Isoflavone Supplements Do Not Affect Thyroid Function in Iodine-Replete Postmenopausal Women

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1 JOURNAL OF MEDICINAL FOOD J Med Food 6 (4) 2003, Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition Isoflavone Supplements Do Not Affect Thyroid Function in Iodine-Replete Postmenopausal Women Bonnie Bruce, 1 Mark Messina, 2,3 and Gene A. Spiller 4 1 Division of Immunology & Rheumatology, Department of Medicine, Stanford University, Palo Alto; 2 Department of Nutrition, School of Public Health, Loma Linda University, Loma Linda; 4 Health Research and Studies Center, Los Altos, California; and 3 Nutrition Matters, Inc., Port Townsend, Washington ABSTRACT Despite the safety review conducted by the U.S. Food and Drug Administration in the process of awarding a health claim for the cholesterol-lowering properties of soy protein, concerns about the possible goitrogenic effects of soybean isoflavones persist. Concerns are based primarily on in vitro research, animal studies, and older reports of goiter in infants fed soy formula not fortified with iodine. In a randomized, double blind, placebo-controlled study, we investigated the effect on thyroid function of a daily supplement containing 90 mg (aglycone weight) of total isoflavones/day versus placebo in 38 postmenopausal women, years old, not on hormone replacement therapy. Serum thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) were measured at baseline and after 90 and 180 days. In the supplement group, at baseline and 6 months, TSH (mu/ml), T4 (nm), and T3 (nm) levels (mean 6 SE) were , , and , respectively, and , , and , respectively. In the control group, levels at baseline and at 6 months were , , and , respectively, and , , and , respectively. Intragroup differences for all three measures were statistically indistinguishable at 6 months, and levels were similar between the isoflavone supplement and placebo groups at each measurement. These results indicate that in this group of healthy iodine-replete subjects, soy isoflavones do not adversely affect thyroid function. KEY WORDS isoflavones phytoestrogens soy thyroid function thyroid hormones INTRODUCTION SOYFOODS HAVE PLAYED an important role in the diets of many Asian countries for centuries. Recently, the soybean has attracted widespread attention in the West for its purported health benefits. These include but are not limited to protection against coronary heart disease, 1 3 certain forms of cancer, 4 7 and osteoporosis. 8 Particularly noteworthy is the health claim issued by the U.S. Food and Drug Administration (FDA) in 1999 for the cholesterol-lowering properties of soy protein 9 and the subsequent endorsement of the use of soy protein for cholesterol reduction by the American Heart Association 1 year later. 3 The FDA has set 25 g of soy protein/day as the target intake goal for cholesterol reduction. However, despite the FDA-approved health claim, which required a review of all safety issues, concerns about possible adverse effects of soy have arisen. Most notable is that soy, especially in susceptible individuals, may adversely affect thyroid function. 10,11 Received 12 June Accepted 17 July Address reprint requests to: Bonnie Bruce, Dr.P.H., M.P.H., R.D., Research Associate, Division of Immunology & Rheumatology, Department of Medicine, Stanford University, 1000 Welch Road, Suite 203, Palo Alto, CA 94304, bbruce@stanford.edu Investigation into the effects of soy on thyroid function began with animal research in 1933, which found that the addition of soybeans to a laboratory rat diet led to almost a tripling of the thyroid gland weight. 12 In the late 1950s and early 1960s 12 cases of goiter reported in infants fed a soybased formula helped to further draw attention to the relationship between soy and thyroid function However, since the introduction of iodine-fortified soy-based formula, no additional cases of goiter have been reported. More recently, investigators from the National Center for Toxicology Research have expressed concern about the possible adverse effects of soybean isoflavones, which are a subclass of flavonoids, on thyroid function. 10 Isoflavones have a chemical structure similar to estrogen and exhibit both hormonal and non-hormonal properties in a variety of experimental conditions The soybean is the only nutritionally relevant natural food source of isoflavones. The primary isoflavones in soybeans are genistein (49,5,7-trihydroxyisoflavone) and daidzein (49,7-dihydroxyisoflavone), and their respective b-glycosides, genistin and daidzin. 19 Given concerns regarding the anti-thyroid effects of soyfoods and isoflavones, we studied the effects of an isoflavone-rich supplement on thyroid function in postmenopausal women. We hypothesized that isoflavone supplementation would have no effect on the thyroid. 309

