UT\lIVERSllY OF HAWAJllIBRARV

Transcription

1 UT\lIVERSllY OF HAWAJllIBRARV ASSESSMENT OF URINARY ISOFLAVONES AMONG PREMENOPAUSAL WOMEN A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN NUTRITIONAL SCIENCES DECEMBER 2003 By Linda M. Yeh Thesis Committee: Gertraud Maskarinec, Chairperson Adrian Franke Michael Dunn

2 iii ACKNO~EDGEMENTS I would like to acknowledge Dr. Gertraud Maskarinec, Dr. Adrian Franke, and Dr. Michael Dunn for their expertise, advice, and guidance in my research, laurie Custer for her technical expertise in HPlC and the creatinine assay, Caryn Oshiro and Sandra Hebshi for their assistance in subject recruitment, and Andrew Williams for statistical assistance.

3 ABSTRACT iv Epidemiological and clinical studies researching the effects of soy food intake require a high compliance to a soy diet protocol. Measuring isoflavones in the bodily fluids is the most objective method in determining dietary compliance because isoflavones are highly and positively correlated with soy food intake and specific to soy foods. Urine analysis is preferred over plasma due to its noninvasiveness, which aids in compliance. Our study investigated the accuracy of collecting weekly and single day samples from 19 premenopausal women on a daily soy diet protocol in comparison to monthly samples for determining dietary compliance. We compared urinary isoflavone excretion rates (UIER) of weekly samples and single day samples to the UIER of a monthly sample that consisted of urine collected daily. Correlations were high between all samples and the monthly UIER. The correlations of the mean of all weeks UIER and single day UIER with the monthly UIER were 0.96 and 0.89, respectively. No large differences were seen when samples were stratified by ethnicity, 8MI, and equol excretor status. The small degree of increased accuracy in determining dietary compliance in measuring monthly UIER compared to weekly does not justify the extra time and effort required by the subjects and staff. Therefore, we conclude that analyzing weekly UIER is an accurate and feasible method of determining dietary compliance with a soy-based diet.

4 v TABLE OF CONTENTS Acknowledgements, iii Abstract.,,.iv List of Tables vii L IS t 0 f F' Igures VIII... Chapter 1: Introduction 1 Significance 1 Background of Isoflavones 1 Structure of Isoflavones 2 Dietary Sources and Concentration of Isoflavones 3 Isoflavone Composition of Soy Foods.4 Isoflavones and Disease 5 Cardiovascular Disease 6 Osteoporosis 7 Prostate Cancer 7 Breast Cancer 9 Mechanisms of Isoflavones in Breast Cancer Development.. 10 Hormone-Related Mechanisms 10 Other Mechanisms 12 Mechanisms of Genistein 14 Metabolism of Isoflavones 15 The Metabolite Equol 17 Equol Production 18 Isoflavones in the Bodily Fluids 19 Isoflavone Levels in Plasma 19 Bioavailability of Isoflavones 20 Half-Life of Isoflavones.'" 21 Excretion of Isoflavones 21 Dietary Assessment 22 Traditional Methods 22 Biochemical Methods 23 Analysis of Isoflavones in Bodily Fluids 24 Urine Analysis 25 Purpose 27 Chapter 2: Methods 29 Participants 29 Recruitment 30 Urine Collection 31 Chemicals 32 Apparatus 33 Creatinine Assay 33 Pooling of Samples 34 Enzymatic Hydrolysis and Extraction of Isoflavones From Human Urine 34

5 HPLC Analysis of Isoflavones 35 Data Analysis 35 Chapter 3: Results 37 Subject Characteristics 37 Monthly UIER 39 Mean UIER of Each Isoflavone 39 Weekly UIER Compared to the Monthly UIER.40 Single Day UIER Compared to the Monthly UIER. 43 Single Day UIER Compared to Weekly UIER.45 Stratifications of UIER Comparisons.48 Stratified by Ethnicity.48 Stratified by BMI Status 48 Stratified by Equol Excretor Status.48 Chapter 4: Discussion 53 Summary 53 Comparison With Past Research 54 Strengths of This Study 55 Limitations to This Study 55 Implications 56 Applications 59 Conclusion 62 References 64 vi

6 LIST OF TABLES vii 1. Serving Sizes and Isoflavone Content of Soy Foods Used in the Breast, Estrogens, and Nutrition Study Daily Nutrient Intake and Demographic Information of 19 Women Means, Standard Deviations, and Ranges of UIER of Totallsoflavones and the Individuallsoflavones, Percent Differences in Means and Medians, and Correlation Comparisons Between Weekly and Single Day UIER to Monthly UIER., '", Correlation Comparisons of UIER From Single Day Samples to the UIER of the Week They Were Obtained From and Weekly UIER. '" Means, Standard Deviations, and Ranges of UIER of Totallsoflavones Stratified by Ethnicity, BMI, and Equol Excretor Status, Percent Differences in Means and Medians, and Correlation Comparisons Between Weekly and Single Day UIER to Monthly UIER Stratified by Ethnicity, BMI, and Equol Excretor Status '" Mean Monthly Daidzein, ODMA, and Equol Excretion Rates of Equol and Non-Equol Excretors 52

7 LIST OF FIGURES viii Figure 1. The Aglycone and Conjugate Forms of Genistein 2 2. Structures of the Primary Isoflavones and 17J3-Estradiol Proposed Metabolic Pathways of Daidzin and Genistin Catabolism by Gastrointestinal Bacteria Mean Urinary Isoflavone Excretion Rates (UIER) of Each Isoflavone Over a Time Period of 3 Weeks in Comparison to Their Mean Total Monthly UIER and Mean Total Single Day UIER Correlations of Total Urinary Isoflavone Excretion Rates (UIER) From Weeks 1, 2, and 3 and Mean ofall Weeks Combined to Total Monthly U/ER Correlation of Total Urinary Isoflavone Excretion Rates (UIER) From Single Day Samples to Total Monthly UIER, and to the Week the Single Sample Was Obtained From Correlations Between Total Urinary Isoflavone Excretion Rates (UIER) From Single Day Samples and Total UIER From Weeks 1, 2, and 3, and Mean of All Weeks Combined 47

