetoprotein. Biochim Biophys Acta. 1991;1094:339–45. Natl Acad Sci USA. 1996;93:5925–30. 24. Martin ME, Haourigui M, Pelissero C, Benassayag C, Nunez EA. 4. Morito K, Hirose T, Kinjo J, Hirakawa T, Okawa M, Nohara T, Ogawa Interactions between phytoestrogens and human sex steroid binding S, Inoue S, Muramatsu M, et al. Interaction of phytoestrogens with protein. Life Sci. 1996;58:429–36. estrogen receptors a and b. Biol Pharm Bull. 2001;24:351–6. 25. Marrian GF, Haslewood GA. Equol, a new inactive phenol isolated 5. Muthyala RS, Ju YH, Sheng S, Williams LD, Doerge DR, Katzenellenbogen from the ketohydroxyoestrin fraction of mares’ urine. Biochem BS, Helferich WG, Katzenellenbogen JA. Equol, a natural estrogenic J. 1932;26:1227–32. metabolite from soy isoflavones: convenient preparation and resolution of R- and S-equols and their differing binding and biological activity 26. Shutt DA, Cox RI. Steroid and phyto-oestrogen binding to sheep uterine through estrogen receptors alpha and beta. Bioorg Med Chem. 2004; receptors in vitro. J Endocrinol. 1972;52:299–310. 12:1559–67. 27. Tang BY, Adams NR. Effect of equol on oestrogen receptors and on 6. Setchell KDR, Clerici C, Lephart ED, Cole SJ, Heenan C, Castellani D, synthesis of DNA and protein in the immature rat uterus. J Endocrinol. Wolfe BE, Nechemias-Zimmer L, Brown NM, et al. S-equol, a potent 1980;85:291–7. ligand for estrogen receptor beta, is the exclusive enantiomeric form of 28. Thompson MA, Lasley BL, Rideout BA, Kasman LH. Characterization the soy isoflavone metabolite produced by human intestinal bacterial of the estrogenic properties of a nonsteroidal estrogen, equol, extracted flora. Am J Clin Nutr. 2005;81:1072–9. from urine of pregnant macaques. Biol Reprod. 1984;31:705–13. 7. Lund TD, Munson DJ, Haldy ME, Setchell KDR, Lephart ED, Handa 29. Markiewicz L, Garey J, Adlercreutz H, Gurpide E. In vitro bioassays of RJ. Equol is a novel anti-androgen that inhibits prostate growth and non-steroidal phytoestrogens. J Steroid Biochem Mol Biol. 1993;45: hormone feedback. Biol Reprod. 2004;70:1188–95. 399–405.

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78. Liu J, Ho SC, Su YX, Chen WQ, Zhang CX, Chen YM. Effect of long- 88. Ishiwata N, Melby MK, Mizuno S, Watanabe S. New equol supplement jn.nutrition.org term inervention of soy isoflavones on bone mineral density in women: a for relieving menopausal symptoms: randomized, placebo-controlled meta-analysis of randomized controll trials. Bone. 2009;44:948–53. trial of Japanese women. Menopause. 2009;16:141–8. 79. Wong WW, Lewis RD, Steinberg FM, Murray MJ, Cramer MA, Amato 89. Jou HJ, Wu SC, Chang FW, Ling PY, Chu KS, Wu WH. Effect of P, Young RL, Barnes S, Ellis KJ, et al. Soy isoflavone supplementation intestinal production of equol on menopausal symptoms in women and bone mineral density in menopausal women: a 2-y multicenter treated with soy isoflavones. Int J Gynaecol Obstet. 2008;102:44–9. at American Society for Nutrition on June 18, 2010 clinical trial. Am J Clin Nutr. 2009;90:1433–9. 90. Setchell KDR, Borriello SP, Hulme P, Kirk DN, Axelson M. Nonsteroi- 80. Alekel DL, Van Loan MD, Koehler KJ, Hanson LN, Stewart JW, dal estrogens of dietary origin: possible roles in hormone-dependent Hanson KB, Kurzer MS, Peterson CT. The Soy Isoflavones for Reducing disease. Am J Clin Nutr. 1984;40:569–78.

1368S Supplement The Journal of Nutrition Supplement: Equol, Soy, and Menopause

Emerging Research on Equol and Cancer1–3

Johanna W. Lampe*

Cancer Prevention Program, Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109

Abstract

Mechanisms of action of equol described using in vitro studies suggest possible effects of this compound in relation to cancer risk. However, experimental data are lacking with regard to the effects of S-(-)-equol (a gut bacterial product of daidzein), racemic equol, or even daidzein on tumorigenesis in vivo. Rodent studies, using racemic equol or daidzein in equol-producing animals, suggest that equol exposure does not stimulate mammary tumor growth, but there is little Downloaded from evidence that it is protective either. Racemic equol has been shown to inhibit skin carcinogenesis in hairless mice. Epidemiologic studies of associations between urinary or plasma isoflavone concentrations and breast cancer risk in women have reported no association nor increased risk associated with higher equol measures in low-soy–consuming populations but have reported a trend toward decreased cancer risk with increased equol in Asian populations. These population-based differences have been reported for prostate cancer too. Several studies in Asian men report lower equol jn.nutrition.org concentrations or a lower prevalence of equol-producers among men with prostate cancer compared with controls, whereas studies in European populations report no association. Studies using intermediate biomarkers of cancer risk and susceptibility in humans also have examined the effects the equol-producer phenotype in relation to soy intake with varying results. Overall, the role of equol in relation to cancer remains unclear. With the availability of R- and S-equol, at American Society for Nutrition on June 18, 2010 animal studies of carcinogenesis and human intervention studies addressing effects of the equol enantiomers on intermediate biomarkers may help to ascertain the role of equol in cancer risk. J. Nutr. 140: 1369S–1372S, 2010.

Introduction the racemic mixture are used. For example, in binding assays, S- Equol is a chiral molecule that can exist as 2 distinct optically equol had a high and preferential binding affinity for estrogen active isomers, R- and S-equol. The enantiomer S-(-)-equol, is receptor (ER)4 b, whereas R-equol bound more weakly with a the product of gut bacterial metabolism of the soy isoflavone preference for ERa (3). Further, compared with the racemic daidzein. Several actions of equol, including its estrogenic and mixture, S-equol had no antigenotoxic or antioxidant effects in antioxidant properties and its proliferative and antiproliferative breast cancer cell lines (2). The objective here was to summarize effects, suggest that exposure to the compound may have the available animal studies of equol and tumorigenesis, to implications for cancer risk [reviewed in (1,2)]. However, results update our 2005 review of the epidemiologic literature of equol of in vitro studies can be influenced by whether R- or S-equol or exposure and cancer risk (1), and to discuss the complexities of conducting research in this area. 1 Published in a supplement to The Journal of Nutrition. Presented at the “Equol, Soy, and Menopause Research Leadership Conference”, held in Washington, Animal studies DC, June 16, 2009. The Supplement Coordinator for this supplement is Kara Experimental data are lacking with regard to effects of S- Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The (-)-equol, racemic equol, or even daidzein on tumorigenesis. supplement is the responsibility of the guest editors to whom the Editor of The Journal of Nutrition has delegated supervision of both technical conformity Rodent studies, using racemic equol or daidzein in equol- to the published regulations of The Journal of Nutrition and general oversight of producing animals, suggest that R- and S-equol combined do not the scientific merit of each article. Publication costs for this supplement were stimulate mammary tumor growth, but there is little evidence defrayed in part by the payment of page charges. This publication must therefore that these compounds provide a protective effect either. be hereby marked "advertisement" in accordance with 18 USC section 1734 Lamartiniere et al. (4) reported that in rats, dimethylbenz(a) solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement anthracene-induced mammary tumorigenesis was not affected Coordinator disclosure: Kara Lewis is currently under contract with and receives by feeding daidzein-containing diets. Further, Ju et al. (5) compensation from the supplement sponsor. She was also compensated for showed that dietary racemic equol administered at 3 doses [250, attending and organizing the Equol, Soy, and Menopause Research Leadership 500, and 1000 ppm (1.03, 2.06, and 4.12 mmol/kg diet, Conference and for organizing, writing, editing, or reviewing, and collection of supplemental manuscripts. The opinions expressed in this publication are those respectively)] to ovariectomized athymic mice did not stimulate of the authors and are not attributable to the sponsors or the publisher, Editor, or growth of implanted estrogen-dependent human breast tumor Editorial Board of The Journal of Nutrition. 2 Supported in part by US NIH grants R01 CA120560 and R01 CA097366. 3 Author disclosure: J. W. Lampe, no conflict of interest. 4 Abbreviations used: EPIC, European Prospective Investigation into Cancer and * To whom correspondence should be addressed. E-mail: [email protected]. Nutrition; ER, estrogen receptor; OR, odds ratio.

ã 2010 American Society for Nutrition. 1369S First published online May 26, 2010; doi:10.3945/jn.109.118323. (MCF-7) cells, increase tumor cell proliferation, or induce the highest relative to the lowest quartile level (15). Some of the estrogen-responsive pS2 expression, despite stimulating growth limitations of these studies included small sample sizes, insuf- of MCF-7 cells in vitro. Findings such as these point to the ficient statistical power, and lack of controlled evaluation of challenges of translating in vitro results to effects in vivo and equol-producer status. speak to the need for more in vivo studies that allow for the Since 2005, additional, larger studies have examined the integration of pharmacokinetic and other factors that may affect relationship between equol measures and risk of prostate, colon, the biologic response. and breast cancer (Table 1). They continue to report mixed Topical application of racemic equol has been shown to results. A recent study in Japanese men reported reduced risk of reduce the proportion of tumors progressing from benign prostate cancer across tertiles of plasma equol and genistein (16). papillomas to malignant squamous cell carcinoma and reduce This association was limited to men with localized disease. In a the average diameter of lesions in hairless mice treated with study of the Multiethnic Cohort, a cohort including men in 5 solar-simulated UV radiation and/or dimethylbenz(a)anthracene ethnic/multi-racial groups (i.e., African Americans, Native Ha- (6). Further, in mice treated with solar-simulated UV radiation, waiians, Japanese Americans, Latinos, and Whites), Park et al. racemic equol topically applied prior to UV treatment reduced (17) reported a nonsignificant association between prostate DNA damage as measured by cyclobutane pyrimidine dimers, cancer risk and tertiles of urinary equol. Odds ratios (OR) whereas equol applied after UV treatment did not increase the (95% CI) for the second and 3rd tertiles compared with the rate of dimer removal (7). Whether there are differences in lowest tertile were 0.89 (0.58–1.37) and 1.32 (0.84–2.08), effects of the specific equol enantiomers on tumorigenesis respectively (P-trend = 0.08). There was no significant interaction remains to be established. of urinary equol by race/ethnicity or any difference by tumor Downloaded from characteristics. Epidemiologic studies of equol and cancer In European populations, 2 large studies conducted in the The association between equol production and cancer risk in European Prospective Investigation into Cancer and Nutrition humans has not been extensively characterized. Because of the (EPIC) cohorts reported no association between equol and risk lack of commercially available dietary equol supplements, of prostate cancer and colon cancer (18,19). Two studies of human exposure to equol historically has been exposure to S- breast cancer, also in EPIC cohorts, similarly reported no jn.nutrition.org (-)-equol as a result of gut bacterial conversion of daidzein to association between equol measures and overall breast cancer equol. In 2005 in a review of the literature, Atkinson et al. (1) risk (20,21); however, among ER-positive cases in the Norfolk identified 8 studies of equol and cancer. The studies in Asian men cohort, urinary equol was associated with a slightly higher risk tended to report lower equol concentrations or a lower [OR (95% CI) = 1.07 (1.01–1.112); P = 0.013] in the 95 cases at American Society for Nutrition on June 18, 2010 prevalence of equol producers among men with prostate cancer compared with the 329 controls. compared with controls (8–10). The studies of breast cancer Observational studies of equol and disease outcomes, such as yielded inconsistent results, with reports of nonsignificant lower the ones described above, present particular challenges. They equol excretion in breast cancer cases than controls in Asian and require sufficient habitual exposure to daidzein to allow for Asian-American populations (11,12), a significant trend toward bacterial production of equol. In Asian cohorts, the primary lower risk of breast cancer across increasing quartiles of equol source of daidzein in observational studies is soyfoods and, excretion in an Australian study (13), and higher urine and among individuals excreting measurable amounts of equol, serum equol associated with breast cancer in the UK (14). In a equol excretion is soy protein dose-dependent (22). Therefore, in case-control study of women with histologically confirmed a population with a range of soy intakes, it is often difficult to cervical squamous intraepithelial lesions (cases) and normal tease out whether equol itself is associated with disease risk, cytology (controls), plasma equol concentrations were positively whether equol is serving as an additional measure of daidzein or associated with cervical squamous intraepithelial lesions risk for genistein exposure or of soy food intake in general, or whether

TABLE 1 Summary of studies since 2005 evaluating associations between urinary or circulating equol measures and cancer risk1

Study population Cases Controls Findings Reference

Japanese Prostate: 201 402 Highest tertile for plasma equol associated with decreased risk (16) of total cancer [OR (95% CI) = 0.60 (0.36–0.99); P-trend = 0.04]; association stronger when confined to cases with localized disease [OR (95% CI) = 0.43 (0.22–0.82); P-trend = 0.02]. Multiethnic cohort, US Prostate: 249 404 NS trend for higher prostate cancer risk with higher urinary equol (17) (nmol mg21 creatinine; (P-trend = 0.08). EPIC cohort, Europe Prostate: 950 1042 No association between plasma equol and prostate cancer risk. (18) EPIC cohort, Norfolk, UK Prostate: 191 cases (serum); 815 (serum); No association between serum or urine equol and prostate (19) 152 (urine) 665 (urine) cancer risk. EPIC cohort, Norfolk, UK Colon and rectum: 214 (serum); 877 (serum), No association between serum or urine equol and colorectal (19) 146 (urine) 686 (urine) cancer risk. EPIC cohort, The Netherlands Breast: 383 (296 postmenopausal) 383 No association between plasma equol and breast cancer risk. (20) EPIC cohort, Norfolk, UK Breast: 219 (serum); 198 (urine) 891 (serum), No association between serum or urine equol and breast (21) 7971 (urine) cancer risk overall; however, among ER+ cases, urinary equol (mg mmol21 creatinine) associated with higher risk [OR (95% CI) = 1.07 (1.01–1.112); P = 0.013].