2 310 BRUCE ET AL. Subjects MATERIALS AND METHODS Postmenopausal women already participating in a 6- month, placebo-controlled, randomized trial that was designed to evaluate the effects of daily isoflavone supplementation on bone density were recruited for this nested study. To be eligible for the randomized trial, subjects had to have been at least 60 years of age at study entry, postmenopausal, and not on hormone replacement therapy (HRT). Eligibility criteria for entry into this nested study additionally stipulated absence of history of breast cancer and a willingness to make an extra clinic visit for phlebotomy and assessment of thyroid function. As part of the randomized trial all subjects were instructed to take daily a multivitamin and mineral supplement that included the recommended dietary allowance of 150 mg of iodine. Subjects were withdrawn from the study if they began using HRT, developed a major illness, such as cancer, parathyroid disease, diabetes, or kidney or heart disease, experienced a major clinical event, such as myocardial infarction, had a mental condition that would make them unsuitable to participate in the study, or a body mass index (BMI).35. All subjects signed a consent form approved by the Administrative Panel on Human Subjects in Medical Research at Stanford University School of Medicine before participating in this study. Study supplement For the primary trial, subjects had been randomly assigned to receive three times per day either a placebo composed of maltodextrin with 10% caramel color or an isoflavone supplement (Novasoy ; provided by Archer Daniels Midland Co., Decatur, IL), containing 50 mg (approximately 30 mg expressed as aglycone weight) of soyderived isoflavones. The supplement consisted of 40% isoflavones (1.3:1.0:0.3 ratio of the glycosides genistin, daidzin, and glycitin), 40% natural soy components (mostly saponins), 7 12% protein, 1% fat, and 4% ash. The 90 mg of total isoflavones taken daily by subjects is the amount contained in approximately 9 12 ounces of tofu 19 and represents approximately two to three times the typical Japanese adult intake. 20 Study visits Subjects were seen at the study center at baseline and at 3 and 6 months. Blood was drawn at each visit after a 12- hour fast. At the study visits, subjects met one of the investigators or trained study personnel, to receive more tablets and to answer questions about general health status and medication use. Diet and lifestyle habits Subjects were asked to maintain their usual diet and lifestyle habits, with the exception of limiting intake of soycontaining foods, if consumed, to one serving, once a week. They received verbal and written dietary instructions about soy-containing foods from a registered dietitian. Thyroid function analyses Serum was analyzed for thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3). Serum samples were frozen at 272 C and batch-analyzed at 6 months to minimize interassay variations. T3 and T4 were analyzed using radioimmunoassay and TSH by immunoradiometric assay. Statistical analyses All data are presented as mean 6 SE. Standard statistical techniques were used to assess two-sample means. A nonpaired t test was used to compare differences in thyroid function between the supplement and placebo groups. Generalized linear modeling was used to compare fasting serum levels of thyroid function at baseline and at 3 and 6 months. Level of significance was set at P,.05. Data were analyzed using SAS version 8.2. RESULTS Thirty-eight of the 42 subjects completed the trial. Of the four who did not complete the protocol, three were from the isoflavone group and one from the placebo group. Three withdrew for reasons unrelated to the trial, and one was lost to follow-up. There were no statistical differences in subject characteristics between the two groups, indicating that randomization was successful (Table 1). With the exception of one subject in the placebo group, all of the women were Caucasian (97.4%). Overall, in both groups subjects were on average in their late 60s, were well-educated, and had a normal BMI. The number of current smokers and women taking thyroid medication upon entrance did not differ between groups. As shown in Table 2, after 6 months there were no statistically significant intrasubject differences in serum levels of TSH, T4, and T3, and levels were similar between the isoflavone supplement and placebo groups at