8 1 CHAPTER 1: INTRODUCTION SIGNIFICANCE Epidemiological and clinical studies investigating the effects of soy food intake on disease risk require high compliance to the soy food protocol, which needs to be evaluated. Various methods can be used to assess dietary compliance, such as maintaining food records, using 24-hour recalls, or food frequency questionnaires. However, these methods have limitations and are subjective. Food records require literacy and a high degree of subject cooperation. 24-hour recalls rely on memory and are often not representative of the usual diet. Food frequency questionnaires may not include all foods in the usual diet. More objective methods are preferred for measuring dietary compliance in studies investigating soy intake and disease risk. Analyzing urinary isoflavones is a valid method for diet assessment because dietary isoflavones are found primarily in soy foods and have been shown to be highly correlated to soy intake. Moreover, isoflavones are readily excreted in the urine and have a short half-life. BACKGROUND OF ISOFLAVONES Isoflavones are naturally-occurring plant chemicals found in legumes, but are especially high in soy foods (1, 2). They are classified as phytoestrogens

9 2 due to their similarity in structure to estrogens (Figure 1) and their weakly estrogenic or anti-estrogenic properties (3, 4). There are 12 isoflavone derivatives (4, 5) found in soy foods, which occur as aglycones and conjugates. The primary derivatives are the J3-glucoside conjugates (genistin and daidzin) and the malonylglucosides (6"-O-malonylgenistin and 6"-O-malonyldaidzin), with the aglycones (genistein and daidzein) and acetylglucosides (6'-O-acetylgenistin and 6'-O-acetyldaidzin) being the other less common forms (5, 6, 7) (Figure 1). aglycon Figure 1. The aglycone and conjugate forms of genistein Structure of Isoflavones Isoflavones are heterocyclic phenols, possessing the structural features of flavonoid compounds. The benzene ring is linked to the 3-position of the chroman ring (3-phenyl benzopyran), while for flavonoids it is linked to the 2 position (2-phenyl benzopyran) (1, 3, 8) (Figure 2). Additionally, isoflavones are structurally similar to the hormone estradiol. The presence of 2 phenolic hydroxyl

10 groups with a spatial distribution similar to the 2 hydroxyl groups in estradiol is a key structural feature that enables them to bind to estrogen receptors (9). 3 Daidzein Genistein HO HO 17r3-Estradiol OH HO Figure 2. Structures of the primary isoflavones and 1713-estradiol Dietary Sources and Concentration of Isoflavones Isoflavones are found in legumes, but primarily in soybeans and their products. The hypocotyl portion of the soybean seed contains the highest amounts of isoflavones, followed by the cotyledon and hull (10). Soybeans contain approximately 1-4 mg/g protein (5, 11, 12). Other legumes were found to contain little to undetectable amounts of isoflavones (2, 13, 14). Soy products will have varying isoflavone content depending on the soybeans they were made from. Most soy foods contain approximately

11 4 mg/g protein oftotal isoflavones (2,5,6, 15, 16). Ofthe unfermented soy foods, soybeans and tofu contain the highest concentration of isoflavones. Miso and tempeh have the highest levels ofisoflavones amongst the fermented soy foods (5, 16). Second-generation soy foods (i.e., soy hot dogs, tempeh burgers, and soy cheese) have low amounts of isoflavones because soy is not the main ingredient. Soy is used in these foods primarily to replace animal protein and/or reduce fat. However, the food needs to maintain the characteristics of the original food, thus, the amount of soy that can be incorporated into the food is limited (6). Isoflavone concentration varies between different sources of soy (5, 8, 17). Concentrations also depends on factors such as geographic region of the soybean crop (11, 18), time of harvest (11, 18), and seed maturity (2). Isoflavone concentration increases with seed maturation (19), which has been seen to be highest when soybeans are grown in lower temperatures during seed development (18, 20). Moreover, concentration also depends on genetics (11, 18) and processing conditions of the soybeans (10, 11, 18,20,21). Isoflavone Composition of Soy Foods The primary isoflavone forms found in soybeans and soy foods are the ~ glucosides of genistein and daidzein and their 6"-O-malonyl derivatives (6"-0 malonylgenistin and 6"-D-malonyldaidzin), which account for 83-93% of total isoflavone content in soybeans (11). The remaining consist of acetylglucosides (6'-O-acetylgenistin and 6'-0-acetyldaidzin) and aglycones (Figure 2). Glycitein

12 5 (7,4'-dihydroxy-6-methoxyisoflavone) and its glucoside glycitin are also present in low amounts of approximately 5-10% of total isoflavones (11,22). One study found the average ratio of daidzein:genistein:glycitein in soy foods to be approximately 1:1:0.2 (16). The aglycone forms, genistein (4',5,7 trihydroxyisoflavone) and daidzein (4',7-dihydroxyisoflavone) are mainly found in the highly fermented soy foods (i.e., miso and soy sauce). The type of soy product determines the isoflavone composition. The malonyl derivatives are less stable in heat than the glucosides and, therefore, are converted to the more stable ~-glucosides. Soymilks that undergo longer thermal processing treatment have higher amounts of glucosides and lower levels of the malonyl derivatives than other soymilks that are treated for a shorter amount of time. Similarly, tofu that undergoes greater heat processing has higher concentrations ofglucosides than malonyl derivatives (5). In contrast, fermented foods such as miso, tempeh, and bean paste contain mainly aglycones due to the action of the native glucosidases of the fermentation organisms (5, 6). 1S0FLAVONES AND DISEASE Isoflavones are considered weakly estrogenic when bound to the estrogen receptor, possessing approximately 1 x 10-5 to 10-3 ofthe activity of 1713-estradiol on a molar basis (23,24). In several studies, plasma isoflavone levels after soy consumption have reached high nanomolar to low micromolar

13 6 concentrations (25, 26, 27, 28, 29). Plasma estrogen throughout life ranges from approximately 10 pg/ml to 10,000 pg/ml while plasma isoflavone levels after soy consumption have been reported to range from 100,000 pg/ml to 500,000 pg/ml (9). Although the activity of isoflavones is weak, their high plasma levels have the potential to exert physiological effects in humans consuming soy foods (1, 4). As a result, researchers have been investigating the role of these compounds in various diseases such as cardiovascular disease, osteoporosis, prostate and breast cancer, and in treating menopausal symptoms. Cardiovascular Disease Cardiovascular disease is the leading cause of death of men and women in the U.S. In 1998, black males had the highest death rate and Asians and Pacific Islanders had one of the lowest (30). Isoflavones have been shown to exert a hypocholesterolemic effect, which decreases the overall risk of cardiovascular disease. A strong inverse relationship was found between serum cholesterol and daily intake of soy (32). Also, a meta-analysis of 38 clinical studies concluded that the mean total serum cholesterol was reduced by 9.3% and serum LDL-cholesterol decreased 12.9% with a mean intake of 47g of soy protein (33). Several other studies also found significant decreases in hypercholesterolemic or hyperlipidemic subjects for

