Plasma isoflavone levels versus self-reported soy isoflavone levels in Asian-American women in Los Angeles County

Anna H. Wu2, Mimi C. Yu, Chui-Chen Tseng, Nathan C. Twaddle1 and Daniel R. Doerge1

Department of Preventive Medicine, University of Southern California, Keck School of Medicine, Los Angeles, CA, USA and 1 National Center for Toxicological Research, Jefferson, AR, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a case–control study conducted among Asian-American women in Los Angeles County, we reported that the risk of breast cancer was significantly reduced in association with soy intake [Wu,A.H., Wan,P., Hankin,J. et al. (2002) Carcinogenesis, 23, 1491–1496]. In a subset of cases (n = 97) and controls (n = 97) we investigated the relationship between self-reported usual adult intake of soy isoflavones which was determined from a food frequency questionnaire and levels of plasma isoflavones (genistein and daidzein) and isoflavone metabolites (equol, dihydrogenistein and dihydrodaidzein) from a randomly timed blood specimen. In analyses conducted in cases and controls separately, levels of plasma genistein, daidzein and total isoflavones increased with increasing levels of self-reported intake of soy isoflavones. Breast cancer cases and control subjects did not differ in their respective associations between total plasma isoflavone levels and self-reported intake (P = 0.48). Among all subjects, there was a 3-fold difference in geometric mean plasma levels of total isoflavones [81.8 (95% CI = 53.4, 125.1) versus 26.4 nmol/l (95% CI = 16.6, 41.8)] between women in the highest quartile of soy isoflavone intake (>12.68 mg isoflavones/1000 kcal) compared with those in the lowest quartile of intake (<=1.79 mg isoflavones/1000 kcal), a difference that was statistically significant (P = 0.002). The present study provides independent corroboration that breast cancer cases and control subjects can reliably recall their usual soy intake and that there is no evidence of selective recall biases between breast cancer cases and controls. These results further strengthen our previous observation of an inverse association between soy intake and breast cancer risk in the Los Angeles Asian Breast Cancer Study.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In 1991, Lee and colleagues (1) first reported a reduced risk of breast cancer in association with soy intake among premenopausal Chinese women in Singapore. Since then, at least a dozen epidemiological studies have investigated the association between self-reported soy intake and breast cancer risk and the results are mixed (2). Two recent case–control studies, in populations with substantial soy intake, were conducted in Shanghai, China (3) and among Asian-Americans in Los Angeles County, California (4), and provided support for a significant inverse relationship between soy intake and breast cancer risk. A newly published prospective study from Japan further strengthens the overall evidence that soy intake may have protective effects against breast cancer (5).

Recall bias, particularly in the assessment of diet prior to cancer diagnosis, remains a concern in case–control studies (6). In studies of healthy participants in the Singapore Chinese Health Study (7), control subjects from a case–control study of breast cancer in Shanghai, China (8) and healthy women in Hawaii (9), urinary excretion of isoflavones correlated significantly with self-reported soy intake. Plasma levels of isoflavones also correlated with self-reported soy intake among female participants selected from the Oxford arm of the European Prospective Investigation into Cancer and Nutrition (10). While these results are reassuring that questionnaire assessment of soy intake provides a reasonable estimate of intake when compared with a biomarker of soy exposure, it is not known whether subjects with cancer (cases) and those without cancer (controls) are comparable in terms of their respective self-report of soy intake in relation to a biomarker (either blood- or urine-based). We now report the relationships between plasma levels of genistein, daidzein and several isoflavone metabolites determined in blood specimens collected at interview and self-reported intake of soy isoflavone among case and control participants in a multi-ethnic (Chinese, Japanese and Filipino) case–control study of breast cancer (the Los Angeles Asian Breast Cancer Study) (4). In addition, we examined whether blood isoflavone levels vary by Asian ethnicity among women of similar ages and self-reported intake levels of soy isoflavone. We included in this investigation blood measurements of genistein and daidzein, the parent isoflavone compounds, as well as the main metabolites of genistein (dihydrogenistein) and daidzein (equol and dihyrodaidzein) (11).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study participants and specimen collection
Between 1995 and 1998, we conducted a population-based case–control study of breast cancer among Asian-American (Chinese, Filipino and Japanese) women in Los Angeles County, California (4). In brief, the study included women between the ages of 25 and 74 years who were newly diagnosed with breast cancer. Women in the control group were selected from the neighborhood of residence of breast cancer cases and they were frequency matched to cases on age and Asian ethnicity. All participants were interviewed in person using a standardized, structured questionnaire that covered demographic characteristics and migration history, menstrual and reproductive history, body size, physical activity and usual adult diet. Dietary intake during the year prior to cancer diagnosis (for cases) and interview (for controls) was characterized. Total soy intake was estimated based on the intake pattern of 14 foods that are rich in soy [miso soup or soups with tofu, tofu with pork or beef, tofu with chicken, fish or shellfish, tofu and vegetables (no meat), fresh green soy beans, dried soy beans, natto, fresh tofu, fried tofu, dried or pressed tofu, Chinese vegetarian meats, western vegetarian meats, soymilk and soy bean desserts]. The questionnaire asked for frequencies of intake and the usual amounts consumed. Using the Hawaii Food Composition Database (12), total intake of isoflavones (mg/day) was estimated based on total combined levels of daidzein, genistein and glycitein measured in these soy foods. Quartile cut points for isoflavone intake were selected based on intake of isoflavones among all control women. Isoflavone density expressed as intake per 1000 kcal was used and the quartile cut points were <=1.79, >1.79–6.24, >6.24–12.68 and >12.68 mg/1000 kcal (4).