1 EP, Equol producer; NS, nonsignificant.

1370S Supplement equol is a marker of harboring a particular gut bacterial Literature Cited community (1). To address some of these issues, statistical 1. Atkinson C, Frankenfeld CL, Lampe JW. Gut bacterial metabolism of approaches are needed that adjust for overall soy or isoflavone the soy isoflavone daidzein: exploring the relevance to human health. exposure before testing for equol effects. Studies of equol Exp Biol Med (Maywood). 2005;230:155–70. exposure are more problematic in Western Europe and the US 2. Magee PJ, Raschke M, Steiner C, Duffin JG, Pool-Zobel BL, Jokela T, where soy intake is very low. Even among individuals in the Wahala K, Rowland IR. Equol: a comparison of the effects of the highest quantiles of exposure in these populations, equol in racemic compound with that of the purified S-enantiomer on the blood and urine is low and likely to be below clinically growth, invasion, and DNA integrity of breast and prostate cells in vitro. Nutr Cancer. 2006;54:232–42. relevant levels (23). Equol exposure in some Western popula- 3. Muthyala RS, Ju YH, Sheng S, Williams LD, Doerge DR, tions also may be due to dietary intake of equol from animal Katzenellenbogen BS, Helferich WG, Katzenellenbogen JA. Equol, a and dairy sources, rather than from daidzein from soy foods natural estrogenic metabolite from soy isoflavones: convenient (24). preparation and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and Equol phenotype and intermediate biomarkers beta. Bioorg Med Chem. 2004;12:1559–67. in human studies 4. Lamartiniere CA, Wang J, Smith-Johnson M, Eltoum IE. Daidzein: bioavailability, potential for reproductive toxicity, and breast cancer Studies using intermediate biomarkers of cancer risk and cancer chemoprevention in female rats. Toxicol Sci. 2002;65:228–38. susceptibility in humans have also examined the effect of equol- 5. Ju YH, Fultz J, Allred KF, Doerge DR, Helferich WG. Effects of dietary producer phenotype. Similar to the studies of cancer outcomes daidzein and its metabolite, equol, at physiological concentrations on in humans, these studies also reflect exposure to S-(-)-equol. In the growth of estrogen-dependent human breast cancer (MCF-7) tumors Downloaded from an observational study of postmenopausal women phenotyped implanted in ovariectomized athymic mice. Carcinogenesis. 2006; for equol production, Fuhrman et al. (25) reported a significant 27:856–63. interaction between equol-producer phenotype and soy intake 6. Widyarini S, Husband AJ, Reeve VE. Protective effect of the isoflavonoid equol against hairless mouse skin carcinogenesis induced in association with mammographic density (a biomarker of by UV radiation alone or with a chemical cocarcinogen. Photochem breast cancer risk) despite no independent associations of Photobiol. 2005;81:32–7. phenotype or soy intake individually. In contrast, an interven- 7. Widyarini S. Protective effect of the isoflavone equol against DNA jn.nutrition.org tion study testing the effects of soy protein on mammographic damage induced by ultraviolet radiation to hairless mouse skin. J Vet density showed no effect of equol-producer phenotype (26). Sci. 2006;7:217–23. An isoflavone supplement intervention in men with a personal 8. Akaza H, Miyanaga N, Takashima N, Naito S, Hirao Y, Tsukamoto T, Fujioka T, Mori M, Kim WJ, et al. Comparisons of percent equol or family history of colorectal adenoma showed that circulat- producers between prostate cancer patients and controls: case-con- ing insulin-like growth factor-I decreased in equol producers trolled studies of isoflavones in Japanese, Korean and American at American Society for Nutrition on June 18, 2010 but not nonproducers (27). Further, the serum insulin-like residents. Jpn J Clin Oncol. 2004;34:86–9. growth factor-I change was inversely associated with serum 9. Akaza H, Miyanaga N, Takashima N, Naito S, Hirao Y, Tsukamoto T, equol concentration. In another study of postmenopausal Mori M. Is daidzein non-metabolizer a high risk for prostate cancer? A women, a stronger effect of isoflavone supplementation (900 case-controlled study of serum soybean isoflavone concentration. Jpn J Clin Oncol. 2002;32:296–300. mg/d for 84 d) on estrogen-responsive genes in peripheral 10. Ozasa K, Nakao M, Watanabe Y, Hayashi K, Miki T, Mikami K, Mori lymphocytes was observed among equol producers compared M, Sakauchi F, Washio M, et al. Serum phytoestrogens and prostate with nonproducers (28). These studies suggest that there may cancer risk in a nested case-control study among Japanese men. Cancer be a differential response to isoflavones dependent on equol- Sci. 2004;95:65–71. producer phenotype; however, results are not consistent across 11. Wu AH, Yu MC, Tseng CC, Twaddle NC, Doerge DR. Plasma studies. isoflavone levels versus self-reported soy isoflavone levels in Asian- American women in Los Angeles County. Carcinogenesis. 2004;25: 77–81. Summary 12. Zheng W, Dai Q, Custer LJ, Shu XO, Wen WQ, Jin F, Franke AA. The role of equol in relation to cancer remains unclear. To date, Urinary excretion of isoflavonoids and the risk of breast cancer. Cancer animal studies using either daidzein or racemic equol are few Epidemiol Biomarkers Prev. 1999;8:35–40. and there are no studies of S-(-)-equol specifically. The number of 13. Ingram D, Sanders K, Kolybaba M, Lopez D. Case-control study of epidemiologic studies of equol exposure and cancer risk in phyto-oestrogens and breast cancer. Lancet. 1997;350:990–4. humans is also limited and the studies are difficult to interpret. 14. Grace PB, Taylor JI, Low YL, Luben RN, Mulligan AA, Botting NP, These studies have had to rely on measurement of circulating or Dowsett M, Welch AA, Khaw KT, et al. Phytoestrogen concentrations in urinary equol concentrations in populations routinely consum- serum and spot urine as biomarkers for dietary phytoestrogen intake and their relation to breast cancer risk in European prospective ing soy. The ideal test would be a randomized, placebo- investigation of cancer and nutrition-norfolk. Cancer Epidemiol Bio- controlled intervention trial of supplemental equol with cancer markers Prev. 2004;13:698–708. endpoints. However, given the lack of preclinical data and the 15. Hernandez BY, McDuffie K, Franke AA, Killeen J, Goodman MT. lack of consistent effects of equol-producer phenotype in soy- Plasma and dietary phytoestrogens and risk of premalignant lesions of isoflavone intervention studies, such an undertaking is unwar- the cervix. Nutr Cancer. 2004;49:109–24. ranted. With the recent availability of sufficient amounts of 16. Kurahashi N, Iwasaki M, Inoue M, Sasazuki S, Tsugane S. Plasma R- and S-equol, animal studies of carcinogenesis and human isoflavones and subsequent risk of prostate cancer in a nested case- control study: the Japan Public Health Center. J Clin Oncol. intervention studies that address directly the effects of the equol 2008;26:5923–9. enantiomers on intermediate biomarkers of cancer risk may help 17. Park SY, Wilkens LR, Franke AA, Le Marchand L, Kakazu KK, to further ascertain the effects of the equol-producer phenotype Goodman MT, Murphy SP, Henderson BE, Kolonel LN. Urinary and equol itself. phytoestrogen excretion and prostate cancer risk: a nested case-control study in the Multiethnic Cohort. Br J Cancer. 2009;101:185–91. 18. Travis RC, Spencer EA, Allen NE, Appleby PN, Roddam AW, Overvad Acknowledgment K, Johnsen NF, Olsen A, Kaaks R, et al. Plasma phyto-oestrogens and The sole author had responsibility for all parts of the manu- prostate cancer in the European Prospective Investigation into Cancer script. and Nutrition. Br J Cancer. 2009;100:1817–23.

Equol and cancer 1371S 19. Ward H, Chapelais G, Kuhnle GG, Luben R, Khaw KT, Bingham S. products, eggs, meat, fish, and seafood. J Agric Food Chem. Lack of prospective associations between plasma and urinary phytoes- 2008;56:10099–104. trogens and risk of prostate or colorectal cancer in the European 25. Fuhrman BJ, Teter BE, Barba M, Byrne C, Cavalleri A, Grant BJ, Prospective into Cancer-Norfolk study. Cancer Epidemiol Biomarkers Horvath PJ, Morelli D, Venturelli E, et al. Equol status modifies the Prev. 2008;17:2891–4. association of soy intake and mammographic density in a sample of 20. Verheus M, van Gils CH, Keinan-Boker L, Grace PB, Bingham SA, postmenopausal women. Cancer Epidemiol Biomarkers Prev. 2008; Peeters PH. Plasma phytoestrogens and subsequent breast cancer risk. 17:33–42. J Clin Oncol. 2007;25:648–55. 26. Verheus M, van Gils CH, Kreijkamp-Kaspers S, Kok L, Peeters PH, 21. Ward H, Chapelais G, Kuhnle GG, Luben R, Khaw KT, Bingham S. Grobbee DE, van der Schouw YT. Soy protein containing isoflavones European Prospective into Cancer-Norfolk cohort. Breast cancer risk in and mammographic density in a randomized controlled trial in relation to urinary and serum biomarkers of phytoestrogen exposure in postmenopausal women. Cancer Epidemiol Biomarkers Prev. 2008;17: the European Prospective into Cancer-Norfolk cohort study. Breast 2632–8. Cancer Res. 2008;10:R32. 27. Vrieling A, Rookus MA, Kampman E, Bonfrer JM, Korse CM, van 22. Karr SC, Lampe JW, Hutchins AM, Slavin JL. Urinary isoflavonoid Doorn J, Lampe JW, Cats A, Witteman BJ, et al. Isolated isoflavones do excretion in humans is dose dependent at low to moderate levels of soy- not affect the circulating insulin-like growth factor system in men at protein consumption. Am J Clin Nutr. 1997;66:46–51. increased colorectal cancer risk. J Nutr. 2007;137:379–83. 23. Messina M. Western soy intake is too low to produce health effects. Am 28. Niculescu MD, Pop EA, Fischer LM, Zeisel SH. Dietary isoflavones J Clin Nutr. 2004;80:528–9. differentially induce gene expression changes in lymphocytes from 24. Kuhnle GG, Dell’Aquila C, Aspinall SM, Runswick SA, Mulligan AA, postmenopausal women who form equol as compared with those who Bingham SA. Phytoestrogen content of foods of animal origin: dairy do not. J Nutr Biochem. 2007;18:380–90. Downloaded from jn.nutrition.org at American Society for Nutrition on June 18, 2010

1372S Supplement The Journal of Nutrition Supplement: Equol, Soy, and Menopause

Dietary Equol and Bone Metabolism in Postmenopausal Japanese Women and Osteoporotic Mice1,2

Yoshiko Ishimi*

National Institute of Health and Nutrition, Tokyo 162-8636, Japan

Abstract

Equol binds to the estrogen receptor with greater affinity than its precursor, daidzein, an isoflavone found in soybeans. Downloaded from Inter-individual differences in the ability to produce equol may lead to differential effects of isoflavone intervention on human health. Here, we review previously published work from our laboratory on equol producer status and bone health in humans and in a mouse model of osteoporosis. We performed a 1-y, double-blind, randomized trial to compare the effects of isoflavone (75 mg of isoflavone conjugates/d; equivalent to 47 mg/d of the aglycone form) with those of placebo on bone mineral density (BMD), fat mass, and serum isoflavone concentrations in 54 early postmenopausal Japanese women jn.nutrition.org classified by their equol-producer phenotype. Isoflavone intervention increased the serum equol concentration in equol producers but not in nonproducers (P , 0.04). The annualized changes in BMD in the total hip and intertrochanteric regions in the isoflavone-treated equol producers (–0.46 and –0.04%, respectively) were less than in the nonproducers (–2.28 and

–2.61%, respectively). The annualized change in fat mass was lower in the equol producers compared with the at American Society for Nutrition on June 18, 2010 nonproducers in the isoflavone group. The annualized changes in BMD and fat mass did not differ between the equol producers and nonproducers in the placebo group. Equol also inhibited bone loss and fat accumulation in estrogen- deficient osteoporotic mice. Our data suggest that prevention of bone loss and fat accumulation in early postmenopausal women by isoflavones may depend on an individual’s equol-producing capacity. J. Nutr. 140: 1373S–1376S, 2010.

Effects of the intestinal metabolites of daidzein, equol, isoflavones (40–200 mg/d) for 1–2 y and measured bone mineral on bone metabolism density (BMD).3 The results from human studies are inconsistent Epidemiological studies indicate that women who have high soy (5). A recent meta-analysis of 10 randomized, controlled trials intake have less risk for osteoporosis than those consuming a indicated that isoflavone intervention significantly attenuated typical Western diet (1). Additionally, several recently published bone loss in postmenopausal women (6,7). However, another studies report that isoflavone supplementation decreases risk for meta-analysis concluded that isoflavones do not affect bone loss osteoporosis (2–4). In general, these studies administered (8). One potential reason for these inconsistencies is individual differences in isoflavone metabolism. Recent studies suggest that

1 Published in a supplement to The Journal of Nutrition. Presented at the “Equol, the clinical effectiveness of isoflavones on bone metabolism Soy, and Menopause Research Leadership Conference” held in Washington, might be due to individual differences in the ability to produce DC, June 16, 2009. The supplement coordinator for this supplement is Kara the daidzein metabolite, equol, in the intestine (9). Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The Equol is an isoflavone and a nonsteroidal estrogen (9). It supplement is the responsibility of the guest editors to whom the Editor of The Journal of Nutrition binds to both estrogen receptors and induces transcription more has delegated supervision of both technical conformity to a the published regulations of The Journal of Nutrition and general oversight of the strongly than other isoflavones, especially estrogen receptor- scientific merit of each article. Publication costs for this supplement were (10). Moreover, equol is a chiral molecule, which exists as defrayed in part by the payment of page charges. This publication must therefore enantiomers R (+)-equol and S (–)-equol. In humans, the be hereby marked "advertisement" in accordance with 18 USC section 1734 intestinal bacterial metabolism of daidzein to equol results in solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement S-equol production only (11). Humans may be the only species Coordinator disclosure: Kara Lewis is currently under contract with and receives that exhibits inter-individual variability in equol production. compensation from the supplement sponsor. She was also compensated for Several animals, including rats (12), mice (13), and chimpanzees attending and organizing the Equol, Soy, and Menopause Research Leadership (14), excrete equol. O-desmethylangolensin (O-DMA), another Conference and for organizing, writing, editing, or reviewing, and collection of daidzein metabolite, is found in ~80–90% of the human supplemental manuscripts. The opinions expressed in this publication are those of the authors and are not attributable to the sponsors or the publisher, Editor, or Editorial Board of The Journal of Nutrition. 2 Author disclosure: Y. Ishimi, no conflicts of interest. 3 Abbreviations used: BMD, bone mineral density; Lc., Lactococcus; O-DMA, * To whom correspondence should be addressed. E-mail: [email protected]. O-desmethylangolensin; OVX, ovariectomized.

ã 2010 American Society for Nutrition. 1373S First published online May 19, 2010; doi:10.3945/jn.110.124842. population (15,16) and equol is found in ~30–50% (9,17–20). interleukin-1b. Furthermore, Nakamura et al. (35) recently The variability in mammalian equol production is considered to reported apoptosis and Fas ligand expression upregulation in be dependent on individual differences in the intestinal micro- trabecular osteoclasts from wild type but not estrogen receptor D D biota responsible for equol production (9,21). Intestinal bacteria knockout (ERa Oc/ Oc) mice after estrogen treatment. These play an important role in isoflavone metabolism; young infants results support a model in which estrogen regulates the life span of with undeveloped gut microflora do not produce equol (22–24) mature osteoclasts via the induction of the Fas/Fas ligand system and germ-free animals also do not produce equol or O-DMA and help explain the osteoprotective function of estrogen and (25–28). Because some reports suggested a lower disease risk for selective estrogen receptor modulators, including isoflavones. equol producers than for nonproducers (9), there is a growing interest in certain bacterial strains that can produce equol. The role of equol status in the effects of isoflavones on A number of strains involved in daidzein metabolism have bone health in humans been identified (29–31). However, the identification of equol- Equol production depends on an individual’s intestinal flora. producing bacteria is complicated (9,11,21,25,26). Research has shown that ~30–50% of individuals in the Lactococcus (Lc.), 20–92 homologous to Lc. Garvieae, is the population studied are capable of producing equol from only lactic acid bacterium to date known to produce equol daidzein (36). Lydeking-Olsen et al. (36) conducted a 2-y, directly from daidzein without producing O-DMA (32). Inter- randomized, placebo-controlled trial to investigate the effec- estingly, the strain, Lc. 20–92, can also cleave glycosidic bonds tiveness of isoflavone-supplemented soymilk (76 mg/d aglycone of daidzein. Here we review our previously published work on isoflavones content) for prevention of bone loss in postmeno- equol producer status and bone health in humans and a mouse pausal women aged 41–75 y (mean age 58 y). The lumbar spine Downloaded from model of osteoporosis. BMD for women who consumed soymilk with low levels of isoflavones (n = 22) decreased (4.2%; P = 0.01) by the end of the Effects of equol on bone metabolism in animals study; however, the lumbar spine BMD of women who Estrogen deficiency is associated with increased bone turnover consumed soymilk with isoflavones (n = 23) did not change and acceleration of bone loss, which leads to an increased compared with baseline. Equol producers (n = 10), defined by a susceptibility to bone fracture. A number of studies have cutoff level of 10 ng/mL (40 mmol/L) plasma equol, had a 2.4% jn.nutrition.org reported that the soybean isoflavones, genistein and daidzein, increase in lumbar spine BMD compared with the 0.6% increase dosed dependently inhibit bone loss in both female and male for equol nonproducers (n = 12) after the 2-y intervention with osteoporotic animal models without causing notable effects on isoflavone-enriched soymilk. reproductive organs (33). Similarly, equol may inhibit bone loss We assessed the effects of equol-producing activity on BMD due to ovariectomy. We have reported that administration of in postmenopausal Japanese women (37). Study participants at American Society for Nutrition on June 18, 2010 equol (0.5 mg/d subcutaneously) inhibited bone loss of the were 68 healthy women aged 45–60 y who had undergone whole body and femur in ovariectomized mice without uterine natural menopause within the previous 5 y, where menopause hypertrophy (34). Although 17b-estradiol administration (0.03 was defined as at least 12 mo beyond the last menstrual cycle. mg/d subcutaneously) prevented ovariectomized-induced bone Fifty-four women (29/34 in the placebo, 25/34 in the isoflavone loss from all regions, uterine hypertrophy occurred in mice (34). group) completed the 1-y intervention and their data were These results suggest that similar to selective estrogen receptor used for equol analysis. Study participants in the isoflavone modulators, isoflavones, including equol, inhibit bone loss group received a daily dose of 75 mg of isoflavone conjugates without estrogenic activity in the reproductive organs of (38.3 mg daidzin, 0.2 mg malonyl-daidzin, 2.1 mg acetyldaidzin, estrogen-deficient animals. It is now recognized that one of the 0.6 mg daidzein, 8.6 mg genistin, 0.6 mg acetylgenistin, 0.2 mg mechanisms by which estrogen deficiency causes bone loss is via genistein, and 24.4 mg glycitin with glycitein) in capsule form stimulation of osteoclast formation, a process enhanced by several (Fujiflavone P40, Fujicco; 47 mg aglycon form) with dextrin and inflammatory cytokines, such as tumor necrosis factor-a and those in the placebo group received capsules containing only

FIGURE 1 Percent changes in BMD of the whole body (A), hip (B), femoral neck (C), and the inter- trochanter region (D) in postmenopausal Japanese women who were equol (EQ) producers or non- producers after 1 y of isoflavone (75 mg/d) or placebo treatment. Values are means 6 SD, n = 10– 15. *Different from EQ producers, P , 0.05. Adapted from (37) with permission by Wolters Kluwer/Lippincott, Williams & Wilkins.