14 7 serum cholesterol (34, 35), serum LDL-cholesterol (34, 35), and plasma homocysteine levels (34). But these effects were not seen in normolipidemic subjects in another study (36). Isoflavones may also reduce cardiovascular disease risk through inhibition of platelet aggregation (37) and decreasing lipid oxidation (38). Osteoporosis Osteoporosis affects primarily women with Caucasian and Asian ancestry. Asian women in Japan tend to consume more soy and the incidence of osteoporosis appears to be significantly lower in postmenopausal Japanese women than in Western countries, suggesting that soy food intake could be involved (9). Bone-conserving effects of isoflavones have been seen with intakes of soy protein. Isoflavones have a high affinity for the estrogen receptor ~ (ER~) SUbtype, which is found in bone, and can down-regulate osteoclast activity, thereby decreasing bone resorption (15). Animal studies have shown that soy decreases bone loss in ovariectomized rats (39, 40). However, several other studies did not see any changes in bone mineral density in postmenopausal women (41, 42) or in women aged 21 to 25 (43). Prostate Cancer Prostate cancer is the most commonly diagnosed cancer among men in the U.S. The incidence rate from in the U.S. was 172.8/10 4 with 60%

15 8 of new cases in men 70 years of age and older. Blacks in the U.S. have the highest rate of prostate cancer in the world, while Asians and Pacific Islanders had the lowest (44). Prostate cancer incidence rates are also lower in soy-consuming countries of Asia than in the U.S. This fact and that prostate cancer is responsive to estrogen therapy (1, 15) has led to the possibility of isoflavones playing a role in decreasing prostate cancer risk. Isoflavones have been observed to be more concentrated in the prostatic fluid relative to plasma concentrations, with concentrations being highest in men from soyfood-consuming countries (45, 46). Genistein has also been seen in vitro to inhibit growth of androgen-dependent and androgen-independent prostate cancer cells (47, 48) and in vivo to downregulate sex steroid receptor expression and increase cell differentiation in the prostate (49). These all lead to decreased prostate cancer risk by decreasing the uncontrolled cell growth of cancer cells derived from gene mutations. Moreover, genistein inhibits 5-a-reductase and 17-p-hydroxysteroid dehydrogenase, which are enzymes involved in androgen and estrogen synthesis (50, 51). These effects decrease the amount of hormones available to bind to receptors located in the prostate, which decreases the stimulatory effect of hormones on cell proliferation. This is especially important when cancer cells have developed because the stimulation of cancerous cells is undesirable.

16 9 Breast Cancer Breast cancer is the most common cancer among women in the United States (44). In , the incidence rate among females in the U.S. was 137.1/10 4, with 77% of new cases occurring in women aged 50 and older. As of January 2000, Caucasians had the highest risk of breast cancer in the U.S., while Asian/Pacific Islanders had the lowest (52). Several studies have shown increased breast cancer risk among Asian women after immigration to the U.S. from Asia. One study showed a decreased risk when immigration occurred later in life compared to earlier, and Asian Americans born in the U.S. had a 60% greater risk than the non-u.s. born (53). Another study found increasing risk with the number of years of residence in the U.S. (54). Also, low tofu intake was found to be associated with increased years of residence in the U.S. and increased breast cancer risk (55). These studies indicate an environmental factor, such as diet, may be involved in breast cancer development. The high intake of soy and lower rates of breast cancer in Japan and China have suggested a protective effect of isoftavones (3, 56, 57). A longer exposure to steroidal estrogen increases breast cancer risk because of the increased rate of cell proliferation, which can lead to cancer development (58). This is the case with early age at onset of menarche (before age 12), late age at onset of menopause (after age 55), first full term pregnancy after age 30, nulliparity, or exposure to hormone replacement therapy for long periods of time

17 10 (44). Isoflavones, particularly genistein, may decrease breast cancer risk because of its ability to enhance mammary gland differentiation leading to decreased number of target cells susceptible to cancer development, which has prompted numerous studies to investigate the role of isoflavones in breast cancer development (49, 59). Two studies found that regular soy food consumption may be associated with decreased breast cancer risk (60, 61). One study showed a 30% decreased risk among women who reported eating tofu more than once a week (55). Furthermore, two studies showed that soy intake during adolescence was inversely related to breast cancer risk (54, 62), which suggests a protective effect of early life exposure (54). High total isoflavone excretion in urine, which is highly positively associated with consumption of soy foods, was also shown to be associated with decreased risk (63, 64, 65). Other studies reported decreased estrogen levels with soy intake, which may also reduce breast cancer risk (66, 67). MECHANISMS OF ISOFLAVONES IN BREAST CANCER DEVELOPMENT Hormone-Related Mechanisms There have been various mechanisms proposed to explain the chemoprotective effects of isoflavones including hormone-related mechanisms. Isoflavones have been proposed to act as anti-estrogens by competing with

18 11 endogenous estrogen for binding to estrogen receptors due to the structural similarities between estrogen and isoflavones. There are two known estrogen receptor subtypes, estrogen receptor-a (ERa) and estrogen receptor-13 (ERI3). ERa is found in the kidney, testes, breast, uterus, and ovary, while ERI3 is found in the brain, bone, bladder, lung, prostate, ovary, uterus, breast, and vascular epithelia (15). At puberty, estrogen stimulates a variety of female-specific changes. But long-term exposure of certain tissues, such as the breast, to estrogen over the lifetime can lead to cancer development due to estrogen's stimulating effect on cell proliferation. Estrogen elicits its physiological effects by binding to its receptors. When estrogens bind to their receptors, a conformational change in the binding cavity is induced. The new conformation allows coregulator proteins to bind to the estrogen receptor complex, which then activates gene transcription of proteins. Coregulators are required to bind to the complex in order to initiate gene transcription. When gene mutations occur that lead to cancer cell development, transactivation by estrogen is undesirable because estrogen will stimulate uncontrolled cell growth of cancer cells. However, other ligands, such as isoflavones, can bind to estrogen receptors and elicit estrogenic or anti-estrogenic responses. Each isoflavone has a different affinity for the estrogen receptors. Genistein has been observed to bind with similar affinity as 1713-estradiol. Genistein, daidzein and equol have