Study participants were asked to donate a blood specimen at the completion of the interview. From the first 167 breast cancer cases (47 Chinese, 65 Japanese and 55 Filipino) and 224 control subjects (72 Chinese, 77 Japanese and 75 Filipino) who donated blood specimens, we selected a subset of 97 cases and 97 controls as subjects in the present study. By design, roughly 40% of study subjects were in the two extreme quartile categories of soy intake within each of the race and case/control groups (Table I).


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Table I. Self-reported intake of soy isoflavones (mg/1000 kcal) among breast cancer cases and controls included in this analysis

 
Laboratory assays of plasma isoflavones
Plasma levels of total soy isoflavones and their metabolites (i.e. after enzymatic deconjugation) were quantified using a validated isotope dilution electrospray tandem mass spectrometric method (13). Soy isoflavones were quantified using a high throughput method based on liquid chromatography with electrospray tandem mass spectrometry (13). The limit of quantification for all analytes was ~1 nmol/l, and the intra- and inter-assay precision was 3–8%.

Statistical analyses
We used analysis of variance (ANOVA) and analysis of covariance (ANCOVA) to assess the relationships between level of self-reported soy isoflavone intake and plasma levels of genistein and daidzein separately and combined (referred to as parent isoflavones). These analyses were repeated for total isoflavones (sum of genistein, daidzein, equol, dihydrodaidzein and dihydrogenistein). Statistical analysis was performed on logarithmically transformed plasma values and geometric mean plasma levels [and 95% confidence intervals (CI)] are presented. Mean plasma levels of parent and total isoflavones were estimated for the three categories of adult soy intake (quartile 1, quartiles 2 and 3 combined and quartile 4) after adjusting for age (continuous) and race (Chinese, Japanese and Filipino) in cases and controls separately. We investigated whether the relationship between level of soy intake and plasma levels differed between cases and controls (Table III). We tested for linear trends across the categorical levels (0–2; quartile 1 = 0, quartiles 2 + 3 = 1, quartile 4 = 2) of the variables of interest. Since there were no significant case–control differences in the associations between self-reported intake versus plasma levels, cases and controls were combined in subsequent analyses. In addition, we investigated whether mean plasma isoflavone levels differed between Chinese, Filipino and Japanese women after adjustment for age and self-reported soy isoflavone intake. All P values presented are two-tailed and statistical significance was defined as P < 0.05. Calculations were performed using the SAS statistical software system (SAS Institute, Cary, NC).


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Table III. Age- and race-adjusted geometric mean plasma levels of isoflavones (nmol/l) by intake of soy isoflavones in breast cancer cases and control subjects separately and all subjects combined

 

    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Analyses of plasma isoflavones were conducted on samples from 97 controls and 97 cases. By design, roughly 40% of study subjects were in the two extreme quartile categories of soy intake within each of the race and case/control groups (Table I). The daily mean soy isoflavone intakes among cases (13.5 mg/1000 kcal) and controls (13.3 mg/1000 kcal) selected in this analysis were very similar. Soy intake was also similar for cases and controls within each quartile of self-reported soy intake, although intake among cases in the fourth quartile was slightly (and non-significantly) higher than for controls in this quartile (Table I). Table II shows the distribution of blood daidzein, genistein and various isoflavone metabolites in breast cancer cases and control subjects.