each measurement. DISCUSSION To our knowledge this research represents the first clinical trial to examine the effects of isoflavone supplements on thyroid function. Understanding the biological effects of isolated isoflavones is important because these soybean constituents are commercially available as supplements and are being used as food fortificants. These results indicate that in healthy iodine-replete euthyroid postmenopausal women daily isoflavone ingestion for 6 months does not adversely affect thyroid function. The fact that this study found no adverse effects in older women is particularly important because surveys indicate that in the United States as many as 10% of Caucasian women 21 and 5% of all postmenopausal

3 ISOFLAVONES AND THYROID FUNCTION IN FEMALES 311 TABLE 1. BASELINE SUBJECT CHARACTERISTICS BY STUDY GROUP Supplement group Placebo (n 5 22) (n 5 16) Age (years) a 68.9 (1.03) 69.9 (0.98) Education (years) a 15.2 (0.49) 15.9 (0.38) BMI (weight [kg]/height [m 2 ]) a 20.5 (0.68) 20.2 (1.08) Caucasian b 22 (100%) 15 (94%) Current smoker b 1 (5%)0 1 (6%) On thyroid medication at baseline b 3 (14%)0 3 (19%) Subjects had been randomly assigned to receive three times per day either a placebo composed of maltodextrin with 10% caramel color or an isoflavone supplement (Novasoy, provided by Archer Daniels Midland Co.), containing 50 mg (approximately 30 mg expressed as aglycone weight) of soy-derived isoflavones. The supplement was comprised of 40% isoflavones (1.3; 1.0; 0.3 ratio of the glycosides genistin, daidzin, and glycitin), 40% natural soy components (mostly saponins), 7 12% protein, 1% fat, and 4% ash. Subjects in both groups were similar, and differences were statistically indistinguishable (all P..05). a Mean and SE values in parentheses. b Number (percentage). TABLE 2. women 22 have subclinical hypothyroidism. This subgroup may be particularly sensitive to goitrogenic influences. Also, postmenopausal women are heavily targeted by the soyfood industry because of research suggesting that soy may improve menopausal health and serve as an alternative to HRT In women receiving the isoflavone supplement all three measures of thyroid function (TSH, T4, and T3) were within the normal range upon enrollment and remained so and did not differ from the placebo group at baseline or after 3 and 6 months of supplementation. The dose of isoflavones used in this study is approximately two to three times that typically consumed by Japanese adults via the consumption of soyfoods. 20 Thus, the lack of an effect cannot be attributed to low isoflavone exposure. The findings from this study are in agreement with the results of a small 4-week Japanese pilot study in which three women were given isoflavone supplements. 28 However, one limitation of the current trial is that there was no biological verification of compliance, nor was isoflavone bioavailability of the supplement directly determined by assessing serum or urinary isoflavone levels. But, unpublished research indicates isoflavones from this particular supplement are absorbed to approximately the same degree as are isoflavones from soyfoods (Kenneth D. Setchell, personal communication). Previous investigators have found that flavonoids inhibit the activity of thyroid peroxidase (TPO) in vitro. 29 Furthermore, isoflavones have been found to be more potent than the non-isoflavone flavonoids and to inhibit TPO activity at relatively low (1 10 mm) concentrations. 29,30 Of related interest are reports indicating that the flavonoids in millet are thought to contribute to the high incidence of goiter in certain African populations where iodine deficiency is common However, flavonoids are widely distributed in the plant THYROID FUNCTION VALUES AT BASELINE, 90, AND 180 DAYS TSH (mu/ml) T4 (nm) T3 (nm) Number Mean (SE) Number Mean (SE) Number Mean (SE) Supplement group Baseline (0.44) (5.04) (0.13) 90 days (0.50) (5.06) (0.13) 180 days (0.52) (2.09) (0.12) Placebo group Baseline (0.51) (6.69) (0.18) 90 days (0.78) (6.01) (0.12) 180 days (0.57) (6.64) (0.10) Subjects had been randomly assigned to receive three times per day either a placebo composed of maltodextrin with 10% caramel color or an isoflavone supplement (Novasoy, provided by the Archer Daniels Midland Co.), containing 50 mg (approximately 30 mg expressed as aglycone weight) of soy-derived isoflavones. The supplement was comprised of 40% isoflavones (1.3; 1.0; 0.3 ratio of the glycosides genistin, daidzin, and glycitin), 40% natural soy components (mostly saponins), 7 12% protein, 1% fat, and 4% ash. Changes within groups were statistically indistinguishable (all P..05).

