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,,2
* Department of Food Science and Technology, Chungnam National University, Daejeon, 305-764, Korea; Cancer Prevention Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; ** National Cancer Institute, Division of Cancer Prevention, Rockville, MD 20852; Department of Organic Chemistry, University of Helsinki, Helsinki, Finland; and Department of Epidemiology, University of Washington, Seattle, WA 98195
2 To whom correspondence should be addressed. E-mail: jlampe{at}fhcrc.org.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • soy • isoflavone • equol • diet
Soy foods are rich sources of isoflavones, including daidzein and genistein, and are traditionally consumed by many Asian populations. Soy isoflavones have received considerable attention for their potential effects on human health, but interindividual differences in their metabolism exist. Daidzein can be metabolized to equol and O-desmethylangolensin (ODMA)3 by intestinal bacteria, and these metabolites are absorbed, enter the circulation, and are excreted in urine (1). Differences in isoflavone-metabolizing bacteria in the gut are a main criteria contributing to the variation in isoflavonoid profiles among individuals (2,3) and some, but not all, recent studies have suggested that the daidzein-metabolizing phenotypes may be associated with risk biomarkers of several diseases (4).
Soy-challenge studies (1,5), in which individuals are given a dose of soy containing isoflavones, have shown that only 
30–40% of people are capable of producing equol from daidzein. The prevalence of equol producers may be higher in Asian populations than in Caucasians (6,7), but this has not been studied using a standardized soy challenge. Dietary factors, including soy, meat, and carbohydrate intakes (1,2,5,8–10) and host genetics (1) have been suggested as contributing factors to the equol-producer phenotype in humans.
Soy foods are a major component of the traditional Korean diet, and, in the United States, Korean Americans (KA) consume more soy foods than Caucasian Americans (CA) (11). We hypothesized that the prevalence of equol producers in KAs would be higher than in CAs and that this difference would be associated with differences in habitual soy intake. The objectives of this study were to determine the prevalence of the equol-producer phenotype in KA women and girls, compare the prevalence data with those in CA women and girls, and examine whether the dietary habits of these 2 groups were associated with the capacity to produce equol.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Participant procedures.
Each participant was asked to consume one soy protein bar (Revival, Physicians Laboratories; containing 38 mg daidzein as aglycone equivalents per bar) per day, preferably in the afternoon or evening, on 3 consecutive days, and to provide a first-void urine sample on day 4, according to our standard protocol (1). Participants mailed the urine sample to the lab and aliquots were stored at –20°C until analysis. In addition, participants were asked to record how much of the soy bar they ate each day.
Demographic and dietary survey. Participants completed a self-administered questionnaire that gathered information on factors such as date of birth, self-reported height and weight, and dietary characteristics, such as typical meat and grain consumption and frequency of fried food consumption. In addition, participants were asked to indicate the number of servings of certain foods eaten in the past month. Information on consumption of the following 10 most commonly consumed soy foods in Korea was gathered: tofu, soybean sprouts, soy milk, doenjang (soybean paste) soup, chongkukjang, kongkooksoo, miso, aburaage, kongjaban, and kongbiji. We calculated mean daily isoflavone intake using data from several published sources (12–14). Previously collected demographic and dietary-habit data for 222 CA women and girls (1) were available for comparison with the KA women and girls. For the CA study, soy intake was assessed using a single question that encompassed any soy food.
Isoflavonoid analysis. Urine samples were analyzed for isoflavonoids (equol, ODMA, genistein, and daidzein) by gas chromatography-mass spectrometry as described elsewhere (1). The intraassay CV for isoflavonoids in the quality-control sample, measured in duplicate for each batch, were <8%. The interassay CV were <14%. Equol and ODMA producers were defined as individuals with urinary concentrations of equol >44 µg/L (183 nmol/L) and ODMA >44 µg/L (170 nmol/L), respectively. Genistein and daidzein were measured to monitor compliance to the soy challenge. Daidzein concentrations <100 µg/L (394 nmol/L) are considered indicative of noncompliance (1). Urinary creatinine concentrations were measured, as described previously (1), to ensure that urine samples were sufficiently concentrated (>80 mg/L; 0.71 mmol/L). If creatinine concentrations were low, a larger urine volume would be used for the extraction.