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Table II. Blood levels of blood isoflavones (nmol/l) in breast cancer cases and control subjects

 
Table III shows the geometric mean plasma levels of isoflavones among cases and controls by self-reported dietary soy intake. Among breast cancer cases, plasma levels of genistein alone (P trend = 0.17) and in combination with dihydrogenistein (P = 0.04), daidzein alone (P trend = 0.0002) and in combination with dihydrodaidzen and equol (P = 0.0003), genistein and daidzein combined (P trend = 0.01) and total isoflavones (P trend = 0.006) increased with increasing self-reported dietary intake of soy isoflavone. Similar patterns were observed among control subjects, although the associations were statistically significant only for genistein alone (P trend = 0.01) and in combination with dihydrogenistein (P = 0.04). Cases and controls did not differ significantly in any of the associations between specific plasma isoflavones or its metabolites and self-reported intake of total soy isoflavone. In all subjects combined, plasma levels of individual and total isoflavones were all statistically significantly related to total soy isoflavone intake (Table III).

Table IV shows mean plasma isoflavone levels in Chinese, Filipino and Japanese women after adjustment for age (continuous) and dietary soy isoflavone intake (continuous). Age did not significantly influence plasma isoflavone levels, but levels of the parent isoflavones (daidzein and genistein) and total isoflavones (daidzein, genistein, equol, dihydrodaidzein and dihydrogenistein) differed significantly by Asian ethnicity. Levels of parent isoflavones were highest in Chinese, intermediate in Japanese and lowest in Filipinos. The ethnic difference in parent isoflavone levels was due largely to differences in plasma genistein levels.


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Table IV. Adjusteda geometric mean (95% confidence interval) plasma isoflavone levels in Chinese, Japanese and Filipino women

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Before discussing our results, the main objectives of this study should be emphasized. The purpose of this study was not to perform a pharmacokinetic validation of the blood isoflavone assay as this has been done by others (1619). Our purpose was to use this already validated biomarker to evaluate the legitimacy of our food frequency questionnaire (FFQ) in assessing level of usual soy intake in our Los Angeles Asian Breast Cancer Study. To this end, we first evaluated the validity of our FFQ in assessing usual adult intake of soy by examining the correlation between dietary response and biomarker level based on a randomly timed single blood draw. Second, we investigated whether the blood–dietary soy associations differ between breast cancer cases and control subjects, since a persistent concern of case–control studies is the comparability of cases and controls to recall and report their lifestyle exposures, particularly because cases are asked to report on their experiences prior to cancer diagnosis.

To achieve the above stated objectives, we conducted a study using a subset of participants in the parent case–control study (4). We intentionally selected the cases and controls to be comparable in their usual adult soy intake (as estimated from the FFQ) and we oversampled subjects who were low or high soy consumers. The blood specimens were collected at the time of interview, i.e. after diagnosis for breast cancer cases.

In epidemiological studies of exposure (e.g. soy intake) and disease (e.g. breast cancer) associations, the availability of a biomarker as an independent, objective measure of the exposure of interest is appealing, especially in the case of dietary assessment. As shown in Table III, there is convincing evidence that our FFQ is valid in assessing usual adult intake of soy since blood levels of various soy isoflavones measured in a randomly timed blood draw increased significantly with increasing level of usual adult intake of soy. Importantly, case–control status did not significantly influence the association between plasma isoflavone levels and self-reported intake (Table III), providing evidence that cases and controls were comparable in their recall of soy intake and that possible recent dietary changes in subjects (especially among cancer cases) had minimal impact on the quality of dietary information in the parent case–control study. This study gives credibility and strengthens our findings of an inverse relationship between self-reported soy intake and breast cancer risk in Asian- American women (4).