19 12 a stronger affinity to bind to estrogen receptors than OOMA, while glycitein appears to be non-estrogenic (5, 68, 69, 70). Additionally, isoflavones were seen to be effective at triggering transactivation in ERI3 (71). Genistein and equol have been seen to have greater affinity for ERI3 than ERa (71, 72, 73). Genistein bound to ERp induces ERI3's binding cavity to adopt an orientation similar to that induced by estrogen receptor antagonists, which may block estrogen coregulators from binding, in addition to being stronger at triggering transactivational repression than activation (71, 74). 17p-estradiol binds equally to both estrogen receptor SUbtypes and, therefore, is able to activate a wide variety beneficial and adverse responses. Since isoflavones are potent at specifically stimulating ERI3-mediated transactivational pathways, this explains its tissue-specific effects, especially in the breast. Other Mechanisms IsofJavones have also been shown in vitro to inhibit the activity ofvarious enzymes involved in estrogen metabolism. Inhibition of 1713-hydroxysteroid oxidoreductase (1713-HSOR) was seen to alter the availability of the more active form of endogenous estrogen, 1713-estradiol, by preventing the 1713 oxidoreduction ofthe weaker form, estrone (51). Thus, less 1713-estradiol is formed, resulting in less available to bind to the estrogen receptors and less stimulatory responses that may lead to cancer development.

20 13 One study showed isoflavones to increase urinary levels of 2 hydroxyestrone, which is thought to be an anticarcinogenic metabolite of 17Pestradiol. This suggests isoflavones may affect the enzymes involved in the 2 hydroxylation of estrogen (75). Increasing enzyme activity for 2-hydroxylation may decrease formation of the other metabolites, 4- and 16a-hydroxylated forms, which may increase cancer development. Also, 2-hydroxylated estrogens can be further metabolized to 2-methoxy estrogens, which are thought to be antiangiogenic (75). Isoflavones have also been seen to weakly inhibit aromatase (CYP 19), which converts androgen to 17p-estradiol, thereby decreasing circulating endogenous 17p-estradiollevels (76, 77) and, thus, less available to bind estrogen receptors. Additionally, isoflavones have been seen in vitro to inhibit proliferation (78, 79,80,81,82,83, 84, 85), decrease metastasis (81,86), stimulate apoptosis (80, 81, 84, 87, 88), and function as an antioxidant (89, 90, 91). Inhibition of angiogenesis by isoflavones has also been shown in vitro and in vivo (87, 88, 92). Several studies also observed isoflavones in vivo and in vitro to increase sex hormone-binding globulin (SHBG), a plasma transport protein of estradiol. SHBG binds estradiol, making the bound estradiol unavailable for tissue uptake. Therefore, this may contribute to less circulating free estradiol available for tissue uptake and estrogen receptor binding (93, 94, 95). However, other studies did not see any effects on SHBG levels (96, 97, 98).

21 14 Mechanisms of Genistein Genistein, specifically, has been shown in vitro to inhibit several enzymes, such as DNA topoisomerases I and II (99, 100) and tyrosine protein kinases (101), suggesting its effects are not mediated by estrogen receptors. Inhibiting protein tyrosine kinases possibly ceases cell proliferation and initiates the differentiation pathway by altering protein phosphorylation. It has also been show to halt cell cycle progression of cancer cells by interfering with signal transduction pathways (102,103). In one study, genistein inhibited cell growth in vitro by stimulating the transforming growth factor-i3-1-signaling pathway, causing inhibition of the cell cycle (104). Moreover, it was observed in vivo to promote cell differentiation in the mammary gland and to decrease epidermal growth factor receptor expression in adulthood with pre-pubertal genistein exposure (49, 59). Genistein increased the number of lobules in the mammary gland, which are the most differentiated epithelial cells and are less susceptible to carcinogens. However, genistein has been reported to exhibit a biphasic effect in vitro. At concentrations less than 10 Ilmolll, cell growth of estrogen-dependent breast cell lines was seen to be stimulated by genistein (85, 105, 106). At higher concentrations of 10 to 50 ",molll, it was observed to inhibit cell growth (82, 105, 107). Also, the combination of estrogen and genistein exerted no additional proliferation (85), and caused no reduction in 1713-estradiol-stimulated cell proliferation (85,106,107). Furthermore, the addition oftamoxifen, an anti-

22 15 estrogen. to genistein reversed Tamoxifen's inhibitory effects on cell proliferation in vitro (108) and in vivo (109). In contrast, genistein acted synergistically in vitro with Tamoxifen (110). METABOLISM OF ISOFLAVONES After consumption of soy. the daidzein, genistein, and glycitein glucoconjugates are hydrolyzed by brush border membrane and bacterial deconjugating enzymes, ~-glucosidases and arylsulfatases, located along the entire length of the intestinal tract (111, 149). The enzymes cleave the glycosidic bonds to release the aglycones, daidzein, genistein, and glycitein ( ) (Figure 2). They can then be absorbed from the intestinal lumen, likely by passive diffusion, or are further metabolized and degraded by intestinal microflora, presumably in the colon (21, 25, 114, 115). Aglycones are readily absorbed in the small intestine due to its small molecular weight and lower polarity compared to the conjugated form (25). Thus, isoflavones are absorbed faster when consumed as aglycones in fermented soy foods, such as miso, nalto, and soy sauce. Conjugated isoflavones that escape hydrolysis or unabsorbed aglycones are further subjected to bacterial metabolism and degradation in the colon. Genistein has been proposed to be degraded by bacteria to dihydrogenislein and 6' -hydroxy-o-demethylangolensin (6'-OH-ODMA). Daidzein is believed to produce two metabolites, 7-hydroxy-3-(4'-hydroxyphenyl)-chroman (equol), and

23 16 by Coring cleavage, o-demethylangolensin (OOMA) (116). Equol's intermediates are believed to be dehydroequol (Intermediate-E) or dihydrodaidzein, and tetrahydrodaidzein. OOMA's intermediates are likely dihydrodaidzein (lntermediate-o) and 2-dehydro-o-demethylangolensin (2-dehydro-OOMA) (69, 115) (Figure 3). Further metabolism of glycitein appears limited due to the hindering position of its 6-methoxyl group (29, 114). Other minor metabolites are likely produced by the human liver or by other means. Some have been identified as hydroxylated oxidation products (151). / Dihydrodaidzein Daidzin Daidzein,,,, '" Dehydroequol ~ ~ 2-Dehydro-ODMA Tetrahydrodaidzein l ODMA Equol Genistin ~ Genistein ~ Dihydrogenistein ~ 6'-OH-ODMA Figure 3. Proposed metabolic pathways of daidzin and genistin catabolism by gastrointestinal bacteria