Statistical analysis.
Dietary habits and demographic characteristics of 91 KA and 222 CA women and girls were analyzed using Stata (Stata Corp). Differences between groups for categorical variables were analyzed by chi-square analysis or logistic regression and adjusted for age when appropriate. Differences between groups for continuous variables were analyzed by linear regression and adjusted for age when appropriate. We also combined the dietary data from both groups to evaluate, in a larger and more diverse sample, whether dietary habits differed by equol-producer phenotype. This analysis was adjusted for age and used a generalized estimating equation model to account for combining 2 studies conducted separately but using the same questionnaire. P-values 
0.05 were considered significant. The number of ODMA nonproducers was too small to allow for a comparison of demographic and dietary data based on this phenotype. Values are means ± SD unless otherwise indicated.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2 = 13.5, P < 0.004, Table 1).
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18 y), 16 (22%) KA women and 35 (19%) CA women had 12 y of education (P = 0.58). KA women with >12 y of education were 75% less likely, relative to KA with 12 y of education, to be equol producers (odds ratio = 0.25, 95% CI: 0.72, 0.87), whereas CA women with >12 y of education, relative to CA with fewer years of education, were two-times more likely to be equol producers (odds ratio = 2.18, 95% CI: 0.92, 5.11). No KA women were current or former smokers. KA women were less likely to consume alcohol than CA women (50 vs. 84%, P < 0.001), and this was not associated with the equol-producer phenotype within either group of women. Urinary isoflavonoid concentrations, which reflect both habitual dietary intake and the isoflavone exposure from the soy challenge, were significantly higher among the KA.
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3 times that of CA (8.1 servings vs. 2.4 servings per week).
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Among the KA women and girls alone, consumption of traditional Korean and other soy foods did not differ between equol producers and nonproducers (Table 4). In addition, daily soy isoflavone intake was similar between equol producers and nonproducers, 21.2 ± 19.0 and 20.3 ± 17.8 mg/d (P = 0.8), respectively. The most commonly consumed soy foods among KA, in order of frequency of consumption, were tofu, doenjang (soybean paste) soup, and soybean sprouts.
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Asian populations, including Chinese, Japanese, and Korean, generally consume more soy foods than Caucasians. Hedlund et al. (10) reported that long-term dietary habits affect soy isoflavone metabolism in Caucasians; individuals with a long-term high consumption of soy foods were more likely than low consumers to be equol producers. In the context of these findings, we hypothesized that soy intake would be associated with the difference in equol-producer prevalence between KA and CA women and girls. We did not detect a difference in soy intake between equol producers and nonproducers despite a wide range in intakes even among the KAs (Table 4). We also did not detect the positive association between meat intake and equol production that has been reported in several studies (8,10).
Mean daily soy isoflavone intake for KA women and girls was 20.8 mg/d, which is comparable with values reported in the literature for other Asian populations (16,17); however, it is approximately twice that consumed by Japanese American (10.2 mg/d) and ten times that of Caucasian (<2mg/d) residents living in the same geographic region as study participants (17,18). Among residents in Hawaii (16), daily isoflavone intake was 11.9 mg/d for Chinese, 18.9 for Japanese, and 5.2 for Caucasian. Among the KA in our study, the most frequently consumed soy foods were tofu, doenjang (soybean paste) soup, and soybean sprouts. The soy foods most commonly consumed by Japanese American women in Seattle were tofu, miso, and aburaage (17), and the soy foods commonly consumed by other predominantly CA populations in Seattle were tofu, soymilk, and edamame (cooked green soybeans) (18,19).