4 312 BRUCE ET AL. kingdom and are found in a large number of fruits and vegetables, 35 foods that are thought to confer a variety of health benefits. 36 The soybean isoflavones genistein and daidzein block TPO-catalyzed tyrosine iodination in vitro by acting as alternate substrates. 30 Incubation of either genistein or daidzein with TPO in the presence of hydrogen peroxide causes irreversible inactivation of TPO, but the addition of iodide to the media completely abolishes this inactivation. 37 The ability of the iodine ion to prevent inactivation of TPO is consistent with research in rodents indicating that the addition of soy, especially raw soy, to iodine-deficient diets leads to an increase in thyroid weight beyond that observed in rats fed iodine-deficient diets alone, but that increasing the iodine content to about double the amount needed on soy-free diets lowers the weight of the thyroid gland to that of the control animals The role of iodine deficiency in exacerbating the goitrogenic effects of soy on thyroid function in rats is consistent with past reports of goiter in infants who were fed non-iodine fortified soy-based formulas It is also consistent with the lack of reports of goiter in infants fed iodine-fortified soy formula. Since the introduction of iodine-fortifed formula in the 1960s an estimated 20 million U.S. infants have used soy formula. 43 This observation appears to be particularly relevant since recent data indicate that serum isoflavone levels in soy formula-fed infants greatly exceed those in adults consuming soyfoods. 44,45 Additional evidence for lack of goitrogenicity of soy-derived isoflavones in iodine-replete animals is provided by Chang and co-workers: They found that although TPO was partially inactivated (approximately 50%) in vivo in rats fed soy-free diets fortified with 500 ppm of genistein aglycone starting in utero through 20 weeks of age, there were no effects on serum thyroid hormone (T3, T4, and TSH) levels, thyroid weights, and thyroid histopathology. 46,47 Thus, even the feeding of genistein to rats, which are especially sensitive to goitrogenic influences, caused no disruption in thyroid functioning. 48 The lack of effects in the current study does not preclude the possibility that non-isoflavone components of soyfoods adversely affect thyroid function. Son et al. 49 found that in rats defatted soy flour but not a soybean isoflavone mixture exacerbated the effects of iodine deficiency on thyroid function. Over the past few years, numerous clinical trials have examined the impact of soy consumption on thyroid function. 19,50 61 Of the 11 trials identified (one unpublished and two published only as abstracts), three involved both men and women, three only males 62,65 (S. Potter, personal communication), and five only women With one exception, these trials found that soyfoods did not exert any clinically relevant effects on thyroid function. 63 Ham et al. 65 did find that in hypercholesterolemic men who consumed 50 g of soy protein for 4 weeks (approximately five times the adult native Japanese soy protein intake) serum T4 levels increased by about 25% (P,.01) compared with baseline; however, levels remained within the normal range. Fur- thermore, in a second study in which men were fed 25 g of soy protein no increases in T4 were noted (S. Potter, personal communication). In evaluating studies of the effect of soyfoods on thyroid function the amount of soy fed and study duration are clearly paramount considerations. In addition to the two studies cited above, several others fed subjects soy in amounts that greatly exceeded that typically consumed by Japanese adults. For example, Jayagopal et al. 67 fed postmenopausal women 30 g of isolated soy protein containing 132 mg of isoflavones, and Duncan et al. 68,69 fed pre- and postmenopausal women approximately 30 g of soy protein that contained as much as 128 mg of isoflavones. Finally, although most trials were of relatively short (#16 weeks) duration, studies by Thiagarajan et al. 64 and Lydeking-Olsen et al. 70 were all at least 1 year in length. Thus, it is reasonable to conclude that even large amounts of soy over an extended time period likely do not adversely affect thyroid function in healthy adult men and women. However, one trial did conclude that soy adversely affects thyroid function. Since this study is frequently cited as primary evidence for the goitrogenic effects of soy it warrants careful evaluation. Ishizuki et al. 63 fed three groups of Japanese subjects daily for 1 or 3 months 30 g of roasted soybeans that were pickled and stored in rice vinegar. They found that most measures of thyroid function were unaffected, but although TSH values remained within the normal range for all groups, TSH levels increased from 45% to 90%. Three months after discontinuing soy feeding, levels had returned to their initial values. Even more striking was the high incidence of goiter and side effects that were reported among the study subjects: Goiters were found in 15% (three of 20) in group 1, which was composed of men and women with an age range of years, 71% (five of seven), in group 2, which was composed of younger women, and 30% (three of 10), in group 3, which was composed of postmenopausal women. The range of side effects was varied; most notable was diarrhea (35% in group 1), constipation (20% and 50% in groups 1 and 2 1 3, respectively) and malaise/sleepiness (53% and 42%, respectively, in groups 2 and 3). There are, however, aspects to this study that make interpretation of the findings difficult. For example, the soy product used differs from the traditional Japanese soy products such as tofu or the Western soy product isolated soy protein that is most often used in clinical trials. It is not clear how the nutrient and chemical composition of this product was affected by pickling and storing the soybeans in rice vinegar. This concern is highlighted by the high incidence of gastrointestinal side effects reported, since in the current study no side effects were reported, which is typical for most of the clinical trials involving isolated soy protein. Also puzzling are the striking findings in response to the consumption of just 30 g/day of boiled soybeans. This amount of soy yields only about 5 g of soy protein, which represents about half the average adult Japanese soy protein intake. Given the range of soy intake in Japan, the findings