1374S Supplement dextrin. Fifty-six percent of study participants were equol in intestinal microflora influence the effects of isoflavones on producers, as assessed by equol production in fecal suspension bone health. The preventive effects of daidzein on bone loss in incubated with daidzein under anaerobic conditions. In the postmenopausal women might depend on an individual’s equol- placebo group, there were 15 and 14 equol producers and producing capacity. In research from our laboratory, supple- nonproducers, respectively; and in the isoflavone group, there mentation of the normal diet for 1 y with an additional 47 mg/d were 15 and 10 equol producers and nonproducers, respectively. of isoflavone aglycon equivalent did not have adverse effects in Serum daidzein concentrations, as determined by reverse-phase postmenopausal women. Further studies are required to address HPLC, increased to 3-fold the baseline value in equol producers the numerous questions on the potential benefits, mechanisms of and nonproducers who consumed isoflavone for 1 y (P = 0.002). action, and safety of isoflavones for postmenopausal women. However, there was no difference in the serum daidzein concentration between equol producers and nonproducers. On Acknowledgment the other hand, serum equol increased only in equol producers in The sole author had responsibility for all parts of the manu- the isoflavone group after 1 y (P = 0.04). Serum equol did not script. change in equol producers in the placebo group. Because equol is a metabolite of daidzein, it has a slower plasma clearance rate than daidzein (10). Therefore, whereas the serum levels of Literature Cited daidzein did not significantly differ between the equol producers and nonproducers, the equol levels were significantly higher in 1. Messina MJ. Soy foods and soybean isoflavones and menopausal health. the equol producers. Equol producers tended to have lower Nutr Clin Care. 2002;5:272–82. Downloaded from serum concentrations than nonproducers (P = 0.15), but the 2. Huang HY, Yang HP, Yang HT, Yang TC, Shieh MJ, Huang SY. One- longer half-life of equol in the bloodstream could explain why year soy isoflavone supplementation prevents early postmenopausal bone loss but without a dose-dependent effect. J Nutr Biochem. there were no significant differences in daidzein levels. After 1 y, 2006;17:509–17. serum genistein did not differ between equol producers and 3. Wong WW, Lewis RD, Steinberg FM, Murray MJ, Cramer MA, Amato nonproducers in the isoflavone group. P, Young RL, Barnes S, Ellis KJ, et al. Soy isoflavone supplementation

In the isoflavone intervention group, the percent change in and bone mineral density in menopausal women: a 2-y multicenter jn.nutrition.org bone loss in the total hip (–0.46%) and the hip intertrochanteric clinical trial. Am J Clin Nutr. 2009;90:1433–9. region (–0.04%) of equol producers was lower (P , 0.05) than 4. Bannwart C, Adlercreutz H, Fotsis T, Wahala K, Hase T, Brunow G. that of equol nonproducers (–2.28 and –2.61%, respectively) Identification of O-desmethylangolensin, a metabolite of daidzein and of matairesinol, one likely plant precursor of the animal lignan (Student’s t test; Fig. 1) (38). enterolactone in human urine. Finn Chem Lett. 1984;4–5:120–5. A 2-factor analysis of covariance revealed no significant main 5. Brink E, Coxam V, Robins S, Wahala K, Cassidy A, Branca F. Long-term at American Society for Nutrition on June 18, 2010 effect on the percent change in BMD in any region after 1 y. The consumption of isoflavone-enriched foods does not affect bone mineral percent change in bone loss in the total hip and the hip density, bone metabolism, or hormonal status in early postmenopausal intertrochanteric region did not differ between equol producers women: a randomized, double-blind, placebo controlled study. Am J Clin Nutr. 2008;87:761–70. and nonproducers in the placebo group. Equol producers had a 6. Ma DF, Qin LQ, Wang PY, Katoh R. Soy isoflavone intake increases significantly lower annualized change in fat mass than non- bone mineral density in the spine of menopausal women: meta-analysis producers in the isoflavone group, but this was not the case for of randomized controlled trials. Clin Nutr. 2008;27:57–64. the placebo group. Based on these results, the effects of 7. Taku K, Melby MK, Takebayashi J, Mizuno S, Ishimi Y, Omori T, isoflavone on bone and fat mass might depend on equol- Watanabe S. Effect of soy isoflavone extract supplements on bone producing activity in postmenopausal Japanese women (37). mineral density in menopausal women: meta-analysis of randomized Isoflavone supplementation (47 mg/d aglycone equivalent) with controlled trials. Asia Pac J Clin Nutr. 2010;19:33–42. a normal diet for 1 y did not affect serum levels of estrogen, 8. Liu J, Ho SC, Su YX, Chen WQ, Zhang CX, Chen YM. Effect of long- term inervention of soy isoflavones on bone mineral density in women: a follicle-stimulating hormone, luteinizing hormone, progester- meta-analysis of randomized control trials. Bone. 2009;44:948–53. one, triiodothyronine, thyroxine, and thyroid stimulating hor- 9. Setchell KDR, Brown NM, Lydeking-Olsen E. The clinical importance mone in postmenopausal Japanese women (39). of the metabolite equol: a clue to the effectiveness of soy and its isoflavones. J Nutr. 2002;132:3577–84. Isolation of equol-producing bacteria from human feces 10. 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Daidzein: bioavailability, potential for reproductive toxicity, and breast cancer in 35.3% of the postmenopausal Japanese women (39). Inter- chemoprevention in female rats. Toxicol Sci. 2002;65:228–38. estingly, women with Lc. garvieae were not always equol 13. Ohta A, Uehara M, Sakai K, Takasaki M, Adlercreutz H, Morohashi T, producers, suggesting that other bacteria may play a role in Ishimi Y. A combination of dietary fructooligosaccharides and isofla- human equol production. Several other factors such as hydrogen vone conjugates increases femoral bone mineral density and equol pro- gas and short chain fatty acids, which can affect the environ- duction in ovariectomized mice. J Nutr. 2002;132:2048–54. mental conditions in the colon, may also be necessary for equol 14. Adlercreutz H, Musey PI, Fotsis T, Bannwart C, Wahala K, Makela T, production. Brunow G, Hase T. Identification of lignans and phytoestrogens in urine of chimpanzees. Clin Chim Acta. 1986;158:147–54. Conclusions 15. 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1376S Supplement The Journal of Nutrition Supplement: Equol, Soy, and Menopause

Equol, via Dietary Sources or Intestinal Production, May Ameliorate Estrogen Deficiency-Induced Bone Loss1-3

Connie M. Weaver* and LeeCole L. Legette

Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907

Abstract

Equol, a product of intestinal metabolism of daidzein, is chemically similar to estrogen (without the lipophilic moiety) and Downloaded from has higher estrogen receptor-b binding affinity than its parent precursor. In 2004, a long-term, randomized controlled trial that characterized postmenopausal women by their equol-producing status showed stronger advantages to lumbar spine bone mineral density (BMD) in equol- compared with nonequol-producers. Subsequent studies have related equol status of participants to change in bone turnover markers or BMD in response to soy isoflavone interventions. To our knowledge, we are the first to prescreen women for equol-producing status prior to initiating an intervention. In menopausal Western jn.nutrition.org women, equol status did not affect the modest, but significant, reduction in bone resorption achieved with a soy isoflavone intervention. J. Nutr. 140: 1377S–1379S, 2010. at American Society for Nutrition on June 18, 2010

Introduction 50% S-equol) in 6-mo-old ovariectomized (OVX) Sprague Dietary soy isoflavone supplements have been studied for Dawley rats. Doses were selected to achieve similar serum their effect on ameliorating bone loss associated with meno- concentrations in postmenopausal women consuming medium pause. However, studies of dietary equol as a potential dietary and high amounts of isoflavones. We found that tissue (heart, supplement are limited. The reported studies of oral and i.v. intestine, kidney, and liver) equol concentrations reflected the equol administration on bone in animal models used high doses diet, but only the highest dose studied (200 mg/kg) increased the to inhibit bone loss, which caused adverse effects on reproduc- femoral calcium content. Uterine, but not mammary gland, tive tissues. We conducted a dose-ranging study (0, 50, 100, and epithelial tissues were stimulated at this dose. These findings 200 mg equol/kg diet) of dietary racemic equol (50% R-equol, suggested limited benefit with the potential for adverse effects of equol as a dietary supplement.

1 The Journal of Nutrition Published in a supplement to . Presented at the “Equol, Equol may be a bone antiresorption agent Soy, and Menopause Research Leadership Conference” held in Washington, DC, June 16, 2009. The supplement coordinator for this supplement is Kara Endogenous estrogen helps with maintaining bone mass and its Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The deficiency with transition to menopause has been associated supplement is the responsibility of the guest editors to whom the Editor of with rapid bone loss (1,2). When circulating levels decrease, The Journal of Nutrition has delegated supervision of both technical conformity bone resorption rates exceed bone formation rates, leading to to the published regulations of The Journal of Nutrition and general oversight of the scientific merit of each article. Publication costs for this supplement were accelerated bone loss for 3–5 y until bone formation rates catch defrayed in part by the payment of page charges. This publication must therefore up (3). Estrogen therapy was a mainstay of osteoporosis preven- be hereby marked "advertisement" in accordance with 18 USC section 1734 tion in postmenopausal women until adverse cardiovascular and solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. breast cancer health events were reported in women randomized Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement Coordinator disclosure: Kara Lewis is currently under contract with and receives to a conjugated estradiol treatment (premarin) in the Women’s compensation from the supplement sponsor. She was also compensated for Health Initiative trial (4). Replacements for estrogen have been attending and organizing the Equol, Soy, and Menopause Research Leadership highly sought that have the antiresorption properties of estrogen Conference and for organizing, writing, editing, or reviewing, and collection of therapy without the adverse effects on reproductive tissues. supplemental manuscripts. The opinions expressed in this publication are those Soy isoflavones have been studied the most for their potential of the authors and are not attributable to the sponsors or the publisher, Editor, or Editorial Board of The Journal of Nutrition. to replace estrogen therapy. These studies have had mixed results 2 Supported by the Purdue-University of Alabama-Birmingham Botanical Center (5–15). Equol has received much attention in the last 5 y. Equol, for Age Related Diseases sponsored by NIH (grant nos. P50 AT00477-01 and P50 the end product of intestinal metabolism of daidzein, is AT000477-07S1). chemically similar to estrogen (without the lipophilic moiety) 3 Author disclosures: C. M. Weaver is a member of the advisory board for and has 80 times more estrogen receptor-b binding affinity than Pharmavite, LLC; L. L. Legette, no conflicts of interest. b * To whom correspondence should be addressed. E-mail: weavercm@purdue. its parent precursor (16). Estrogen receptor- is dominant in edu. bone and is presumed to be a primary mode of action of estrogen

ã 2010 American Society for Nutrition. 1377S First published online May 26, 2010; doi:10.3945/jn.109.118331. and phytoestrogens such as isoflavones (16). Other modes of genistein content (158 mg/d) was soy derived and suppressed bone action, including suppressing osteoclastogenesis, have also been resorption about one-third as well as either estradiol (1 mg/d) and proposed (14,15). Osteoclast formation was inhibited by equol mederoxyprogestrone (2.5 mg/d) or a bisphosphate (Actonel, 5 in a dose-dependent manner (10–1000 nmol/L) in a coculture mg/d). We are using this method to determine the response to soy system of mouse bone marrow and primary osteoclast cells (17). feeding in equol-producers compared with nonproducers and also All rodents, but not all humans, have gut microflora that to determine the effective dose in a dose-response study of purified convert daidzein to equol. Many approximate that only genistein. This method has promise for determining drug-botan- 30–50% of the population possesses the gut microflora needed ical interactions. for bacteria intestinal conversion of daidzein to equol (18,19). Here, we explore equol as a dietary supplement on bone Effect of equol-producing status on response to soy properties as well as the effect of equol-producing status on bone Recent evidence suggests that the effect of soy isoflavone antiresorptive responsiveness to soy. treatment on health is dependent on the ability to convert daidzein to equol. Soy isoflavone supplementation has a stronger Dietary equol, calcium metabolism, and bone properties effect on the expression of estrogen responsive genes (genes with in estrogen deplete rodent models estrogen response elements in promoter regions) in equol Studies of dietary equol as a potential dietary supplement are producers compared with nonequol producers (30). A long- limited. Early studies administered equol by i.v. injection or term, randomized controlled trial that characterized postmen- osmotic pump, because it was only recently made affordable by opausal women by their equol-producing status showed stronger synthesis in adequate quantities for feeding studies. The reported advantages to lumbar spine BMD after a 2-y intervention in Downloaded from studies of oral and i.v. equol administration on bone in animal equol (2.4% increase) compared with nonequol producers models used high doses, which inhibited bone loss (20,21) but (0.6% increase) (8). Several subsequent studies have related caused adverse effects on reproductive tissues (21). Plasma equol equol status of subjects to change in bone turnover markers or levels ranged from 1550 nmol/L (20) to 8433 nmol/L (21). Daily BMD in response to an intervention containing soy isoflavones subcutaneous injections of 0.5 mg equol/d in mice (20) as well as (19,31). Equol-producing ability determined by fecal equol m ×

oral dosing of 10 g equol/(g body weight d) (21) in rats levels in 68 of 122 postmenopausal Japanese women was jn.nutrition.org maintained bone mineral density (BMD) of OVX animals to associated with reduced total hip BMD after 24 wk of isoflavone similar levels of SHAM rats. Consumption of 1000 mg/kg treatment (31). Both urinary equol and O-desmethylangolensin dietary equol led to increased uterine epithelial proliferation and levels indicating the presence of gut metabolizing microflora plasma equol levels of 6–8 mmol/L (22) in mice. Dietary equol at following 3 d of soy food consumption were related to BMD in