24 17 After absorption from the gut, most of the isoflavones are re-conjugated to glucuronates or sulfates within the enterocyte or the liver. Aglycones and conjugates can then circulate in the plasma and be distributed to the tissues. Approximately 46% of unconjugated genistein, 12% of unconjugated daidzein, and 50% of equol circulated unbound to plasma proteins (117, 118), and therefore available for tissue uptake. Alternatively, some of the isoflavones are released into the bile, go through enterohepatic circulation, and are biliarty excreted. The isoflavones are mostly excreted by the kidney in urine due to their polarity, with only small amounts excreted in the feces (113,115,119,120). THE METABOLITE EQUOl Equol is not classified as a phytoestrogen because it does not originate from plants, but exclusively as a product of intestinal bacterial metabolism (114). However, it does have estrogenic activity and affinity for estrogen receptors (69). It was seen to have similar binding affinity for Era and Er~ as genistein in vitro, but induced transcription more strongly (73) and may down-regulate the mrna expression for estrogen receptors (121). One study showed approximately half of equol in plasma circulates in the unbound form, which is greater than estradiol. This may enhance its potency because it is the unbound form that binds to estrogen receptors (117).

25 18 Eguol Production Equol producers have been observed to have a more favorable plasma estrogen pattern consistent with lowered breast cancer risk (94), thus, they may receive more protection than low equol producers. However, equol production has only been seen in approximately 30% of individuals consuming soy foods (115, 122). This is probably due to the variation of intestinal microflora between individuals (26, 115, 122). Other studies cite possible preferential pathways for daidzein metabolism because equol excretion was inversely related to ODMA and daidzein excretion (115, 123). In contrast, one study did not see an inverse relationship between equol and ODMA excretion (21). It has been proposed that equol production can be affected by the composition of the diet. Several studies have shown that a high carbohydrate environment can stimulate fermentation in the colon and increase the rate of conversion of daidzein to equol (9,15,124). One study found equol production to be associated with a higher intake of dietary fiber and carbohydrates (21). However, another study in which soy diets were supplemented with wheat bran concluded equol production was not strongly associated or altered by diet (122). Decreased equol production may be due to dietary fat decreasing the capacity of the gut microflora to produce equol, rather than carbohydrates or dietary fiber stimulating it (125).

26 19 ISOFLAVONES IN THE BODILY FLUIDS Isoflavones in the urine and plasma are found primarily as conjugates, with urine having higher glucuronide conjugates and plasma having more sulfate conjugates (126,127). Unconjugated forms account for approximately 1-3% of circulating isoflavones (29). Isoflavone Levels in Plasma Ingested aglycones peak in the plasma approximately 4 to 7 hours after soy intake and gluco-conjugates peak later at about 6 to 11 hours after intake (29, 128). The time lag seen between peak concentration levels of aglycones and conjugates is most likely due to the hydrolysis of the conjugates being a ratelimiting step in absorption. In contrast, no significant difference in peak time has been seen between aglycone and conjugate intake in one study (31). Sulfate conjugates have been seen to peak at 4 to 5 hours after intake (126, 127). Oaidzein and genistein appear in plasma as early as minutes after soy consumption and peak at approximately 6 to 8 hours after soy intake (22, 26, 28, 29, 126). Genistein tends to be higher than daidzein in the plasma (29, 111, 128, 129, 130), which is likely due to daidzein's greater distribution in the tissues compared to genistein (130), or due to the higher polarity of daidzein, which could promote its excretion in urine (111,129). Glycitein has been seen to peak in plasma at approximately 4 to 5 hours (29, 126), while equol and OOMA appear later in the plasma, in approximately 6

27 20 to 8 hours after soy intake (28, 29, 130, 131). This is due to the time required and the colonic origin for their formation. Bioavailabilitv of Isoflavones Bioavailability of algycones and conjugates in humans is very similar (29, 130,131) and genistein has been found to be more bioavailable than daidzein (130,131). In contrast, some studies claimed that the bioavailability of daidzein to be greater than genistein (111, 132). However, in those studies daidzein was incorrectly assumed to be more bioavailable because these results were based on urinary excretion, while bioavailability is restricted to measurement in the circulation. Since urinary isoflavones are not recovered completely, bioavailability must be measured from the plasma after oral and intravenous administration of pure isoflavones. Also, the bioavailability of daidzein and genistein has a curvilinear relationship with increasing levels of isoflavone intake. This may be caused by decreased absorption due to the bacterial enzymes in the intestine being overwhelmed by the higher amounts of isoflavones, resulting in hydrolysis of some, but not all, of the isoflavones. Therefore, only a portion of the isoflavones are hydrolyzed and absorbed, while the remaining isoflavones are likely excreted (130, 131).

28 21 Half-Life of Isoflavones The half-life of genistein and daidzein in plasma is approximately 5 to 8 hours (25, 26, 28, 127, 130), with daidzein reaching higher concentrations and having a slightly longer half-life (26). Sulfate conjugates have shorter half -lives of approximately 3 to 6 hours (126, 127). However, renal disease patients exhibit half-lives of up to 53 hours and 99 hours for daidzein and genistein, respectively (27). EXCRETION OF ISOFLAVONES Plasma and urinary isoflavone excretion is completed in approximately hours after soy intake (16, 29,130,131). The long half-life of isoflavones in renal disease patients shows the fact that renal clearance is a major route of isoflavone elimination from the body (27). The excretion patterns of daidzein and genistein are similar, but levels of daidzein have been seen to be higher in urine (26,28,111,70,126,129,130,133,134). Also, inter-individual variation in isoflavone excretion has been noted in several studies (25, 111, 115, 126, 135, 136) causing a wide variation in fractional recoveries of daidzein and genistein (131). The percent of ingested dose recovered in urine was seen to range from 12% to 50% for daidzein and 1% to 39% for genistein (25, 28, 112, 126, 130, 131). Additionally, approximately 1 5% of ingested isoflavones have been observed to be excreted in the feces as a

29 minor route for elimination (26, 28, 111). These low recovery rates are probably due to degradation of isoflavones to metabolites that have yet to be detected. 22 DIETARY ASSESSMENT Traditional Methods There are various methods for determining dietary compliance in nutritional studies, such as food records, 24-hour recalls, and food frequency questionnaires. Food records can provide information on usual intake and do not depend on memory, but require a high degree of cooperation, literacy, and analysis can be time consuming and expensive. 24-hour recalls are easier to administer and can result in faster results, lower respondent burden, and are cheaper than food records. However, one recall does not represent a person's usual intake. Because these rely on the participant's memory, they can result in under- or over-reporting. Food frequency questionnaires can be self-administered, are relatively inexpensive for large-scale studies, and can be tailored to the target population. But, they depend on the subject's ability to relate their diet to the questionnaire and may omit common foods or portion sizes consumed by the target population. Longer questionnaires can be more comprehensive, but may increase respondent burden.