5 ISOFLAVONES AND THYROID FUNCTION IN FEMALES 313 suggest that the incidence of goiter in Japan should be much higher than it currently is. 56,71 Finally, the malaise and sleepiness reported by subjects in groups 2 and 3 are inconsistent with the finding that serum concentrations of total and free T4 and of T3 were not altered. Another concern related to soy intake and the thyroid is the possibility that soy consumption could increase risk of thyroid cancer, perhaps mediated through increased serum TSH levels. 11,37,72 Although Kimura et al. 73 did find that the addition of soybean to iodine-deficient diets increased malignant goiter in Wistar rats, Son et al. 74,75 found no effect of isoflavones on thyroid carcinogenesis in either male 74 or female 75 rats. Furthermore, in a U.S. case-control study conducted by Horn-Ross et al. 76 isoflavone intake was inversely related to risk of thyroid cancer. While soy consumption as estimated by isoflavone intake was quite low in this population study, overall, the notion that soy intake could increase risk of thyroid cancer has little scientific evidence. Finally, critics of soy formula often point to a study by Fort et al. 77 as evidence of the detrimental effects of soy intake on thyroid function in infants. 11,37 This group found that the frequency of feedings with soy-based milk formulas early in life was significantly higher in children with autoimmune thyroid disease (ATD) as compared with siblings and healthy non-related control children. 77 However, even if one accepts this finding, it is not clear that soy formula is causally related to ATD or that the relationship resulted from the direct effects of soy on thyroid function. As discussed by the investigators and as noted by Tuohy, 78 the association between soy formula and ATD may have resulted from the reported relationship between atopy and ATDs 79 and not soy formula per se, since the use of soy formula was initiated by the parents, and such decisions are often made in response to allergic reactions to cow s milk formula. Second, Fort et al. 77 suggested that the reported association may have stemmed from a general immunogenic effect of soy formula that could have resulted in damage to the intestinal barrier and not to direct effects on the thyroid. 80 Third, Setchell and Radd 44 pointed out that in the study by Fort et al., 77 the comparison groups were composed predominantly of males, whereas the ATD group was composed primarily of females. Since ATD occurs primarily in females one would expect a lower incidence of ATD in the control groups. Interestingly, in the ATD group approximately the same percentage of infants was breast-fed or fed soy formula or cow s milk formula. Thus, the study by Fort et al. 77 lacks the basis as evidence for claiming that soy formula contains constituents that directly adversely affect thyroid function. On the other hand, there is evidence from both infants and adult patients on synthetic thyroxine that if soy products are consumed the dose of medication needs to be increased or needs to be separated temporally from the ingestion of the medication 83 in order to maintain therapeutic levels of the hormone. However, this interaction with orally administered thyroxine appears to stem from an increased fecal loss of the medication and not from a systemic effect of soy. 83,84 Parenthetically, fiber supplements also increase the need for larger than expected doses of thyroxine in hypothyroid patients. 85 Worthy of note is that isoflavones did not affect the thyroid hormone levels of two women in the current study on thyroid medication. In conclusion, the results of the current research in conjunction with previous clinical trials indicate that in healthy iodine-replete subjects, neither soyfoods nor isolated soybean isoflavones adversely affect thyroid function. Whether soy consumption or isoflavones adversely affect thyroid function in individuals with a compromised thyroid status or whose iodine intake is marginal remains to be determined. This issue needs to be investigated and is one of the research needs identified by the Committee on Toxicity, which recently released their report Phytoestrogens and Health. 86 Fortunately, through the use of iodized salt iodine intake in the United States is more than adequate, although there is a downward trend in iodine intake, and some subsets of the population such as women of child-bearing age may have marginal intakes Clearly, it is important for all individuals regardless of their soy intake to consume adequate iodine. Those counseling clients who have recently begun to consume large amounts of soy protein may want to discuss the theoretical concerns about the effects of soy on thyroid function, but the emphasis should not be on reducing soy intake but rather on consuming sufficient amounts of iodine. If concerns persist, thyroid functioning can easily be assessed, something the American Thyroid Association recommends that all individuals have done every 5 years beginning at the age of ACKNOWLEDGMENTS This work was supported by a grant from Archer Daniels Midland Company. REFERENCES 1. Anderson JW, Johnstone BM, Cook-Newell ME: Meta-analysis of the effects of soy protein intake on serum lipids. N Engl J Med 1995;333: Clarkson TB: Soy, soy phytoestrogens and cardiovascular disease. J Nutr 2002;132(Suppl):566S 569S. 3. Erdman JW Jr: Soy protein and cardiovascular disease: a statement for healthcare professionals from the Nutrition Committee of the AHA. Circulation 2000;102: Jacobsen BK, Knutsen SF, Fraser GE: Does high soy milk intake reduce prostate cancer incidence? The Adventist Health Study (United States). Cancer Causes Control 1998;9: Messina MJ, Persky V, Setchell KD, Barnes S: Soy intake and cancer risk: a review of the in vitro and in vivo data. 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