400 mg/kg diet attenuated OVX-induced trabecular bone loss at 92 postmenopausal women (18). O-desmethylangolensin, but at American Society for Nutrition on June 18, 2010 the lumbar spine but had a mild uterotropic activity, decreased not equol, producers had greater total, leg, and head BMD weight gain at these concentrations, and circulating equol compared with nonproducers (P , 0.05). concentrations of 2460 nmol/L (23). Equol supplementation also improved biomechanical and histomorphometric measures Equol metabolism in young adults of femurs in OVX rats (24) as well as aided in healing of The form of equol provided for supplementation also plays a key osteoporotic fractures in OVX rats (25). role in metabolism and could alter potential health benefits of We conducted a dose-ranging study (0, 50, 100, and 200 mg equol. Currently, there is only one pharmacokinetic study to our equol/kg diet) of dietary racemic equol (50% R-equol, 50% knowledge that examined the effect of S-equol, R-equol, and S-equol) in 6-mo-old OVX Sprague Dawley rats. Doses were racemic equol on circulating equol levels in adults (32). A single selected to achieve serum concentrations similar to those of oral dose of 20 mg of S-13C-equol, R-13C-equol, or racemic 13C- postmenopausal women consuming medium (56 mg/d, 3–39 equol was administered to healthy young men (n = 6) and mmol/L) or high (90 mg/d, 5–49 mmol/L) amounts of isoflavones women (n = 6) and plasma was collected for 48 h. Compared (26). We found that heart, intestine, kidney, and liver tissue with other phytoestrogens (genistein and daidzein), equol was equol concentrations reflected the diet, but only the highest dose more bioavailable and rapidly absorbed. Differences in bioa- studied (200 mg/kg) increased femoral calcium content (27). vailability and absorption were associated with the form of Calcium absorption and retention tended (P = 0.07) to be higher equol consumed. R-13C-equol had higher bioavailability and at equol concentrations .100 mg/kg. Mammary gland epithelial fractional absorption than S-13C-equol. However, both enanti- tissues were not stimulated, but uterine epithelial tissues were omers had higher absorption, plasma concentrations, and stimulated at this dose. These findings suggest limited benefit bioavailability than the racemic mixture. with the potential for adverse effects of equol as a dietary supplement. Future directions Although evidence suggests that equol may attenuate estrogen New method for examining effect of soy phytoestrogens deficiency-related bone loss, there are various factors that could on bone influence the health benefit potential of equol, such as dose, form We have developed a novel method using the rare isotope, 41Ca, of equol, and the ability for intestinal production of equol. Other to prelabel the skeleton and then monitor bone resorption by lifestyle traits could also affect the health protective actions of measuring urinary 41Ca excretion by accelerator MS (28). The equol. For instance, our dose-response equol study revealed that advantages of this method are its extreme sensitivity and equol had a detrimental effect on tibia calcium content that specificity compared with traditional methods. We have used could be contributed to the estrogen-like effects during mechan- this method to study the effect of dose-ranging intervention of ical loading (27). Future research should determine the bone soy isoflavones in soy protein isolates (28) as well as to compare health effect of equol supplementation in physically active several botanical supplements with traditional therapies of postmenopausal women. Overall, a deeper understanding of osteoporosis (29). Response to the botanical supplements equol and interaction with lifestyle factors is needed to reflected their genistein content. The supplement with the highest thoroughly assess the bone health benefit of equol.

1378S Supplement Acknowledgments biological activity through estrogen receptors alpha and beta. Bioorg C.M.W. and L.L.L. wrote the paper. Both authors read and Med Chem. 2004;12:1559–67. approved the final manuscript. 17. Ohtomo T, Uehara M, Penalvo JL, Adlercreutz H, Katsumata S, Suzuki K, Takeda K, Masuyama R, Ishimi Y. Comparative activities of daidzein metabolites, equol and O-desmethylangolensin, on bone mineral density and lipid metabolism in ovariectomized mice and in osteoclast cell Literature Cited cultures. Eur J Nutr. 2008;47:273–9. 18. Setchell KD, Brown NM, Lydeking-Olsen E. The clinical importance of 1. Reinwald S, Weaver CM. Soy isoflavones and bone health: a double- the metabolite equol-a clue to the effectiveness of soy and its edged sword? J Nat Prod. 2006;69:450–9. isoflavones. J Nutr. 2002;132:3577–84. 2. Kalu DN. Evolution of the pathogenesis of postmenopausal bone loss. 19. Frankenfeld CL, McTiernan A, Thomas WK, LaCroix K, McVarish L, Bone. 1995;17:S135–144. Holt VL, Schwartz SM, Lampe JW. Postmenopausal bone mineral 3. Weaver CM, Spence LA, Lipscomb ER. Phytoestrogens and bone density in relation to soy isoflavone-metabolizing phenotypes. Matur- health. In: Burckhardt P, Dawson-Hughes B, Heaney RP, editors. itas. 2006;53:315–24. Nutritional aspects of osteoporosis. Proceeding of the Symposium on 20. Fujioka M, Uehara M, Wu J, Adlercreutz H, Suzuki K, Kanazawa K, Nutritional Aspects of Osteoporosis, Lausanne, Switzerland, 2000. Takeda K, Yamada K, Ishimi Y. Equol, a metabolite of daidzein, inhibits New York: Academic Press, Inc.; 2001. p. 315–24. bone loss in ovariectomized mice. J Nutr. 2004;134:2623–7. 4. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, 21. Mathey J, Mardon J, Fokialakis N, Puel C, Kati-Coulibaly S, Mitakou Stefanick ML, Jackson RD, Beresford SA, Howard BV, et al. Risks and S, Bennetau-Pelissero C, Lamothe V, Davicco MJ, et al. Modulation of benefits of estrogen plus progestin in healthy postmenopausal women: soy isoflavones bioavailability and subsequent effects on bone health in principal results from the Women’s Health Initiative randomized ovariectomized rats: the case for equol. Osteoporos Int. 2007;18:671–9. controlled trial. JAMA. 2002;288:321–33.

22. Selvaraj V, Zakroczymski MA, Naaz A, Mukai M, Ju YH, Doerge DR, Downloaded from 5. Alekel DL, Germain AS, Peterson CT, Hanson KB, Stewart JW, Toda T. Katzenellenbogen JA, Helferich WG, Cooke PS. Estrogenicity of the Isoflavone-rich soy protein isolate attenuates bone loss in the lumbar isoflavone metabolite equol on reproductive and non-reproductive spine of perimenopausal women. Am J Clin Nutr. 2000;72:844–52. organs in mice. Biol Reprod. 2004;71:966–72. 6. Potter SM, Baum JA, Teng H, Stillman RJ, Shay NF, Erdman JW Jr. Soy 23. Rachon D, Menche A, Vortherms T, Seidlova-Wuttke D, Wuttke W. protein and isoflavones: their effects on blood lipids and bone density in Effects of dietary equol administration on the mammary gland in postmenopausal women. Am J Clin Nutr. 1998;68:S1375–9. ovariectomized Sprague-Dawley rats. Menopause. 2008;15:340–5.

7. Wangen KE, Duncan AM, Merz-Demlow BE, Xu X, Marcus R, Phipps 24. Tezval M, Sehmisch S, Seidlova´-Wuttke D, Rack T, Kolios L, Wuttke W, jn.nutrition.org WR, Kurzer MS. Effects of soy isoflavones on markers of bone turnover Stuermer KM, Stuermer EK. Changes in the histomorphometric and in premenopausal and postmenopausal women. J Clin Endocrinol biomechanical properties of the proximal femur of ovariectomized rat Metab. 2000;85:3043–8. after treatment with the phytoestrogens genistein and equol. Planta 8. Lydeking-Olsen E, Beck-Jensen JE, Setchell KD, Holm-Jensen T. Med. 2010;76:235–40. Soymilk or progesterone for prevention of bone loss–a 2 year random- 25. Kolios L, Sehmisch S, Daub F, Rack T, Tezval M, Stuermer KM, at American Society for Nutrition on June 18, 2010 ized, placebo-controlled trial. Eur J Nutr. 2004;43:246–57. Stuermer EK. Equol but not genistein improves early metaphyseal 9. Clifton-Bligh PB, Baber RJ, Fulcher GR, Nery ML, Moreton T. The fracture healing in osteoporotic rats. Planta Med. 2009;75:459–65. effect of isoflavones extracted from red clover (Rimostil) on lipid and 26. Persky VW, Turyk ME, Wang L, Freels S, Chatterton R Jr, Barnes S, bone metabolism. Menopause. 2001;8:259–65. Erdman J Jr, Sepkovic DW, Bradlow HL, et al. Effect of soy protein on 10. Anderson JJ, Chen X, Boass A, Symons M, Kohlmeier M, Renner JB, endogenous hormones in postmenopausal women. Am J Clin Nutr. Garner SC. Soy isoflavones: no effects on bone mineral content and 2002;75:145–53. bone mineral density in healthy, menstruating young adult women after 27. Legette LL, Martin BR, Shahanazari M, Lee W, Helferich WG, Qian J, one year. J Am Coll Nutr. 2002;21:388–93. Waters DJ, Arabshahi A, Barnes S, et al. Supplemental dietary racemic 11. Gallagher JC, Satpathy R, Rafferty K, Haynatzka V. The effect of soy equol has modest benefits to bone but has mild uterotropic activity in protein isolate on bone metabolism. Menopause. 2004;11:290–8. ovariectomized rats. J Nutr. 2009;139:1908–13. 12. Kreijkamp-Kaspers S, Kok L, Grobbee DE, de Haan EH, Aleman A, 28. Cheong JM, Martin BR, Jackson GS, Elmore D, McCabe GP, Nolan JR, Lampe JW, van der Schouw YT. Effect of soy protein containing Barnes S, Peacock M, Weaver CM. Soy isoflavones do not affect bone isoflavones on cognitive function, bone mineral density, and plasma resorption in postmenopausal women: a dose-response study using a lipids in postmenopausal women: a randomized controlled trial. JAMA. novel approach with 41Ca. J Clin Endocrinol Metab. 2007;92:577–82. 2004;292:65–74. 29. Weaver CM, Martin BR, Jackson GS, McCabe GP, Nolan JR, McCabe 13. Morabito N, Crisafulli A, Vergara C, Gaudio A, Lasco A, Frisina N, LD, Barnes S, Reinwald S, Boris ME, et al. Antiresorptive effects of D’Anna R, Corrado F, Pizzoleo MA, et al. Effects of genistein and phytoestrogen supplements compared to estradiol or risedronate in hormone-replacement therapy on bone loss in early postmenopausal postmenopausal women using 41Ca methodology. J Clin Endocrinol women: a randomized double-blind placebo-controlled study. J Bone Metab. 2009;94:3798–805. Miner Res. 2002;17:1904–12. 30. Niculescu MD, Pop EA, Fischer LM, Zeisel SH. Dietary isoflavones 14. Marini H, Minutoli L, Polito F, Bitto A, Altavilla D, Atteritano M, differentially induce gene expression changes in lymphocytes from Gaudio A, Mazzaferro S, Frisina A, et al. Effects of the phytoestrogen postmenopausal women who form equol as compared with those who genistein on bone metabolism in osteopenic postmenopausal women: a do not. J Nutr Biochem. 2007;18:380–90. randomized trial. Ann Intern Med. 2007;146:839–47. 31. Wu J, Oka J, Higuchi M, Tabata I, Toda T, Fujioka M, Fuku N, 15. Marini H, Minutoli L, Polito F, Bitto A, Altavilla D, Atteritano M, Teramoto T, Okuhira T, et al. Cooperative effects of isoflavones and Gaudio A, Mazzaferro S, Frisina A, et al. OPG and sRANKL serum exercise on bone and lipid metabolism in postmenopausal Japanese concentrations in osteopenic, postmenopausal women after 2-year women: a randomized placebo-controlled trial. Metabolism. 2006;55: genistein administration. J Bone Miner Res. 2008;23:715–20. 423–33. 16. Muthyala RS, Ju YH, Sheng S, Williams LD, Doerge DR, 32. Setchell KD, Zhao X, Jha P, Heubi JE, Brown NM. The pharmacoki- Katzenellenbogen BS, Helferich WG, Katzenellenbogen JA. Equol, a netic behavior of the soy isoflavone metabolite S-(-)equol and its natural estrogenic metabolite from soy isoflavones: convenient prepa- diastereoisomer R-(+)equol in healthy adults determined by using ration and resolution of R- and S-equols and their differing binding and stable-isotope-labeled tracers. Am J Clin Nutr. 2009;90:1029–37.

Dietary equol status and bone loss 1379S The Journal of Nutrition Supplement: Equol, Soy, and Menopause

Menopausal Hot Flashes: A Review of Physiology and Biosociocultural Perspective on Methods of Assessment1,2

Fredi Kronenberg*

Clayman Institute for Gender Research, Stanford University, Stanford, CA 94305

Abstract

Hot flashes continue to be a troublesome problem for menopausal women the world over. After .50 y of research, we still Downloaded from do not understand the etiology and mechanism of hot flashes, nor do we know how estrogen, the major pharmaceutical treatment, works to reduce hot flashes. We are gaining insight into sociocultural complexities that may affect how and whether women report hot flashes. And we are becoming more sophisticated in our research tools (be it questionnaires, physiological monitors, or brain imaging techniques). New aspects of hot flash research, including neuroimaging and the study of genetic polymorphisms, when combined with increasingly nuanced ways of asking questions of culturally distinct jn.nutrition.org populations, provide challenges but rich complexity from which a better understanding will emerge. J. Nutr. 140: 1380S– 1385S, 2010. at American Society for Nutrition on June 18, 2010

Introduction flashes they thought they had left far behind. Yet, while women Hot flashes are one of the most common symptoms experienced have complained of these “spells” of heat sensation from time by women around the world during the transition to and immemorial and despite this considerable public health prob- through menopause. Whereas prevalence rates tend to be higher lem, at the start of the 21st century, we still do not understand in Western countries than, e.g., in Asian countries, rates vary the etiology or physiology, nor do we know how the most widely widely and are likely influenced by a range of factors (1). In the used pharmaceutical treatment to date, estrogen, works to U.S., hot flashes are one of the main reasons women at relieve hot flashes. menopause seek medical help or look for dietary supplements This artice aims to provide a brief overview of some aspects and over-the-counter remedies for relief (2,3). In 2002, after of the biology of hot flashes and methods used in research, given publication of the results from the large NIH-funded Women’s insights we now have into biosociocultural factors that influence Health Initiative study, which indicated that estrogen was how we observe and the phenomenon being observed. Emerging harmful on a number of indices, many women stopped taking areas of research are also discussed. this hormone therapy (4) and for .30%, bothersome, often severe hot flashes returned (5). These women re-experienced hot Hot flashes: the phenomenon The terms hot flash and hot flush are used interchangeably and are typically synonymous, referring to a sudden sensation of heat 1 The Journal of Nutrition Published in a supplement to . Presented at the “Equol, and sweating, most notably on the upper body. Hot flashes occur Soy, and Menopause Research Leadership Conference” held in Washington, DC, June 16, 2009. The supplement coordinator for this supplement is Kara primarily and most intensively in peri- and postmenopausal Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The women. They also may occur when estrogen drops suddenly and supplement is the responsibility of the guest editors to whom the Editor of The rapidly, such as after removal of the ovaries of premenopausal Journal of Nutrition has delegated supervision of both technical conformity to women, with chemically induced menopause, and also in breast the published regulations of The Journal of Nutrition and general oversight of the scientific merit of each article. Publication costs for this supplement were cancer patients treated with selective estrogen receptor modifiers defrayed in part by the payment of page charges. This publication must therefore such as tamoxifen. Men can experience hot flashes, particularly be hereby marked "advertisement" in accordance with 18 USC section 1734 when testosterone levels fall rapidly, such as in men with solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. prostate cancer treated medically or surgically. In both women Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement and men, these are situations where there is an abrupt drop in Coordinator disclosure: Kara Lewis is currently under contract with and receives compensation from the supplement sponsor. She was also compensated for sex steroid hormones, resulting in hot flashes. attending and organizing the Equol, Soy, and Menopause Research Leadership Hot flashes can occur day or night; when they occur at night, Conference and for organizing, writing, editing, or reviewing, and collection of they are called night sweats. Each particular episode lasts supplemental manuscripts. The opinions expressed in this publication are those between 3 and 10 min and can recur with varying frequency (6). of the authors and are not attributable to the sponsors or the publisher, Editor, or Editorial Board of The Journal of Nutrition. Some women experience hot flashes hourly or daily, while for 2 Author disclosure: F. Kronenberg, no conflicts of interest. others they may occur only occasionally; a small percent of * To whom correspondence should be addressed. E-mail: [email protected]. women report not having any hot flashes. Whether this is a