30 23 Biochemical Methods More objective methods for measuring dietary compliance analyze the participant's blood or urine for a nutrient or its metabolite of interest. These methods can be used as an adjunct to the traditional techniques mentioned above to determine if subjects are under- or over-reporting their intakes or to validate their accuracy in estimating intake (137, 138). However, measuring bodily fluids for the intake of a specific food item is valid only ifthe nutrient or metabolite being measured is specific to the food being investigated. Nutrients such as vitamin C cannot be used as a biomarker for intake of a specific food item because it is also found in a variety of fruits and vegetables that may also be consumed by the subjects. Therefore, it would difficult to quantify the amount of carotenoids found in the body fluids that are strictly from one food item, which would consequently make it difficult to determine if subjects are adhering to the recommended diet. However, in contrast to isoflavones, carotenoids have a long half-life which makes them a more ideal biomarker because one measurement better reflects usual intake. Additionally, levels of the nutrient or metabolite in the body fluids should be correlated to the intake of the food item and detectable by laboratory instruments. Techniques used to measure the analytes should also be accurate and validated and collections should be done at the same time of the day every time.

31 24 Using either urine or plasma samples require specific storage conditions and minimal handling to maintain the stability of the analytes. Plasma analysis can be inconvenient and uncomfortable to the participants, especially if multiple samples are needed from each person. Measuring urine is non-invasive, reflects exposure, and is better for analytes with short half lives. Information on analyte concentration is obtained when urine is collected over longer times such as 24 hours or longer, but longer collection periods are inconvenient for subjects. Nevertheless, subjects are more likely to participate and to be compliant in clinical studies using urine analysis. ANALYSIS OF ISOFLAVONES IN BODILY FLUIDS Quantification of isoflavones in humans is helpful in assessing the benefits of soy consumption on breast cancer risk. This can be done because urinary excretion of isoflavones and soy intake are highly correlated (16, 133, 134, 135, 139, 140). Also, isoflavones appear in the plasma soon after soy consumption and are readily excreted in urine. The short half-life of isoflavones allows for prompt assessment of recent intake. However, the short half-life also requires multiple sample collections to analyze dietary intake accurately, which can be inconvenient to participants and more time consuming and costly for staff. Several studies have shown a dose-dependent relationship between urinary isoflavone levels and soy intake among the Asian population where soy intake is high (134, 141), and in the U.S. where intake is lower (135). Also, one

32 25 study observed that specifically daidzein, genistein, and glycitein were dosedependent with soy intake (141). Moreover, food frequency questionnaires and diet records have been validated by urinary and plasma isoflavone analysis (137, 138, 142). These observations and the specificity of isoflavones to soy foods allow the use of isoflavone levels in body fluids as a biomarker of soy intake. This method can also be used to determine dietary compliance in clinical and epidemiological studies. Past studies investigating nutritional factors in relation to breast cancer risk have used various methods to determine dietary compliance. Some studies used 24-hour recalls (140), or diet records (26,94, 112, 123, 135, 143) in addition to analyzing urinary isoflavones from overnight (140), 24-hour (26), or 72-hour urine (135, 94, 112, 123) collections. Other studies used only a 24-hour urine collection (122), while several studies solely implemented a strict diet for the intervention group (146), or implemented a strict diet in addition to collecting urine and food records (112, 123). Other studies used 24-hour daily urine collections while on a strict diet (144) or monitored diet (75), urine collections alone (145), or diet records alone (143). Urine Analvsis Urine analysis is a better method than plasma collections because of its non-invasive approach. This facilitates subject recruitment and compliance.

33 26 Overnight urine is preferred because of higher analyte concentration and better compliance. Urinary isoflavone analysis can be carried out easily using highperformance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS). HPLC was the method in this study because it is faster than GC-MS and analyzes all isoflavones in a single run. It also uses less expensive instruments and less technician time, and is as accurate as GC-MS (70). GC-MS is more sensitive than HPLC, but requires more steps. Both methods have been shown to achieve similar results (16, 70). Urine analysis can be performed from 24-hour collections or from smaller sample volumes. Twenty-four hour collections have the advantage of providing the total volume of urine excreted, therefore, analyte excretion amount per day can be determined. When urine analysis is performed on small volumes by sampling an aliquot of urine from a one-time urine collection sample within a 24-hour time period, total urine excretion volume per day is unknown. However, isoflavone excretion rates can be determined but analyte excretion rate must be adjusted to creatinine excretion rate. Creatinine is a degradation product of muscle contraction when creatine is continuously dehydrated to creatinine. This results in creatinine being excreted by the kidney into the urine at a constant rate, thus, creatinine can be used as a

34 27 standard for excretion of analytes in urine, assuming creatinine excretion is similar to muscle mass and exercise. However, since creatinine excretion depends on the muscle mass of the individual, using creatinine is not the most accurate standard, but it is sufficiently accurate. PURPOSE Past studies using urinary isoflavones for dietary assessment and compliance have collected one to three overnight or 24-hour urine samples (123, 133, 135). A single measurement may not be sufficient in assessing soy intake and a 72-hour collection is impractical for participants. One study has investigated the accuracy of sampling one overnight urine collection in comparison to two overnight collections 48-hours apart and found that one was as accurate as both (140). Otherwise, it has not been shown which urine time collection periods provide the optimal assessment of soy intake. The gold standard of assessing soy intake would be daily urinary isoflavone assessment. The purpose of this study was to compare urinary isoflavone excretion from various time collection periods during daily soy consumption to determine a feasible number of urine samples to collect, while being accurate in assessing soy intake and leading to satisfactory compliance. One-day urine samples and weekly samples were compared to the gold standard of 24-consecutive day samples to determine if measuring samples less than daily will reflect true

35 28 dietary soy intake and to establish a practical method for measuring dietary compliance.