The daidzein-metabolizing phenotypes are determined by gut microbial community structure. Because diet plays an integral part in defining the gut microbial community, it has been hypothesized that dietary differences among individuals may contribute to the observed differences in phenotype. Observational studies suggest that several macronutrient patterns (e.g., high carbohydrate, high fiber, high fat) may be associated with the capacity to produce equol; however, these associations are not observed consistently in all cross-sectional studies, nor have interventions, albeit relatively short-term, been successful in altering phenotypes (reviewed in 4). Recently, Clavel et al. (20) reported that a 1-mo isoflavone intervention (100 mg/d aglycone equivalents) in Europeans significantly altered the dominant fecal microbiota, increasing the percentage of microorganisms in certain groups. However, these changes appeared to be transient; by the end of month 2 of the study, bacterial compositions, with the exception of Bifidobacterium species, returned to near original values. The Clostridium coccoides-Eubacterium rectale cluster, which was associated with equol production, was stimulated with isoflavone supplementation, but only in equol producers (20). This cluster did not change quantitatively in nonproducers, further supporting previous findings that it is difficult to convert an equol nonproducer to a producer and that these phenotypes are relatively stable (21). Many differences, in addition to differences in soy intake, exist between the traditional Korean diet and a Western diet (11,22,23). These may contribute to pre- and probiotic exposures that influence the gut microbial community, but this has not been evaluated.
As in previous studies of daidzein-metabolizing phenotypes in other populations (1,5,10,24), we observed that few demographic factors are associated with the capacity to produce equol in the KA population; however, in this study, greater education was inversely associated with being an equol producer in the KA but positively associated with being an equol producer in the CA. This may be a chance association, or, alternatively, it may reflect lifestyle or dietary factors not captured by our questionnaire that are differentially associated with level of education in the 2 racial groups. The positive association between years of education and equol production was also observed among CA men in our previous study (1).
The overall implications for effects of the daidzein-metabolizing phenotypes on human health have not been established (4); however, few studies have been designed specifically to evaluate the association between these phenotypes and health outcomes or risk biomarkers. Further, comparisons of the prevalence of equol producers across populations have not been conducted systematically with a consistent phenotyping method. The strength of this study was that we evaluated the prevalence and potential determinants of the daidzein-metabolizing phenotypes in KAs and CAs using the same protocol: we used a soy challenge, which assured sufficient exposure to daidzein so as to phenotype accurately; we used the same exclusion criteria in the recruitment of both groups; and we collected the same data on both groups, with a minor change in the KA study to collect additional data on traditional Korean soy foods. Having only a general question about frequency of soy food intake among CAs may have underestimated the true intake among CAs relative to KAs. However, the mean weekly soy food intake of 2.4 servings among the CA is within the range of soy food intake we observed previously using 20- and 40-item soy food questionnaires (18,25). Our study also was limited in its sample size and the extent of the questionnaire administered. Although the 2 groups were sampled 3 y apart, it is unlikely that the racial difference in prevalence reflects a secular trend because the phenotypes are relatively stable in individuals over time (21).
There is also a potential limitation related to our phenotyping method. Because we classify individuals on the basis of urinary isoflavonoid concentration, rather than total output over 24 h, there is the possibility of phenotype misclassification depending on how concentrated the urine sample was. Our previous work suggests that this is uncommon (21,26) and several approaches were used to minimize this: 3 d of soy challenge to ensure sufficient time for equol and ODMA production and excretion; a first-void urine to obtain a concentrated sample; and urinary daidzein and creatinine monitoring to evaluate compliance and urine concentration, respectively. All participants in both groups had daidzein concentrations >394 nmol/L and urinary creatinine concentrations >0.71 mmol/L, and there was no difference in creatinine concentrations between the groups. Thus, misclassification, if it occurred, would be similar across the groups and would not contribute to racial differences in phenotype prevalence.
In our assessment of the daidzein metabolizing phenotypes using a soy-isoflavone challenge, we observed that the prevalence of the equol-producer phenotype was higher, and the ODMA-producer phenotype was lower, in KA than in CA women and girls. There was no indication from our study that habitual consumption of soy foods is associated with the equol-producer phenotype. Future phenotyping studies with larger sample sizes and more comprehensive assessment of longer-term dietary patterns and nondietary factors, as well as evaluation of the gut microbial communities associated with these phenotypes, may help elucidate the factors that contribute to daidzein-metabolizing phenotypes in populations that habitually consume soy.
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FOOTNOTES |
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3 Abbreviations used: CA, Caucasian American; KA, Korean American; ODMA, O-desmethylangolensin.
Manuscript received 28 November 2005. Initial review completed 8 January 2006. Revision accepted 8 February 2006.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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