1380S ã 2010 American Society for Nutrition. First published online May 26, 2010; doi:10.3945/jn.109.120840. matter of a threshold of perception or there are genetic, occurs and blood flow to the skin increases, evident in an environmental, or lifestyle factors that preclude hot flashes is a increase in finger skin temperature. With the sudden and rapid subject of increasing interest. increase in heat loss (sweating and cutaneous vasodilation), Although for most women, hot flashes begin and have their internal body temperature drops. The forehead temperature also peak occurrence during the peri- and early postmenopausal cools given the sweating and subsequent evaporative cooling periods, typically when a woman is in her late 40s and early 50s, that occurs. These physiological phenomena can be reliably at times they may begin when menstrual cycles are still regular measured in the laboratory (Fig. 2) (7,8). These are labor- (6). The majority of women have hot flashes for a year or two, intensive studies and are not appropriate for clinical trials of but ~15% may have them nonstop for 10, 20, or 30 y (6). And various hot flash treatments that involve ambulatory, free living while every woman’s estrogen level decreases during the individuals. For this purpose, portable monitors that record transition to and through menopause, it remains an enigma as changes in sweating, as measured by changes in the electrical to why some women’s bodies seem to adjust and hot flashes conductivity of the skin (skin conductance), are used to correlate taper off, while others continue having hot flashes for many subjective reports of hot flash occurrence with this objective years. In addition, for some women whose hot flashes were physiological change. treated successfully by estrogen, the hot flashes resume after cessation of estrogen treatment. Mechanisms of hot flashes Estrogen has been studied and used to treat hot flashes for .60 y, Physiological changes during a hot flash but the mechanism by which it works is still in question. This is Thermography provides a visual snapshot of the skin surface due to relatively little examination of basic hot flash physiology, Downloaded from manifestation of the pattern of thermoregulatory changes during which has not received the research attention that has been a hot flash and provides insights into underlying physiological accorded clinical trials of therapeutic agents. Thus, the body of changes (Supplemental Fig. 1). One can see the skin areas that knowledge on the endocrinology, neurophysiology, thermoreg- warm during the hot flash (fingers, neck, face) and cool as the ulatory physiology, and other aspects of hot flash biology is hot flash subsides. limited. Figure 1 graphically illustrates some of the primary physio- One long-standing assumption has been that hot flashes jn.nutrition.org logical changes that occur during a hot flash (7). “Sensation” is a involve transient dysregulation of the thermoregulatory system, subjective rating of the sense of hot flash intensity on a scale of triggering homeostatic heat loss mechanisms to return the system 0–10 (10 being the most intense). At the onset of a hot flash, to normal. During a hot flash, many of the easily observed there is a sudden increase in sweating. Heart rate increases physiological changes involve the thermoregulatory and vascular anywhere from 5 to 25 beats/min. Cutaneous vasodilation systems. This remains an area of incomplete research. at American Society for Nutrition on June 18, 2010 Clearly, estrogen plays some role as a mediator of hot flashes. Hot flashes occur as estrogen levels decline and they are alleviated for the most part by treatment with estrogen (9). Estrogen priming is likely important. Young women with low estrogen levels do not have hot flashes, but if given estrogen and then withdrawn from it, they will have hot flashes. And premeno- pausal women whose ovaries are removed most often experience hot flashes almost immediately. However, low estrogen levels alone do not explain the presence of hot flashes. Investigators have searched for correlations between estrogen levels and the occurrence of hot flashes in women with and without hot flashes as well as with individual hot flashes with conflicting results. Some population studies have demonstrated inverse relationships between estrogen levels and hot flashes (10,11), as have more recent studies (12). Others, including Freedman et al. (13), have not corroborated these results when small groups of symptomatic and asymptomatic women were compared. There has been examination of the levels of cir- culating hormones, including sex steroids and gonadotropins, among others. Substances such as luteinizing hormone (14,15), B-endorphin, adrenocorticotropic hormone (16), and others show pulsatile activity that is temporally correlated with hot flash occurrence, but not causally related (7). Freedman et al. (17) have proposed, based on several studies, that elevated brain norepinephrine plays a role in the etiology of hot flashes. Examining the complex interactions of the multiple systems that are involved has not been substantively undertaken. Thus, at this time, there is no definitive determination of what it is that triggers individual hot flashes or explains why some women do FIGURE 1 Physiological changes during a hot flash. Characteristic physiological changes during a hot flash: sensation (SENS), heart rate, or do not experience them. internal body temperature (Teso ¼ esophageal), skin temperatures (Tfor ¼ forehead, Tfin ¼ finger), and ambient temperature (Ta ¼ Sociocultural issues in measuring hot flashes ambient). Reproduced from Kronenberg and Downey (7) ã 2008 NRC Hot flashes occur worldwide, and starting in the 1970s, research Canada or its licensors. Reproduced with permission. documenting such occurrence increased substantially. A wide

Hot flashes: physiology and sociocultural perspective 1381S FIGURE 2 Multiple hot flashes: physiological changes. Sensation (SENS), heart rate, finger blood flow, forearm sweating rate, internal body temperature (Teso ¼ esophageal), skin temperatures

(Tfor ¼ forehead, Tfin ¼ finger) and ambient temperature (Ta ¼

ambient). Adapted from Downloaded from Kronenberg and Downey (7). ã 2008 NRC Canada or its licensors. Reproduced with permission. jn.nutrition.org at American Society for Nutrition on June 18, 2010

distribution of the prevalence of hot flashes around the globe Further complexity may relate to a critical period for soy continues to be examined, with reports, particularly in Asian consumption relative to its beneficial health effects. This has countries, of prevalence less than that in the US and other come to light while trying to understand the observation of Western countries (1,18). Interest in understanding these differ- lower breast cancer rates in populations with high soy food ences has raised questions about whether these differences are consumption. Exposure during adulthood did not support these due to genetic, cultural, environmental, or lifestyle factors such observations. Data are accumulating to support a protective as diet and exercise. effect of soy when it is consumed during early life (childhood/ Research in Japan has provided particular insight. Japanese adolescence) with regard to breast cancer later in life (24). Soy women have a high dietary intake of soy (relative to Western foods are a staple in Asian cuisine and are consumed throughout countries) and it was hypothesized that this might explain why life, suggesting their role in breast cancer prevention. Consump- they have fewer hot flashes than women in the US, Canada, and tion of soy or genistein affects gene expression and activates Europe. Basic science research has established that isoflavones estrogen receptor a, thereby affecting BRCA1 (25,26). Similarly, (compounds in soy and other plants) have estrogen-like activity it is possible that consuming soy during puberty may affect (19). Interest in the relationship between the soy consumption whether one experiences hot flashes during menopause; initiat- of different populations and hot flash prevalence led to ing soy consumption or isoflavone supplementation at age 50 y epidemiological studies comparing level of dietary soy intake may not have the same outcome of reducing hot flashes. and frequency of hot flashes in countries such as Japan, where an inverse association between soy intake and hot flashes has The Japanese experience been demonstrated (20,21). Clinical studies of soy foods and In the 1980s, Lock et al. (27) studied menopause among soy isoflavones to treat hot flashes proliferated, with mixed Japanese women and asked women about symptoms, including results, although there was a tendency toward a beneficial those typically experienced by American and Canadian women; effect (22). they also asked the Japanese women (and their physicians) how However, it is now known that there are confounding factors they would describe this time of life and the symptoms they that make this a more complex issue and might contribute to experienced. The respondents used a combination of 3 terms explaining the differing results among the clinical trials. The (sudden feeling of heat; feeling hot or flushed; rush of blood to isoflavone daidzein is biotransformed by intestinal microflora to the head) to encompass what Western women and physicians the active metabolite, equol. The presence of the particular mean by the single term hot flash. The Japanese women reported bacterial species responsible for this conversion differs among fewer hot flashes than their Western counterparts. The re- people in different countries and thus some people are “equol searchers concluded that fewer Japanese women were experi- producers” and some are not (19,23). encing hot flashes and night sweats.

1382S Supplement TABLE 1 Symptoms recalled in previous 2-wk period in Japanese women: comparison across 20 y1

Romanized Japanese symptom English symptom Lock, n = 1141 Melby, n = 140 Incidence

% Katakori Shoulder stiffness 51.7 62.1 20 Zutsuu Headache 27.7 39.3 42 Youtsuu Lumbago 22.4 35.0 56 Benpi Constipation 21.1 25.0 18 Hieshou Chilliness 16.3 29.3 80 Iraira Irritability 11.9 28.6 140 Fumin Insomnia 11.4 11.4 0 Kansetsutsuu Aches and pains in the joints 10.9 15.0 38 Kaze o yoku hiku Frequent colds 10.4 15.0 44 Kyuu na nekkan nobose, hoteri Any hot flash 9.5 22.1 133 Ki ga meiru Depression 7.9 22.9 190 Memai Dizziness 7.2 10.7 49 Kyuu na hakkan Sudden perspiration 4.2 8.6 105 Ne ase Night sweats 3.2 6.4 100 Downloaded from 1 Adapted from Melby 2005 (28) and Lock et al. (27). Reprinted by permission of Elsevier.

Twenty years later, Melby (28) conducted a follow-up study Interestingly, Melby (31) administered to both men and

in Japan, this time focusing on the more linguistically nuanced women in Japan the same symptom questionnaire. Both groups jn.nutrition.org terminologies as she came to understand how Japanese women reported shoulder stiffness (the most prevalent symptom spoke about their sensations of heat and sweating during reported by women) and men reported more night sweats than the menopausal years. Melby included Lock’s symptom list in did the women (Table 2) (31). How do we determine which her questionnaire but asked about hot flash terms separately, symptoms are influenced by cultural factors and expectations and found there was an increase in symptom reporting in the and which are a function of hormonal or other physiological at American Society for Nutrition on June 18, 2010 time since Lock’s study. There was an increase in those reporting change? each of the symptoms comprising the nuanced components hot flashes (Table 1) (28). Some of the explanation for increased Do women of Japanese ancestry really have fewer symptom reporting may involve the globalization of the Western hot flashes than western women or do they just not approach to and discussion of menopause. Zeserson (29) report them? commented on the media’s impact on how women discuss and Theories have been offered to explain the differences in hot flash view their symptoms. In the case of hot flashes, Zeserson (29) prevalence around the world, including genetics, physiology, noted that Japanese women began using the Americanized expectation, lifestyle (soy foods, exercise), or other environmen- expression ‘hotto furasshu’ and became conversant with tal factors. Brown et al. (32) looked elsewhere to examine the Western terminology of “hot flash,” having heard this in whether part of the explanation for fewer hot flashes among the media. As described earlier, there are a number of measur- Japanese-American women might involve a lack of reporting able physiological changes that constitute a hot flash. rather than a lack of occurrence of hot flashes. Their study, Some women’s heart rate increases, some women sweat, conducted in Hawaii, compared objectively measured hot while others flush and feel a sensation of heat. In Western countries, women describe whether they flush or sweat during a hot flash; they name this constellation of changes as a “hot TABLE 2 Two-week symptom prevalence in Japanese women flash” or “hot flush.” Japanese women describe an individual and men1 component of hot flash sensation if they experience it. In Traditional Chinese medicine (and its Kampo counterpart in Romanized Japanese English Females, Males, 22 Japan), a practitioner would identify the particular character- symptom symptom n =32 n =22 x istics of an individual woman presenting with the Western % diagnosis of hot flashes, whether she sweats (sweat effusion or Katakori Shoulder stiffness 43.8 40.9 fa han has many different presentations), flushes (mian jia chao Sutoresu Stress 12.5 36.4 4.2963 hong, e.g., characterizes a visible malar flush while fa re Kyuu na hakkan Sudden perspiration 12.5 0.0 2.9704 represents a generalized sensation of heat), or has rapid heart Iraira Irritability 6.3 27.3 4.5663 rate (jing ji or palpitations) [(30); K.V. Ergil, personal Ne ase Night sweats 6.3 13.6 communication], and the diagnostic process would lead to Shinkei no shinchou Nervous tension 0.0 13.6 4.6203 treatment related to that pattern of symptom presentation. Kentaikan Exhaustion 0.0 9.1 3.0214 Thus, not all women with a Western diagnosis of menopausal hot flashes would receive the same suggested treatment. 1 Adapted from Melby 2006 (31). Reprinted by permission of Taylor and Francis Group, Western investigators, too, can apply these distinctions and http://www.informaworld.com. 2 The nonparametric chi-square test was used to assess sex difference in symptoms stratify clinical trials to explore whether some treatments are experienced in the previous 2 wk and reported by .9% of the sample. more appropriate for certain subsets of women with specific 3 Significant at the 0.05 level (2-tailed). patterns of symptoms. 4 Significant at the 0.10 level (2-tailed).

Hot flashes: physiology and sociocultural perspective 1383S flashes (using the measure of skin conductance) with self-report in women of Japanese descent and European descent living in Hilo, Hawaii. They administered a postal questionnaire asking about symptoms experienced in the past 2 wk and followed that with 24-h objective monitoring with diary reporting in a subset of women. The postal survey indicated that Japanese women had fewer symptoms such as backache, hot flashes, night sweats, depression, and trouble sleeping (Fig. 3). However, the ambu- latory monitor revealed no difference between the groups in the percentage of women exhibiting objective hot flashes (Fig. 4). There was a distinct difference between what was reported in the postal questionnaires and the ambulatory monitoring. Japanese- American women were significantly less likely to report hot flashes in the previous 2 wk, yet, they had the same frequency of objectively recorded hot flashes as the European-Americans. The authors concluded that fewer reported hot flashes in women of FIGURE 4 Incidence and mean number of hot flashes among Japanese ancestry might be a consequence of reporting bias Japanese- and European-American women wearing a hot flash likely due to cultural perceptions of what is acceptable to discuss monitor. Adapted from Brown et al. (32) with permission from Wolters Kluwer. and report. The study of Brown et al. (33) highlights the value of Downloaded from objectively measuring hot flashes. There can be cultural differ- ences in how and whether one reports hot flashes; diary use often Emerging areas of interest underreports hot flashes such as those that occur during sleep or Brain imaging techniques such as functional MRI are being used during a busy period when a hot flash is not noticed or goes to examine brain function during hot flashes. Initial studies of unreported. brain activation during hot flashes have found that the insula

Clinical research has been hampered by the lack of a hot flash and anterior cingulated cortex are activated during hot flashes jn.nutrition.org monitor that is accurate and easy-to-use in ambulatory subjects. (36). Better understanding of the neural control of hot flashes Most of the portable monitors available for measuring skin will provide further insight into mechanisms. conductance (or other physiological signals) are cumbersome to Another area of growing interest is the relationship between wear and have wires that do not facilitate ease of use. A new, hot flashes and polymorphisms of genes involved in estrogen miniature, wireless monitor, developed by Bahr Management in function, such as sex steroid metabolizing enzymes and estrogen at American Society for Nutrition on June 18, 2010 collaboration with researchers at the University of California, receptors. Given that estrogen plays some role in the hot flash San Francisco, promises increased accuracy and ease of use phenomenon, investigators are examining variation in genes (34,35). coding for enzymes involved in estrogen synthesis and hormone interconversion for a possible role in the variance in observed circulating hormone levels (37). Genetic polymorphisms are also being studied in an attempt to explain observations of race/ ethnic differences in hot flash prevalences (38), such as seen in the Study of Women Across the Nation in the US. Two studies indicate that there are certain race/ethnicity associations be- tween polymorphisms for sex metabolizing hormones (39,40). This line of research is in its infancy but may provide new insights into the often conflicting and variable results of studies examining factors that might predict who most is at risk for hot flashes. The study of equol for the treatment of hot flashes holds promise and clinical and basic science studies are underway in the US, Japan, and elsewhere. To better study soy, equol, or any herbal or pharmaceutical agent for the treatment of hot flashes, researchers need better measurement techniques, including better questionnaires that ask questions in a culturally sensitive context. More research on the basic underlying physiology and brain function during hot flashes is needed to better understand mechanisms that can lead to the design of treatments targeting these mechanisms.