36 29 CHAPTER 2: METHODS PARTICIPANTS The urinary isoflavone assessment study was approved by the Committee on Human Studies at the University of Hawaii. Twenty eligible women were selected for this study from 110 women currently in the intervention group of the Breast, Estrogens, and Nutrition (BEAN) Study. The BEAN Study is an on-going two-year randomized dietary intervention trial in Honolulu, Hawaii investigating the effects of soy intake on breast cancer risk. It involves 220 premenopausal healthy women with the aim of examining the effect of a soy diet on circulating estrogen levels and mammographic density patterns among healthy premenopausal women. The study also investigates the feasibility of achieving a long-term dietary change. The criteria for participation in the BEAN study include being at an age between 35 to 46 years, premenopausal with intact ovaries, regular dietary soy intake less than 5 grams per day, residence on Oahu, and dietary fat intake between 20% to 40% of total calories. Excluded are those with previous oophorectomy or history of cancer (except basal cell skin cancer), use of oral contraceptives or other steroidal hormones, suspicious lesion in the mammogram at last screening, pregnancy or intent to become pregnant within the next 2 years, intent to leave Hawaii within the next 2 years, and presence of a serious condition that might adversely affect compliance.

37 30 The intervention group was advised to maintain a daily diet with approximately 30% of calories from fat, and consume at least 5 servings of fruits and vegetables and an adequate intake of other nutrients. They were also instructed to incorporate 45 mg of isoflavones as 2 daily servings of tofu, soymilk, roasted soy nuts, soy protein powder, or soy protein bars anytime within the day (Table 1). Table 1. Serving Sizes and Isoflavone Content" of Soy Foods Used in the Breast, Estrogen, and Nutrition Study Serving Size Isoflavones (9) (mg) Tofu Soymilk 180 ml Roasted Soy Nuts Soy Protein Powder Soy Protein Bars As measured 10 the laboratory of Dr. Adnan A Franke, cancer Research center of Hawaii RECRUITMENT Women in the BEAN study intervention group were contacted by phone and invited to participate in this supplemental study. A meeting was arranged with each participant at a time and location of their convenience. During the meeting, collection procedures were explained. Then participants signed two copies of a consent form to participate in this sub-study after thoroughly reading

38 31 the forms. One copy was retained in each woman's file at the Cancer Research Center of Hawaii (CRCH) and the other was given to the participant. Also, supplies required for the urine collection were distributed to the participants at this meeting. The supplies include 25 pre-labeled cryogenic vials, 25 transfer tubes, 25 latex gloves, 25 cups, a cardboard box to store the empty vials, a freezable plastic box, and a plastic storage container for the freezable plastic box. The women also received a copy ofthe instructions, a small sheet of paper with a summarized version of the instructions, and a soy log sheet. The cryogenic vials were labeled with an identification number and numbered for each day. The women continued consumption of 45 mg of isoflavones per day while recording the approximate time of intake on their soy log sheet. They were instructed to record any occurrence of antibiotic use, indigestion or other side effects. The women received a reminder phone call prior to the initial day of collection and a follow-up call during the collection time period to inquire on their progress. URINE COLLECTION Two milliliters of urine were collected daily first thing in the morning prior to any food intake starting on the first day after menstrual bleeding had stopped. Each participant wrote the date of collection on the label of each vial beginning with the vial labeled 'Day 1', and continued using them in numerical order, ending

39 32 with 'Day 25'. Samples were first collected in a cup and then 2 ml were transferred to cryogenic vials with a plastic transfer tube. The filled vials were placed inside a freezable box, then placed inside a plastic storage container, and stored in the participant's freezer until their collection period ended. The participant stopped collecting urine when she began menstruating. If the participant forgot to collect urine one day, she discarded the unused vial and continued collecting urine. If spot bleeding occurred, no collection for that day was necessary and the unused vial was discarded. The collection was to be done for one menstrual cycle anytime within a 3-month period. Arrangements were made to have the samples picked up when the collection period ended, kept frozen in transit in a cooler, and brought directly to the CRCH to be stored at -Boac. CHEMICALS Methanol, acetic acid, dimethyl sulfoxide (DMSO), and all solvents used for HPLC and absorbance readings were analytic grade or HPLC grade from Fisher Scientific (Fair Lawn, NJ). Butylated hydroxytoluene, sodium acetate, and flavone, creatinine (3 mg/dl) and Accutrol Normal (catalog no. A2034) were purchased from Sigma Chemical Co. (St. Louis, MO). Il-Glucuronidase isolated from Escherichia coli (200 X 10 3 UlL) and arylsulfatase isolated from Helix pomatia (1-5 U/mL) were purchased from Roche (Indianapolis, IN).

40 33 APPARATUS HPLC analyses were performed on a System Gold chromatograph with an autosampler (model 507), a dual-channel diode-array detector (model 168) (both from Beckman; FUllerton, CA), and a NovaPak C 1 8 (300 mm X 3.9 mm, i.d.; 4 f.tm) reversed-phase column (Waters; Milford, MA). Absorbance readings were obtained with a spectrophotometer (Bio Spec 1601, Shimadzu; Columbia, MD). CREATININE ASSAY Urinary creatinine concentrations for each sample were determined as a standard for isoflavone excretion rate using the Quantitative Colorimetric Determination Creatinine Kit, Procedure No. 555 from Sigma Diagnostics (St. Louis, MO). This was used because of its ease and specificity in detecting creatinine concentration. A yellow-orange color forms when creatinine reacts with an alkaline reagent, but fades faster upon acidification than the other interfering substances found in urine. Therefore, this allows creatinine concentration to be determined because it is proportional to the difference in color intensity before and after acidification. 3 mg/dl creatinine was used as the standard and Accutrol was used as the control. Creatinine concentrations of samples were calculated as mg/dl using the absorbance readings of the creatinine standard with the following equation: Initial Sample Absorbance - Final Sample Absorbance x 3 Initial Standard Absorbance - Final Standard Absorbance