Among non-soy consumers (i.e. quartile 1 of adult soy isoflavone intake) in this study, the geometric mean blood level of daidzein and genistein combined was 7 nmol/l, compatible with results found in other non-soy consumers in studies conducted in the US (14) and Finland (15). Among high soy consumers (i.e. quartile 4, mean daily isoflavone intake was 26.4 mg) in this study, the mean blood levels of daidzein and genistein combined (30 nmol/l) and total isoflavones (82 nmol/l) were modest, but not unlike levels that have been reported for subjects with similar self-reported soy isoflavone intake. In a British study, plasma daidzein levels were 5, 8, 39 and 132 nmol/l, while plasma genistein levels were 14, 17, 119 and 378 nmol/l for women whose respective soy isoflavone intakes were 0.2, 20.0, 17.1 and 48.9 mg/day, based on FFQs (the corresponding estimated isoflavone intakes based on 7 day food diaries were 0.6, 5.9, 19.3 and 33.7 mg/day) (10). Thus, Asian-American women in this study with a daily intake of 26.4 mg isoflavone displayed blood genistein and daidzein levels that were comparable to plasma levels of British women who consumed ~20 mg isoflavones/day. In a Japanese study, mean blood daidzein and genistein levels were 120 and 475 nmol/l, respectively, in correspondence with estimated intakes of 19 mg daidzein/day and 31 mg genistein/day (16). Given that peak plasma daidzein and genistein levels occur 6–8 h after ingestion of pure genistein or daidzein (17), soybean powder (18) or stable isotope-labeled analogs of daidzein and genistein (19), variations in plasma isoflavones are to be expected unless the specific time interval between last soy meal and blood draw is known and can be considered in the analysis. In addition, other factors, including the specific types of soy products consumed, host factors (e.g. intestinal bacteria), other components of the diet and use of medications (e.g. antibiotics), also influence the concentrations of isoflavones in blood/urine samples (17,23) (see below).

Some intriguing ethnic differences in plasma isoflavone levels, specifically genistein and dihydrogenistein levels, emerged in this study. Levels of genistein and genistein and daidzein combined were highest in Chinese, intermediate in Japanese and lowest in Filipinos. Combined levels of genistein and its metabolite dihydrogenistein were also highest in Chinese women. Ethnic differences in plasma genistein and dihydrogenistein levels may be related at least partly to differences in the specific types of soy products consumed and host differences in the metabolism of these compounds by intestinal microflora. In fact, data from our Los Angeles Asian Breast Cancer Study show that sources of soy isoflavones differed considerably between the three Asian groups. Tofu eaten alone or in mixed dishes was the predominant source of soy isoflavones, accounting for 74, 57 and 54% of the intake in Japanese, Filipino and Chinese women, respectively. Soy milk accounted for 20% of the soy isoflavone intake among Chinese, but was not commonly consumed by Japanese (5%) or Filipino (6%) women. Miso represented 13% of the soy isoflavone intake among Japanese, but only 7% of the intake in Filipinos and 6% in Chinese. Other soy products, including fried, dried or pressed tofu and vegetarian meats, accounted for 29% of the soy isoflavone consumed by Filipinos, 20% by Chinese and 8% by Japanese. Previous studies conducted in Finland (20), Australia (21) and the USA (22) determined urinary levels of dihydrogenistein and dihydrodaidzen after known amounts of soy bar, soy flour and soy milk, respectively, were consumed. While urinary dihydrogenistein was found to be the major metabolite of genistein and dihydrodaidzen was the major metabolite of daidzein in the Finnish study (20), these metabolites were less important in the other two studies (21,22). Differences in these results suggest that isoflavone metabolism may differ between countries and/or racial/ethnic groups because of differences in intestinal microflora. In this study, 49% of controls had measurable levels of plasma equol compared with 39% of cases; the difference was not statistically significant. Most previous studies reported that ~30–50% of the adult population do not excrete equol in urine when challenged daily with soy foods (23,24). Given that equol is not produced in all subjects in response to dietary challenge with soy or daidzein, it is not surprising that plasma equol was not significantly associated with self-reported soy food intake in this study.

In conclusion, in this study we found that self-reported usual adult intake of soy isoflavones is significantly associated with plasma levels of isoflavones. Importantly, both breast cancer cases and controls can reliably recall their usual soy intake and there is no evidence of selective recall biases between breast cancer cases and controls. These results strengthen our previous observations of an inverse association between soy intake and breast cancer in the Los Angeles Asian Breast Cancer Study.


    Notes
 
2 To whom correspondence should be addressed at: University of Southern California/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, MC 9175, Los Angeles, CA 90089-9175, USA Email: annawu{at}hsc.usc.edu Back


    Acknowledgments
 
We are grateful to all the study participants for their contributions and support. We thank the entire data collection team, especially Betty DeBorja, Annie Fung, Diem Tran, Lydia Tran and June Yashiki. This work was supported by grants (1RB-0287 and 3PB-0102) from the California Breast Cancer Research Program. Incident breast cancer cases for this study were collected by the USC Cancer Surveillance Program (CSP), which is supported under subcontract by the California Department of Health. The CSP is also part of the National Cancer Institute's Division of Cancer Prevention and Control Surveillance, Epidemiology, and End Results Program, under contract no. N01CN25403.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 1, 2003; revised September 3, 2003; accepted September 25, 2003.