Summary Hot flashes remain a particularly bothersome problem for a majority of menopausal women in the US and elsewhere and a primary reason for which women in the menopausal years seek treatment. Yet, after .40 y of studying hot flash physiology, we FIGURE 3 Symptoms reported for previous 2 wk by Japanese- and still do not understand the etiology, nor do we understand how European-American Women in Hawaii surveyed by postal question- the most effective treatment to date, estrogen, works to reduce hot naire. Adapted from Brown et al. (32) with permission from Wolters flashes. There has been substantial examination of and insight into Kluwer. cross-cultural differences, the nuances of linguistic expressions,

1384S Supplement and dietary and other factors that are providing means of Lomax P, Schonbaum E, editors. Thermoregulation: the pathophysio- stratifying results to better understand the variability in responses. logical basis of clinical disorders. Basel: Karger; 1992. p. 6–9. Newer research is focusing on brain function and genetic 18. Obermeyer CM. Menopause across cultures: a review of the evidence. Menopause. 2000;7:184–92. polymorphisms, both of which add new levels of complexity to 19. Mazur W, Adlercreutz H. Overview of naturally occurring endocrine- what is obviously a very rich and compelling area for investiga- active substances in the human diet in relation to human health. tion. After so many years with insufficient research attention, the Nutrition. 2000;16:654–8. time and measurement tools are now ready for a more sophis- 20. Nagata C, Shimizu H, Takami R, Hayashi M, Takeda N, Yasuda K. Hot ticated examination of hot flashes. Any additional understanding flushes and other menopausal symptoms in relation to soy product of mechanism would provide valuable guidance for those wishing intake in Japanese women. Climacteric. 1999;2:6–12. to study and develop new treatments for this age-old problem. 21. Nagata C, Takatsuka N, Kawakami N, Shimizu H. Soy product intake and hot flashes in Japanese women: results from a community-based prospective study. Am J Epidemiol. 2001;153:790–3. Acknowledgments 22. Jacobs A, Wegewitz U, Sommerfeld C, Grossklaus R, Lampen A. Thanks to Kevin V. Ergil, Associate Professor, Finger Lakes Efficacy of isoflavones in relieving vasomotor menopausal symptoms: a School of Acupuncture and Oriental Medicine at New York systematic review. Mol Nutr Food Res. 2009;53:1084–97. Chiropractic College, for personal communication about tradi- 23. Setchell KDR, Brown NM, Lydeking-Olsen E. The clinical importance tional Chinese medicine. The sole author had responsibility for of the metabolite equol: a clue to the effectiveness of soy and its all parts of the manuscript. isoflavones. J Nutr. 2002;132:3577–84. 24. Korde LA, Wu AH, Fears T, Nomura AM, West DW, Kolonel LN, Pike MC, Hoover RN, Ziegler RG. Childhood soy intake and breast cancer

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Hot flashes: physiology and sociocultural perspective 1385S The Journal of Nutrition Supplement: Equol, Soy, and Menopause

Equol Improves Menopausal Symptoms in Japanese Women1,2

Takeshi Aso*

Comprehensive Reproductive Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan

Abstract

It has been well documented that the frequency of vasomotor menopausal symptoms, such as hot flashes and night sweats, of Japanese menopausal women is less than that of Western women. High intake of soy isoflavones in the traditional Japanese diet has been postulated as the possible explanation of the difference. Epidemiological studies have Downloaded from reported that the content of equol, which is a biologically active metabolite of the isoflavone, daidzein, is lower in the women who complain of severe vasomotor symptoms. To investigate the involvement of equol in the manifestation of menopausal symptoms, especially vasomotor symptoms, and the possible therapeutic role of a supplement containing equol (natural S-equol developed by Otsuka Pharmaceutical) on the menopausal symptoms of Japanese women, 3 randomized clinical trials were conducted. The studies indicated that a daily dose of 10 mg of natural S-equol improved jn.nutrition.org menopausal symptoms. In the confirmation study, menopausal women who were equol nonproducers who consumed 10 mg/d of natural S-equol for 12 wk had significantly reduced severity and frequency of hot flashes as well as a significant reduction in the severity of neck or shoulder stiffness. The equol-ingesting group also showed trends of improvement in sweating and irritability and a significant improvement in the somatic category symptoms. Thus, it is concluded that the at American Society for Nutrition on June 18, 2010 supplement containing natural S-equol, a novel soybean-derived functional component, has a promising role as an alternative remedy in the management of menopausal symptoms. J. Nutr. 140: 1386S–1389S, 2010.

Introduction motor symptoms such as hot flashes and night sweating are the Menopause health care is one of the hot issues in aging societies. typical and common complains of women in Western societies; The majority of health problems seen during menopause are a on the contrary, Japanese women in general experience milder consequence of the health conditions in previous stages of life. In vasomotor symptoms during menopause than women in West- addition, the state of health during menopause shows ethnic and ern countries. They mainly complain of shoulder stiffness and individual variations and fluctuates during the transition from general fatigue, which are classified as somatic symptoms (1–3). pre- to postmenopause in the same individual. In particular, it As one of the factors relating to such differences, dietary habits has been widely recognized that the type, severity, and pattern of of consuming soy-rich products, including phytoestrogen and soy menopausal symptoms exhibit typical ethnic variation. Vaso- isoflavones, in the traditional Japanese diet have been postulated (4,5). In Japan it has been reported that isoflavone intake is ~31.7 mg/d (4). In the practice of menopausal health care, dietary 1 Published in a supplement to The Journal of Nutrition. Presented at the “Equol, Soy, and Menopause Research Leadership Conference” held in Washington, soy isoflavones, which potentially have properties of phytoestro- DC, June 16, 2009. The supplement coordinator for this supplement is Kara gens, have been empirically proposed as an alternative in medical Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The therapy, but their efficacies have not been fully elucidated. supplement is the responsibility of the guest editors to whom the Editor of The The principles of health care are the prevention of disorders Journal of Nutrition has delegated supervision of both technical conformity to and diseases and the promotion of health conditions to provide a the published regulations of The Journal of Nutrition and general oversight of the scientific merit of each article. Publication costs for this supplement were better quality of life on an individual basis. To conduct menopausal defrayed in part by the payment of page charges. This publication must therefore medical care appropriately, it is necessary to provide evidence- be hereby marked "advertisement" in accordance with 18 USC section 1734 based alternative medicines as much as possible. Thus, the current solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. situation of menopause medical care strongly indicates that the Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement Coordinator disclosure: Kara Lewis is currently under contract with and receives exclusive investigation into the possibility of soy products as an compensation from the supplement sponsor. She was also compensated for alternative should be conducted. Based on results from a basic attending and organizing the Equol, Soy, and Menopause Research Leadership investigation, clinical trials are needed to prove the efficacy of soy Conference and for organizing, writing, editing, or reviewing, and collection of isoflavones. supplemental manuscripts. The opinions expressed in this publication are those of the authors and are not attributable to the sponsors or the publisher, Editor, or Equol and menopausal symptoms in Japanese women Editorial Board of The Journal of Nutrition. 2 Author disclosure: T. Aso, no conflicts of interest. Soy isoflavone contains 3 compounds: daidzein, genistein, and * To whom correspondence should be addressed. E-mail: [email protected]. glycitein. An active daidzein metabolite, equol, is one of the

1386S ã 2010 American Society for Nutrition. First published online May 19, 2010; doi:10.3945/jn.109.118307. principal isoflavones found in soybeans and most soy foods (6). Development of natural S-equol supplement It was reported that approximately one-half of the Japanese To access the efficacy of equol as an alternative treatment for population and 70–80% of the U.S. population cannot produce menopausal symptoms, a natural S-equol supplement was equol after ingesting soy foods or consuming isoflavones directly developed. Equol exists as an enantiomer, S-isomer and R- (7–9). One-half of middle-aged Japanese women (n = 95) isomer, but only the S-isomer is biologically produced in vivo. A excreted detectable equol levels in 24-h urine, but ~70–80% of lactic acid bacterium, Lactococcus garvieae (Lactococcus 20–92 the younger generation of women (n = 68) and men (n = 75) strain), with equol-producing capabilities was identified and showed undetectable levels [10 mg/L (41.3 nmol/L) of equol was isolated from human feces (15,16). Recently, a standardized the detectable limit by HPLC in the current study] (unpublished product of natural S-equol supplement was developed by Otsuka data). The inability of some humans to produce equol has been Pharmaceutical (17). The supplement was formed from fermen- attributed to individual differences in gut microflora (10). A tation of a soy germ solution by Lactococcus 20–92. The study on urinary isoflavone excretion of Japanese women (n = concentration of S-equol in the supplement ranged from 0.5 to 106, aged 29–78 y) reported that 24-h urinary equol excretion 0.8% of natural S-equol and the supplement does not contain ranged from 0.4 to 123.6 mmol (11). the R-equol isomer. The micronutrient content of the supple- Uchiyama et al. (12) investigated the relationship between ment was not significantly different from the original material. urinary excretion of isoflavones and menopausal symptoms in Fermentation with Lactococcus 20–92 revealed that except for peri- and postmenopausal Japanese women. Surveys were isoflavones and some amino acids, the levels of protein, fat, mailed to 116 dietitians (aged 40–60 y) living in Fukuoka carbohydrate, and other components were not changed by prefecture located in Kyushu Island in southwest Japan, and 108 fermentation. The levels of protein, fat, and carbohydrate in the Downloaded from participated in the study (93.1% response rate). The 24-h urine supplement were 38.3, 14.5, and 26.8%, respectively (17). The samples were collected for the measurement of isoflavonoids by safety of the supplement was confirmed in genotoxicity, acute HPLC using methods reported elsewhere (13) and the simplified and subchronic toxicity, and reproductive and development menopausal index (SMI) (14) was used to assess 10 menopausal toxicity tests (17,18). symptoms. Urinary excreted amounts of total isoflavonoids, 6 Clinical trials on the effects of natural S-equol on genistein, daidzein, and equol of all participants were 38.9 jn.nutrition.org 29.2, 19.6 6 15.1, 10.0 6 8.9, and 9.3 6 14.1 mmol/24 h menopausal symptoms of Japanese women (mean 6 SD), respectively. Genistein and daidzein were detected Three randomized clinical studies (pilot study, dose-finding in all samples, but equol was only detected in the urine of 51.6% study, and confirmation study) were conducted to investigate the of participants. ability of the natural S-equol supplement to relieve menopausal

From the total participants, 46 postmenopausal women symptoms in Japanese women. In this section, the results of 3 at American Society for Nutrition on June 18, 2010 (whose last menstrual period had occurred .6 mo prior) were clinical trials are discussed. The protocols were approved by the selected for the analysis of menopausal symptoms using SMI Institutional Review Board of each site and the studies were (14). The participants were divided into 2 groups according to carried out according to the guidelines of the Declaration of their SMI score: a high score group (SMI score .15, daily life Helsinki. All women provided written informed consent to was disturbed, n = 23) and a low score group (SMI score ,15, participate in the study. The first study has been previously daily activities were not disturbed, n = 23). Urinary excretion of published (13) and the other studies will be submitted for genistein and daidzein for women in the high and low score publication separately. groups did not differ; however, the amount of equol excreted in the urine was lower in the high score group than in the low score Pilot study of natural S-equol supplement group (P , 0.05) (Fig. 1). These results indicate that women Ishiwata et al. (13) conducted a randomized, double-blind, who can metabolize daidzein into equol by their own intestinal placebo-controlled trial to investigate the effects of the natural S- bacteria report milder menopausal symptoms. equol supplement on menopausal symptoms and mood states in pre-, peri-, and postmenopausal Japanese women. Each package of supplement contained 10, 0.8, and 2.0 mg of equol, daidzein, and genistein, respectively. A total of 134 healthy Japanese female participants (aged 40–59 y) were divided into 3 groups: EQ-1 (n = 44, 10 mg of equol/d), EQ-3 (n = 46, 30 mg of equol/ d), and placebo (n = 44). The women were allowed to consume a maximum of 20 mg soy isoflavones from their diet each day. Menopausal symptom score and mood score were self-reported by filling out a menopausal symptom scale and the Profile of Mood States questionnaires. The 24-h urine samples were collected for measurement of isoflavones. Physical examinations and biochemical analyses were conducted at baseline and at the end of the 12-wk period. It was described in the report (13) that 127 participants (94.8%) completed the trials. Participants who excreted ,10 mg/L (41.28 nmol/L) of equol (HPLC detection limit) in 24-h urine FIGURE 1 Relation between urinary equol excretion and menopau- 6 n samples were defined as equol nonproducers. At baseline, equol sal symptoms in Japanese women. Data are means SEM, = 23. , Low: SMI score ,15; daily activities were not apparently disturbed. producers (n = 46) had fewer total menopausal symptoms (P High: SMI score .15; daily life was disturbed. Statistical analysis was 0.05) than nonproducers (n = 81). Somatic symptom scores were carried out using Wilcoxon’s Rank Sum test. *Different from low reduced in the EQ-3 group compared with the placebo group of group, P , 0.05. Adapted from Uchiyama et al. (12) with permission postmenopausal equol nonproducers (P , 0.05) (Fig. 2). Post- from The Japan Menopause Society. menopausal nonproducers also reported beneficial effects on

Natural S-equol and menopause 1387S Confirmation study of natural S-equol supplement To confirm the physiological effects of the natural S-equol supplement in menopausal Japanese women who were equol nonproducers, an intervention trial (randomized double-blind, placebo-controlled, parallel-group) was conducted. This study consisted of a screening period, ingestion period for 12 wk, and follow-up period for 6 wk. One hundred and sixty postmeno- pausal women with normal BMI who reported having hot flashes at least once per day were enrolled in the study. Before enrollment to the trial, all participants were asked to take a placebo for 4 wk (screening period). Participants who showed placebo effects of 50% reduction of SMI after the period were excluded from the final enrollment. During the ingestion period, participants were divided into placebo (n = 60) and equol (n = 66, 10 mg equol/d) groups. The supplement containing equol (5 mg), daidzein (1.2 mg), and genistein (1.4 mg) was orally ingested twice per day (morning and evening) for 12 wk. The baseline frequencies of hot flashes in the placebo and equol groups were 2.9 6 2.1 and 3.2 6 2.4/d (mean 6 SEM), Downloaded from respectively. The difference between the 2 groups was not significant. Women who ingested 10 mg/d of natural S-equol for 12 wk had reductions in the frequency and severity of hot flashes compared with those in the placebo group (P = 0.0092 and P = 0.0154, respectively). A greater reduction of the severity of neck

or shoulder stiffness was observed for the equol group than that jn.nutrition.org of placebo group, when evaluated using both the questionnaire FIGURE 2 Effects of natural S-equol on the menopausal symptom (P = 0.0149) and the visual analogue scale method (P = 0.0070). total score (A) and somatic score (Greene climacteric scale) (B)in After the 6-wk follow-up, the ingested supplement was washed , Japanese women. Values are means 6 SD, n = 18 or 19. * **Different out and rebound responses were observed in hot flashes and neck P , P , from wk 0, 0.05 and 0.01 (Wilcoxon’s Rank Sum test). or shoulder stiffness. No serious adverse events were reported. at American Society for Nutrition on June 18, 2010 #Different from placebo in wk 12 changes, P , 0.05 (Mann-Whitney The results of vital sign and clinical laboratory tests indicated no test). Adapted from Ishiwata et al. (13) with permission from Wolters . Kluwer/Lippincott Williams & Wilkins. changes (P 0.10) after ingestion of 10 mg equol/d for 12 wk. Discussion and conclusion depression and fatigue following the ingestion of 30 mg of Because the mental and physical conditions of women in equol/d. Neither serious adverse events nor abnormal laboratory menopause are complicated, health care in this life stage has to tests were observed. The authors concluded that natural S-equol be done holistically. By the systemic evaluation of these supplementation reduced menopausal symptoms in Japanese conditions, the personal management for each woman should women who were postmenopausal equol nonproducers. be selected. Lifestyle improvements, including diet habits, exercise, and relaxation, should be conducted before medical Dose-finding study of natural S-equol supplement treatment. This study aimed to determine the optimal dose of equol for Among the health problems in menopause, menopausal clinical use. A double-blind, placebo-controlled, parallel-group, symptoms, especially vasomotor symptoms like hot flashes and interventional trial was conducted to investigate the physiolog- sweating, disturb daily activities and quality of life seriously in ical effects of natural S-equol supplementation on Japanese the great portion of women in Western societies and also for a menopausal women who were equol nonproducers. The healthy certain number of Japanese women. In the majority of such Japanese menopausal women of 105 were divided into 4 groups: women, the symptoms cannot be controlled by nonmedical EQ-2 group (n = 26, 2 mg equol/d), EQ-6 group (n = 25, 6 mg management and need to be treated by some medical equol/d), EQ-10 group (n = 27, 10 mg equol/d), and placebo interventions. group (n = 27). Each supplement tablet contained 1.0 mg equol, Although the mechanism underlying vasomotor symptom 0.1 mg daidzein, and 0.1 mg genistein and 2, 6, and 10 tablets/d has not been fully clarified, the results of various basic and were ingested orally in the EQ-2, EQ-6, and EQ-10 groups for clinical studies have indicated that dramatic changes in the 12 wk, respectively. hormonal environment, especially the sharp decline of estrogen, A total of 99 participants (94% response rate) completed the during menopause play key roles. Hormone replacement ther- study. Daily consumption of 10 mg of S-equol improved apy (HRT) has been widely adapted as a potent evidence-based menopausal symptoms of Japanese postmenopausal equol medicine for menopausal symptoms. The report of a large-scaled non-producing women, especially shoulder stiffness at 6 wk, study in the United States, the Women’s Health Initiative compared with the placebo group (P = 0.034). The measurement published in 2002 (19), indicated increasing risks associated of uterine endometrial thickness by transvaginal ultrasonogra- with a kind of HRT regimen after long-term use. It is, however, phy and cytological analysis of vaginal epithelium revealed no still convincing that the first choice of medical treatment for biological effect of the supplement on reproductive organs. severe vasomotor symptom is tailor-made HRT under appropri- Thus, we concluded that 10 mg/d of natural S-equol was the ate diagnostic and follow-up medical care. effective dose for improving Japanese women’s menopausal At the same time, the development of medical treatments for symptoms. the women suffering from health problems caused by estrogen

1388S Supplement deficiency including severe menopausal symptoms who cannot 3. Kudo Y, Fujiwaki S, Sato S, Hatono Y, Shirota T, Taniuchi A, Hosaka T, use HRT has been a great issue in clinical practice. It is also Ishizuka B. A comparative study on menopausal status and climacteric complaints of Japanese women in urban and rural area by cross- expected in menopausal health care that a medical intervention sectional, community based survey [Japanese]. J Jpn Menopause Soc. will improve the quality of life in the majority of menopausal 2005;13:47–54. Japanese menopausal women who complain of moderate- to 4. Wakai K, Egami I, Kato K, Kawamura T, Tamakoshi A, Lin Y, mild-grade menopausal symptoms that are not severe enough to Nakayama T, Wada M, Ohno Y. Dietary intake and sources of be treated by HRT. isoflavones among Japanese. Nutr Cancer. 1999;33:139–45. In the clinical trial in which a subjective score is used as the 5. Adlercreutz H, Hamalainen E, Gorbach S, Goldin B. Dietary end point of evaluation, how to obtain solid data showing the phytooestrogens and the menopause in Japan. Lancet. 1992;339:1233. efficacy of a product is particularly critical. The severity of 6. Reinli K, Block G. Phytoestrogen content of foods: a compendium of literature values. Nutr Cancer. 1996;26:123–48. menopausal symptoms is one of the typical cases, which is easily 7. Lampe JW, Karr SC, Hutchins AM, Slavin JL. Urinary equol excretion influenced by nonspecific factors inducing placebo effects. with a soy challenge: influence of habitual diet. Proc Soc Exp Biol Med. The series of studies described in this paper were attempts to 1998;217:335–9. investigate whether natural S-equol supplementation can be a 8. Rowland IR, Wiseman H, Sanders TA, Adlercreutz H, Bowey EA. new alternative. To confirm the efficacy of the compound strictly Interindividual variation in metabolism of soy isoflavones and lignans: in clinical trials, the confirmation study was designed to include influence of habitual diet on equol production by the gut microflora. Nutr Cancer. 2000;36:27–32. 3 periods. The screening period aimed to exclude the placebo 9. Setchell KDR, Borriello SP, Hulme P, Kirk DN, Axelson M. Nonsteroi- effects of the intervention. At the same time, equol nonproducers dal estrogens of dietary origin: possible roles in hormone-dependent were selected by the results of 3 separate measurements of disease. Am J Clin Nutr. 1984;40:569–78. Downloaded from urinary equol contents. Furthermore, special attention was paid 10. Bolca S, Possemiers S, Herregat A, Huybrechts I, Heyerick A, De Vriese to minimizing the bias caused by evaluation methods of S, Verbruggen M, Depypere H, De Keukeleire D, et al. Microbial and interviewers who assessed changes of symptoms. In the follow- dietary factors are associated with the equol producer phenotype in up period, it was predicted that a rebound response should healthy postmenopausal women. J Nutr. 2007;137:2242–6. appear if the ingested natural S-equol supplement had actual 11. Kimira M, Arai Y, Shimoi K, Watanabe S. Japanese intake of flavonoids and isoflavonoids from foods. J Epidemiol. 1998;8:168–75.

biological effects suppressing menopausal symptoms. As the jn.nutrition.org 12. Uchiyama S, Ueno T, Masaki K, Shimizu S, Aso T, Shirota T. The cross- result, such rebound patterns were observed in the responses of sectional study of the relationship between soy isoflavones, equol and hot flashes and neck or shoulder stiffness. It is indicative that the menopausal symptoms in Japanese women [Japanese]. J Jpn these procedures for standardizing the enrolled population and Menopause Soc. 2007;15:28–37. arrangements adapted in the study made certain contributions to 13. Ishiwata N, Melby MK, Mizuno S, Watanabe S. New equol supplement clarifying the importance of equol’s effects on certain meno- for relieving menopausal symptoms: randomized, placebo-controlled at American Society for Nutrition on June 18, 2010 trial of Japanese women. Menopause. 2009;16:141–8. pausal symptoms. 14. Fukuhara M, Okuma A, Imaizumi K, Uchiyama S, Nakamura G. The This study confirmed the positive effects of equol on menopausal effects of soy isoflavones on menopausal symptoms in Japanese symptoms and was the first report, to our knowledge, based on a perimenopausal women [Japanese]. J Jpn Menopause Soc. 2003;11: prospective randomized, double-blind, placebo-controlled study. 239–46. Thus, natural S-equol, a novel soybean-derived functional compo- 15. Uchiyama S, Ueno T, Suzuki T. Identification of a newly isolated equol- nent, has a promising role in the management of menopausal producing lactic acid bacterium from the human feces [Japanese]. symptoms as an alternative remedy. J Intestinal Microbiol (Tokyo). 2007;21:217–20. 16. Uchiyama S, Ueno T, Suzuki T. Otsuka Pharmaceutical Co. L, editor. Composition containing lactic acid bacterium producing equol Acknowledgment [Japanese]. Japan patent WO 2005/000042 A1. 2005. Available from: The sole author had responsibility for all parts of the http://patft.uspto.gov/. manuscript. 17. Yee S, Burdock GA, Kurata Y, Enomoto Y, Narumi K, Hamada S, Itoh T, Shimomura Y, Ueno T. Acute and subchronic toxicity and genotoxicity of SE5-OH, an equol-rich product produced by Lacto- Literature Cited coccus garvieae. Food Chem Toxicol. 2008;46:2713–20. 18. Matulka RA, Matsuura I, Uesugi T, Ueno T, Burdock G. Developmental 1. Lock M. Encounters with aging: mythologies of menopause in and reproductive effects of SE5-OH: an equol-rich soy-based ingredient. Japan and North America. Berkeley: University of California J Toxicol. 2009. Epub 2008 Dec 15. Press; 1994. 19. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, 2. Avis NE, Stellato R, Crawford S, Bromberger J, Ganz P, Cain V, Stefanick ML, Jackson RD, Beresford SA, Howard BV, et al. Risks and Kagawa-Singer M. Is there a menopausal syndrome? Menopausal status benefits of estrogen plus progestin in healthy postmenopausal women: and symptoms across racial/ethnic groups. Soc Sci Med. 2001;52: principal results from the Women’s Health Initiative randomized 345–56. controlled trial. JAMA. 2002;288:321–33.

Natural S-equol and menopause 1389S The Journal of Nutrition Supplement: Equol, Soy, and Menopause

Cautions and Research Needs Identified at the Equol, Soy, and Menopause Research Leadership Conference1–3

Stephen Barnes* and Helen Kim

Department of Pharmacology and Toxicology, and Center for Nutrient-Gene Interaction, University of Alabama at Birmingham and Purdue University-University of Alabama at Birmingham Botanicals Research Center for Age-Related Disease, Birmingham, AL 35294

Abstract Downloaded from This summary addresses the progress and limitations of existing research on the physiologic properties of the isoflavone daidzein metabolite equol. Previous research demonstrating that physiological equol is its S-enantiomer has led to the preparation of S-(-)equol–enriched products formed by the bacterial fermentation of soy germ. Although this product has interesting properties as described in this workshop, the following important issues must be addressed: 1) the product should be evaluated against a preparation containing an equal amount of pure S-(-)equol to determine whether other jn.nutrition.org components resulting from the fermentation are contributing to the physiological effects; 2) evaluation of the cellular mechanisms of S-(-)equol using cell culture methods should be conducted at concentrations consistent with those encountered physiologically (in the nmol/L range) and in several cell lines representing a target tissue; and 3) in follow-up studies in animal models and in human clinical trials, standardized preparations of S-(-)equol should be made available. at American Society for Nutrition on June 18, 2010 Research opportunities now exist to determine whether equol’s apparent effects on menopausal symptoms (hot flashes, sleep disturbances, bone health) in equol producers can be extended to equol nonproducers. It will be important to ensure that such research is not complicated by cultural differences, differences in lifetime exposure to soy products, experimental techniques, and other variables. Further areas of research that would benefit from the availability of S-(-) equol preparations include its use in skin care (either as an antioxidant or as an estrogen receptor agonist) and in the treatment of brain injury as well as postmenopausal cognitive decline. J. Nutr. 140: 1390S–1394S, 2010.

Introduction

The presentations at the Equol, Soy and Menopause Research 1 Published in a supplement to The Journal of Nutrition. Presented at the “Equol, Soy, and Menopause Research Leadership Conference”, held in Washington, Leadership Conference, held on June 16, 2009 in Washington DC, June 16, 2009. The Supplement Coordinator for this supplement is Kara DC, were organized into 3 general areas: soy and isoflavone Lewis, Life Sciences Research Organization (LSRO) Senior Staff Scientist. The research and metabolism; the relationship of equol to chronic supplement is the responsibility of the guest editors to whom the Editor of diseases in humans; and the potential benefits of equol to women The Journal of Nutrition has delegated supervision of both technical conformity experiencing adverse symptoms during menopause. The follow- to the published regulations of The Journal of Nutrition and general oversight of the scientific merit of each article. Publication costs for this supplement were ing is a discussion of the cautions associated with conducting defrayed in part by the payment of page charges. This publication must therefore and interpreting research in the area and a summary of the be hereby marked "advertisement" in accordance with 18 USC section 1734 research needs identified at the conference. solely to indicate this fact. The Guest Editor for this supplement is Neil Shay. Guest Editor disclosure: Neil Shay declares no conflict of interest. Supplement Cautions Coordinator disclosure: Kara Lewis is currently under contract with and receives compensation from the supplement sponsor. She was also compensated for Physiological equol vs. synthetic equol. There are 2 distinct attending and organizing the Equol, Soy, and Menopause Research Leadership areas of research on equol: studies on equol manufactured by Conference and for organizing, writing, editing, or reviewing, and collection of fermentation using human intestinal bacteria from materials supplemental manuscripts. The opinions expressed in this publication are those containing its precursor, daidzein, and studies on chemically of the authors and are not attributable to the sponsors or the publisher, Editor, or Editorial Board of The Journal of Nutrition. synthesized equol. Equol can exist as the diastereoisomers S-(-) 2 Supported in part by NIH grants U54 CA100949 (S.B., PI), P50 AT00477 equol and R-(+)equol; however, S-(-)equol is the only form (Connie Weaver, PI), and R21 AT004083-01A2 (H.K., PI). produced from daidzein metabolism in humans (1). Information 3 Author disclosures: S. Barnes is a member of the National Advisory Council for most relevant to the health effects of equol in humans will Complementary and Alternative Medicine, a consultant for Frutarom, and holds a necessarily derive from studies on human intestinal bacteria- U.S. patent on the use of conjugated isoflavones for prevention of osteoporosis. H. Kim, no conflicts of interest. produced S-(-)equol and not synthetic equol (2,3), which is a * To whom correspondence should be addressed. E-mail: [email protected]. racemic mixture of S-(-)equol and R-(+)equol.

1390S ã 2010 American Society for Nutrition. First published online May 26, 2010; doi:10.3945/jn.109.120626. A majority of conference presentations concerned equol- revealed a correlation with the metabolites but not with enriched products containing S-(-)equol that are being used in genistein (18). Consideration of the cell line used in a preclinical preclinical and clinical studies. These studies required Institu- study therefore is crucial to drawing conclusions. tional Review Board and Institutional Animal Use Committee approval as well as consideration of safety issues. Human studies Standardization of analytical methods. Analytical standard- may involve interactions with the FDA, because a specific ization represents another research need. Certified reference manufactured product is involved. values do not exist for any soy isoflavone products. The Office of Dietary Supplements has contracted the National Institutes of Possible contribution of other substances to biological Standards and Technology to manufacture of food matrix effects currently attributed to equol. S-(-)equol produced by standards for several dietary supplements. A standard reference bacterial fermentation of soy germ with human intestinal for soy is currently in development (20). In the past, concerns bacteria contains other isoflavone metabolites, some of which about the variability in the values being reported for estrogen are possibly nonphysiologic. It remains to be determined wheth- receptors previously led the National Cancer Institute to develop er the biological activities currently attributed to S-(-) equol in a pooled standard reference product that was distributed to the fermented product are solely due to S-(-)equol alone. The laboratories performing estrogen receptor analysis. The Na- research need can be addressed by conducting studies on pure S- tional Center for Complimentary and Alternative Medicine (-)equol, which can now be chemically manufactured, and the considers the use of standard products to be an extremely bacterial products of daidzein metabolism. If the same amount important point in its studies; products used in the center-funded of pure S-(-)equol and a bacterially produced S-(-)equol prep- studies have to go through extensive prereview by a Product Downloaded from aration lead to different outcomes, the other components of the Quality Working Group. In the absence of a certified standard, product may contribute to the effects currently attributed to S-(-) there should be a pooled sample that various laboratories can equol alone. measure to check variability.

Experimental design. Although convenient, preclinical studies Research needs

in animals are not necessarily relevant to the human response What follows is a list of some of the most pressing research jn.nutrition.org to isoflavones because of differences in equol metabolism. In questions in the field. rodents and nonhuman primates (4–6) fed soy, the principal circulating metabolic form of the isoflavones is S-(-)equol, being Assuming there is a benefit from S-(-)-equol in equol 75% of the total isoflavones, and 4-5 times larger than the producers, would nonproducers also benefit from equol? concentrations of daidzein and genistein. Experiments in rats Some studies have reported a beneficial effect of S-(-)-equol. at American Society for Nutrition on June 18, 2010 suggest that a slower urinary clearance of equol compared with Equol producer status is determined by the presence of the daidzein and genistein (7) accounts for its increased circulating bacteria and the conditions in the intraluminal space, i.e. the amounts. Indeed, because very high circulating levels of equol correct redox potential. Additional studies are needed to deter- result from fairly modest exposure to isoflavones in animal mine whether acute or chronic administration of S-(-)-equol to models, the relevance of animal studies to human experience is equol producers and nonproducers results in the same outcomes. called into question. Effects of equol on hot flashes, sensitivity to changes in temper- In contrast to most other animals, humans do not make large ature, sleep disturbance, and other symptoms reported as adverse amounts of equol and only ;30% of humans are capable of effects by a majority of postmenopausal women are complicated synthesizing equol (8–13), although in Japan and Korea, a larger by cultural differences, differences in lifetime exposure to soy group of individuals is able to synthesize equol (14). The recent products, study techniques, and other variables. work of Setchell et al. (15,16) on the pharmacokinetics of orally administered S-(-)equol and R-(+)equol in human participants Improved sensitivity of questionnaire instruments. Ques- raises interesting questions about equol’s bioavailability. Using tionnaires were developed for different purposes. Even scales 2-13C-labeled equol, an oral dose of 20 mg of S-(-)equol designed to measure global quality of life should pair with produced peak blood concentrations of all equol forms (conju- symptom profiles appropriate for the topic of interest. There is a gated and unconjugated) that were ~3 times higher than those need for more sensitive questionnaire instruments to measure for daidzein and genistein (after correcting for the different sizes changes in the perception of quality of life to improve our of the doses) (17). It is possible that equol, unlike daidzein and objectives in outcome research. Further advancement will come genistein, is a terminal metabolite and is not dissipated into from studying the underlying neurobiology, metabolic path- other chemical forms during transit through the intestine and ways, and measurable physiologic responses. intestinal wall into the blood stream. Is S-(-)equol a marker for other metabolic events in soy Types of cell cultures and concentrations of isoflavones. consumers? The factors that control bacterial equol produc- Most studies using cell cultures to study mechanisms of action of tion require further investigation. Additional information is isoflavones utilize levels ~2 orders of magnitude above the needed about why there are fewer equol producers in Western concentrations found in human exposure. Also, the use of populations (;30%) compared with Eastern populations unconjugated isoflavones does not match what is found in (;50%), and the sensitivity of equol production to redox human blood (.95% conjugated metabolites). The cell lines potential, both microscopically and to the gut in general. typically used in these studies were developed because of their Whether S-(-)-equol is a marker for a different bioactive ability to be cultured rather than they are fully representative of substance should also be investigated. Equol may be a surrogate the disease of interest. In addition, different types of cancer cells marker for a minor metabolite, perhaps not even derived from have a different pattern of metabolism for isoflavones (18,19). the isoflavonoids. Examples of minor, but incredibly bioactive, Attempts to determine the half maximal inhibitory concentra- compounds are the prostaglandins, thromboxanes, and leuko- tion effects of genistein on human breast cancer cell proliferation trienes, all metabolites of long-chain PUFA.

Equol, soy, and menopause research needs 1391S Are differences in response to equol related to time of other protein targets in the pathway. In contrast to peripheral exposure? In some Asian cultures, soy is a traditional part of targets (brain, breast, heart, etc.), cells lining the intestinal lumen the diet. In the West, the US, and Europe, individuals were not may be exposed to very high concentrations of isoflavones (an typically exposed to dietary soy in large amounts until the early estimated 50 or 60 mmol/L) and thereby other less sensitive 1990s. In contrast, Asian women who were in menopause mechanisms may come into play. Another region of the body between 2005 and 2009 have soy exposures dating back to the where isoflavone concentrations are high (with the exception of 1950s and 1960s. The differential effects of administration of genistein) is prostatic fluid, where isoflavone levels are 20–30 soy or soy-derived materials to individuals with different times higher than that in the blood (27–29). And because urine exposure histories may be due to strong epigenetic phenomena, concentrates excreted compounds, the bladder is also exposed to as is the case with exposure to soy and breast cancer. Adolescent high concentrations of isoflavones. exposure to soy is considered to be extremely important to Other areas of work regarding the mechanisms of action of determine future adult breast cancer risk (21,22). More recent receptors are being pursued. Exosomes are 50- to 90-nm vesicles studies suggest that childhood exposure to isoflavones may secreted by a wide range of mammalian cell types that carry influence responses of adults to soy (23,24). Care should be proteins through the blood and thereby target other cells; this is taken to ensure that the right questions are being asked in an endocrine-like mechanism. Several polyphenols influence the nutritionally based epidemiological studies. way in which these exosomes are created, their content, and Because of differences in exposure history, outcomes of their fusion to other cells (30). Equol may regulate this process. studies conducted in the US may not be comparable to outcomes Other isoflavone-dependent targets are the tumor necrosis in societies with large soy consumption from birth such as Japan factor-a pathway (31–33) and PPARa- and PPARg-mediated Downloaded from or China. Furthermore, soy consumption patterns in the West pathways (34–37). have changed and a finding of undetectable blood concentra- tions of isoflavones today is rare. Controlling for different Importance of aglycones. The aglycones are present exposures in different eras is extremely important. in fermented soy foods. They are more rapidly absorbed in the upper small intestine and thereby cause higher peak con-

Reduction of variation on studies. Policy decisions are being centrations during the first enterohepatic cycle. Because they are jn.nutrition.org made about the risk of specific chronic diseases based on studies products of fermentation with microorganisms, the aglycones on consumption of nutritional components. However, most undergo additional hydroxylation (e.g. to 6- or 8-hydroxyiso- studies for which answers to public health questions are sought flavones) prior to the food being consumed (38). There are are statistically inadequate. An obstacle that needs to be questions about whether this gives rise to increased bioactivity of surmounted is having studies that are sufficiently statistically the fermented soy food. at American Society for Nutrition on June 18, 2010 robust that loss of patients from the study or compliance problems do not corrupt the study conclusion. Clinical trials that Role of intestinal microorganisms in human health. The are designed to provide answers for a whole population may not microbiome is the most populous cell type in the human body. be affordable because of the large number of participants There are more cells in the gastrointestinal tract than in the rest required. In addition, the frequent failure of a trial to execute the of the body. Moreover, recent studies have shown that if study as planned may yield insufficiently clear answers. How- microorganisms from the intestines of an obese mouse are ever, as mechanisms become identified, it will be important to transferred to a lean mouse, the lean mouse becomes obese, use this information to more carefully select the group to be indicating that the metabolic state can affect either the compo- studied. This can be achieved by narrowing the study group sition or metabolic capacity of the microbiome in one animal’s using the best available science about possible mechanisms. For body, which can then directly affect another animal’s health instance, one could identify single-nucleotide polymorphisms (39–41). Additional research is needed to determine whether in a receptor and then group individuals according to their isoflavones alter the microbiome and hence affect human health. responses. Another transforming approach is to use an advanced The effect of high alcohol consumption and polyphenols on the technology such as accelerator MS to be able to examine bone microbiome and whether equol production plays a role in the turnover with the use of calcium-41. The extreme sensitivity and physiological effects of alcohol should be determined. specificity of accelerator MS for this rare isotope and the extended period of observation (5–10 y in the same participant) The skin: another area where equol may have important allows for multiple interventions to be tested in a pairwise bioactivity. Does equol have a beneficial effect in skin health? manner, thereby substantially lowering the person-to-person Many topical applications containing polyphenols that protect variance in the experiment. Such an approach has been used to the skin have been developed. This area has 2 complementary evaluate the effect of different soy supplements and standard areas, one where the added polyphenols have purported activity therapies (residronate or estradiol and medroxyprogesterone in sustaining the physical health and youthfulness of the skin and acetate) on bone turnover in perimenopausal women (25). another where the added polyphenols may actually protect against skin cancer later in life. Both may involve antioxidant Mechanisms of action of isoflavones beyond the estrogen and antiinflammatory activities of the polyphenols. receptor. Isoflavones may also elicit effects through mecha- nisms that do not involve the estrogen receptor (ER).4 ER Estrogen-like compounds in sustaining neuronal health receptor activation is very complex and involves coactivators, and numbers. It is widely accepted that estrogen has benefits in repressors, and .30 other proteins (26). Isoflavones may affect brain health and function, including cognitive function. Given that at least some of the principal benefits of equol may be due to its similarity with estrogen, the effects of equol, especially taken 4 Abbreviations used: AMS, accelerator mass spectrometry; ER, estrogen receptor; GSE, grape seed extract; NCCAM, National Center for Complimentary as an additive, in the brain require further study. However, there and Alternative Medicine; SNP, single-nucleotide polymorphism; TBI, traumatic has been controversy regarding the efficacy of estrogen or brain injury. estrogen-like compounds in protecting cognitive function or

1392S Supplement preventing age- or postmenopause-linked cognitive decline. 6. Stroud FC, Appt SE, Wilson ME, Franke AA, Adams MR, Kaplan JR. Nonetheless, cognitive dysfunction induced by ovariectomy Concentrations of isoflavones in macaques consuming standard clearly was attenuated by added estrogen or estrogen-like com- laboratory monkey diet. J Am Assoc Lab Anim Sci. 2006;45:20–3. pounds, including the soy isoflavones (42). It will be important 7. Barnes S, Grubbs C, Smith M, Kirk M, Lubet R. Greater renal clearance of daidzein and genistein accounts for the accumulation of equol in to determine whether equol alone can have the same benefit as blood of soy-fed rats. J Nutr. 2002;132:S619. the soy isoflavones in animal models of dementia including 8. Setchell KDR, Borriello SP, Hulme P, Kirk DN, Axelson M. Nonsteroi- Alzheimer’s disease. Estrogen and genistein independently dal estrogens of dietary origin: possible roles in hormone-dependent prevented age-related mitochondrial dysfunction (43); in such disease. Am J Clin Nutr. 1984;40:569–78. models, it will be important to determine whether equol mimics 9. Kelly GE, Joannou GE, Reeder AY, Nelson C, Waring MA. The variable or suppresses estrogen or genistein effects. An intriguing brain metabolic response to dietary isoflavones in humans. Proc Soc Exp Biol area where phytoestrogens and/or phytochemicals enriched in Med. 1995;208:40–3. antioxidant activity may have activity is in modulating neuro- 10. Karr SC, Lampe JW, Hutchins AM, Slavin JL. Urinary isoflavonoid excretion in humans is dose dependent at low to moderate levels of soy- genesis. In brain regions involved in cognition, neurogenesis is protein consumption. Am J Clin Nutr. 1997;66:46–51. restricted to the dentate gyrus of the hippocampus [see (44)]. 11. Lampe JW, Karr SC, Hutchins AM, Slavin JL. Urinary equol excretion While there is complexity, recent studies showed that ablating with a soy challenge: influence of habitual diet. Proc Soc Exp Biol Med. neurogenesis prevented normal learning and memory, suggesting 1998;217:335–9. a requirement for neurogenesis in normal hippocampal func- 12. Rowland IR, Wiseman H, Sanders TA, Adlercreutz H, Bowey EA. tions in learning (45). If ablation of neurogenesis inhibits Interindividual variation in metabolism of soy isoflavones and lignans: learning and memory, it is reasonable to propose that any influence of habitual diet on equol production by the gut microflora. Nutr Cancer. 2000;36:27–32. Downloaded from compounds that enhance learning and memory have such ac- 13. Atkinson C, Newton KM, Bowles EJ, Yong M, Lampe JW. tions by enhancing or protecting neurogenesis. Previous studies Demographic, anthropometric, and lifestyle factors and dietary reported that grape seed extract enriched in the oligomeric intakes in relation to daidzein-metabolizing phenotypes among proanthocyanidins enhanced cognitive function in young adult premenopausal women in the United States. Am J Clin Nutr. 2008; rats (46). Thus, it was not surprising that grape seed extract 87:679–87. 14. Fujimoto K, Tanaka M, Hirao Y, Nagata Y, Mori M, Miyanaga N,

modulated neurogenesis in developmentally immature mouse jn.nutrition.org Akaza H, Kim WJ. Age-stratified serum levels of isoflavones and brain (J. Cutts, L. Overstreet-Wadiche, and H. Kim, unpublished proportion of equol producers in Japanese and Korean healthy men. observations); however, it is also clear that not all increased Prostate Cancer Prostatic Dis. 2008;11:252–7. neurogenesis is associated with “beneficial” effects in the brain, 15. Setchell KDR, Zhao X, Jha P, Heubi JE, Brown NM. The pharmaco- because increased neurogenesis was correlated with chemically kinetic behavior of the soy isoflavone metabolite S-(-)equol and its induced seizures (47). Thus, neurogenesis will be a critically diastereoisomer R-(+)equol in healthy adults determined by using at American Society for Nutrition on June 18, 2010 important area to explore for potential activity of the soy stable-isotope-labeled tracers. Am J Clin Nutr. 2009;90:1029–37. isoflavones, including equol. There is particular interest in 16. Setchell KDR, Zhao X, Shoaf SE, Ragland K. The pharmacokinetics of S-(-)equol administered as SE5-OH tablets to healthy postmenopausal identifying compounds that are safe in the brain whether or not women. J Nutr. 2009;139:2037–43. they have the highest activity. The brain and the brain region are 17. Setchell KDR, Faughnan MS, Avades T, Zimmer-Nechemias L, Brown thus organs for much further investigation regarding the actions NM, Wolfe BE, Brashear WT, Desai P, Oldfield MF, et al. Comparing of soy phytoestrogens, including equol. the pharmacokinetics of daidzein and genistein with the use of 13C- labeled tracers in premenopausal women. Am J Clin Nutr. 2003;77: Recovery from traumatic brain injury. Screens of compounds 411–9. from existing drugs with good safety profiles and from dietary 18. Peterson TG, Coward L, Kirk M, Falany CN, Barnes S. The role of metabolism in mammary epithelial cell growth inhibition by supplements in a search for ‘safe’ agents have recently revealed the isoflavones genistein and biochanin A. Carcinogenesis. 1996;17: that daidzein and biochanin A are strongly active in supporting 1861–9. recovery from traumatic brain injury (48). Genistein and 19. Peterson TG, Ji GP, Kirk M, Coward L, Falany CN, Barnes S. daidzein are also being studied for their roles in wound recovery. Metabolism of the isoflavones genistein and biochanin A in human breast cancer cell lines. Am J Clin Nutr. 1998;68:S1505–11. Acknowledgment 20. National Institute of Standards and Technology. Measurements and S.B. and H.K. wrote and approved this manuscript. standards for botanical dietary supplements. NIST; 2009 [cited 2009 Nov 4]. Available at: http://www nist gov. 21. Shu XO, Jin F, Dai Q, Wen W, Potter JD, Kushi LH, Ruan Z, Gao YT, Literature Cited Zheng W. Soyfood intake during adolescence and subsequent risk of breast cancer among Chinese women. Cancer Epidemiol Biomarkers 1. Setchell KD, Clerici C, Lephart ED, Cole SJ, Heenan C, Castellani D, Prev. 2001;10:483–8. Wolfe BE, Nechemias-Zimmer L, Brown NM, et al. S-equol, a potent 22. Wu AH, Wan P, Hankin J, Tseng CC, Yu MC, Pike MC. Adolescent and ligand for estrogen receptor beta, is the exclusive enantiomeric form of adult soy intake and risk of breast cancer in Asian-Americans. the soy isoflavone metabolite produced by human intestinal bacterial Carcinogenesis. 2002;23:1491–6. flora. Am J Clin Nutr. 2005;81:1072–9. 23. Lee SA, Shu XO, Li H, Yang G, Cai H, Wen W, Ji BT, Gao J, Gao YT, 2. Gharpure SJ, Sathiyanarayanan AM, Jonnalagadda P. o-Quinone et al. Adolescent and adult soy food intake and breast cancer risk: methide based approach to isoflavans: application to the total syntheses results from the Shanghai Women’s Health Study. Am J Clin Nutr. of equol, 39-hydroxyequol and vestitol. Tetrahedron Lett. 2008;49: 2009;89:1920–6. 2974–8. 24. Korde LA, Wu AH, Fears T, Nomura AM, West DW, Kolonel LN, Pike 3. Takashima Y, Kobayashi Y. New synthetic route to (S)-(-)-equol through MC, Hoover RN, Ziegler RG. Childhood soy intake and breast cancer allylic substitution. Tetrahedron Lett. 2008;49:5156–8. risk in Asian American women. Cancer Epidemiol Biomarkers Prev. 4. Blair RM, Appt SE, Franke AA, Clarkson TB. Treatment with 2009;18:1050–9. antibiotics reduces plasma equol concentration in cynomolgus monkeys 25. Weaver CM, Martin BR, Jackson GS, McCabe GP, Nolan JR, McCabe (Macaca fascicularis). J Nutr. 2003;133:2262–7. LD, Barnes S, Reinwald S, Boris ME, et al. Antiresorptive effects of 5. Gu L, House SE, Prior RL, Fang N, Ronis MJ, Clarkson TB, Wilson phytoestrogen supplements compared to estradiol or risedronate in ME, Badger TM. Metabolic phenotype of isoflavones differ among postmenopausal women using 41Ca methodology. J Clin Endocrinol female rats, pigs, monkeys, and women. J Nutr. 2006;136:1215–21. Metab. 2009;94:3798–805.

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