41 34 POOLING OF SAMPLES Urine from each subject was aliquoted into 3 to 5 different pools depending on the total number ofsamples collected. The 'monthly' pool consisted of combined aliquots of 100 Ilg creatinine from every sample. The 'weekly' pools consisted of combined aliquots of 100 Ilg creatinine per day from 3 days for each week. This included one weekend and two weekday urine samples at least one day apart from each other. Each woman had between 1 to 4 'weekly' pools. The 'single' sample was taken from the same day, in mid-week, as one of the three samples used in one of the weekly pools. The single sample did not come from the same week for every subject. Overall, each subject has one monthly pool, one single day pool, and 2 to 4 weekly pools. ENZVMATIC HYDROLYSIS AND EXTRACTION OF ISOFLAVONES FROM HUMAN URINE From each sample, 750 III was mixed with 200 III triethylamine acetate buffer (0.5 M triethylamine acetate in 0.15 M acetate buffer, ph 7) and 5 III each of ~-glucuronidaseand arylsulfatase, and incubated at 37 C for one hour. Then, 15 III flavone (120 ppm) as internal standard was added and the samples were extracted 2 times in 2 ml diethyl ether. The combined organic phases were dried under a steam of nitrogen and redissolved in 75 III methanol and 75 III of 0.2 M sodium acetate buffer (ph 4).

42 35 In a parallel experiment, 15 III flavone (120 ppm) mixed in 150 III methanol and 0.2 M sodium acetate buffer (ph 4) and was run through the column for internal standard recovery calculation purposes. Flavone was used as the internal standard due to its structural similarity to the analytes and its stability in heat and acid. HPLC ANALYSIS OF ISOFLAVONES Samples were analyzed by injecting 20 III into the HPlC system. Elution was performed at a flow rate of0.8 ml per minute. The linear gradient was as follows: A =10% acetic acid:water (10:90, v/v), B =water:methanol:acetonitrile (1:0.8:1, vlvlv); 10% B to 50% B in 25 minutes, 50% B to 95% B in 9 minutes, 95% B to 10% Bin 3 minutes, with equilibration for 13 minutes before subsequent injection. Analyses were monitored during the entire HPlC run by photodiode array detection at 260 nm and 280 nm. Observed signals were scanned between 190 and 600 nm for identification purposes. DATA ANALYSIS Means were calculated for the women's age, BMI, energy intake from fat and protein, fiber intake, and daily servings of fruit and vegetables. Urinary isoflavone data were calculated as nmollmg creatinine. Correlations in Tables 2, 3, and 4 were performed on the natural log of data due to non-normal distribution ofvalues. Correlations (r) were calculated for total

43 36 urinary isoflavone excretion rates (UIER) and for the individual isoflavones to assess the degree of correlation between the monthly total isoflavone UIER and UIER of: 1. Each week 2. All weeks combined 3. Single day Correlations of UIER were also determined between single day UIER of total isoflavones and UIER of: 1. Each week 2. All weeks combined 3. The corresponding week the single day sample was derived from Additionally, correlations were estimated for subgroups of women according to ethnicity, 8MI, and equol excretor status. Two-tailed z-tests and P-values were calculated to determine the level of significance of the above correlations. Percent differences of the means and medians of single and weekly UIER as compared to the monthly means and medians for all isoflavones and the subgroups of ethnicity, 8MI, and equal excretor status were computed to further examine variations.

44 37 CHAPTER 3: RESULTS SUBJECT CHARACTERISTICS Of the 20 women enrolled, 19 completed the study. One woman did not submit her urine samples in time for urine analysis. Table 2 shows the demographic and daily nutrient intake of the SUbjects. Seven of the women were Caucasian, 1 was Filipino, 3 were Japanese, 6 were of mixed race, and 2 women did not specify. The mean age of the women was 44 years and the mean BMI was 25.3 kg/m 2 On average, they consumed 30% kcal from fat, 14.7% kcal from protein, and 17.5 g fiber daily. They also consumed an average of 3 daily servings of fruit and vegetables. Eight women reported taking multi-vitamin supplements on a daily or weekly basis. Table 2. Daily nutrient intake and demographic information of 19 women Age (years) BMI Energy From Fat (%) Energy From Protein (%) Fiber (gjday) Daily Servings of Fruit Daily Servings of Vegetables Multi-vitamin Supplement Use Mean (SD) 44.4 (2.7) 25.3 (6.0) 30 (5.7) 14.7 (2.1) 17.5 (7.8) 1.2 (1.1) 2.2 (1.0) 42% Ethnicity White Filipino Japanese Mixed Race Other No. of Women (%) 7 (37%) 1 (5%) 3 (16%) 6 (32%) 2 (10%)

45 38 Table 3. Urinary isoflavone excretion rates for total and individual isoflavones r otal lsoflavones Mean" SD" Range" Monthly Week Week Week All Weeks Single Daidzein Monthly Week Week Week All Weeks Single Genistein Monthly Week Week Week All Weeks Single Glycitein Monthly Week Week Week All Weeks Single ODMA Monthly Week Week Week All Weeks Single Equol b Monthly Week Week Week All Weeks Single " Values in nmol/mg creatinine b Results of 7 women

46 39 MONTHLY URINARY ISOFLAVONE EXCRETION RATES The subjects collected urine samples between 17 to 25 days. The mean excretion rates for the monthly pool of total parent isoflavones were as follows: daidzein > genistein> glycitein. On average, the total isoflavone monthly UIER was nmol/mg creatinine and ranged from 6.71 to nmol/mg creatinine (Table 3). The means of the total isoflavone UIER for individual weeks ranged from 22.5 to 27.4 nmol/mg creatinine. MEAN URINARY ISOFLAVONE EXCRETION RATES OF EACH ISOFLAVONE Figure 4 shows the mean UIER of each isoflavone over a time period of 3 weeks in comparison to their mean monthly UIER and mean single day UIER. The UIER means for daidzein, genistein, and glycitein increased over the 3 weeks, whereas the means for equol decreased and OOMA fluctuated ~ B c :E 6 (> : 4 X, ",,~ 0 0 ~ '" )C X " w... co -c&dzein -.-GenislHl -+-G1ycitein --><-- O!>N<..."" ~_' Figure 4. Mean urinary isoflavone excretion rates (UIER) ofeach isoflavone over a time period of 3 weeks in comparison to their mean total monthly UIER and mean total single day UIER, expressed as nmol/mg creatinine.

47 40 WEEKLY URINARY ISOFLAVONE EXCRETION RATES COMPARED TO MONTHLY URINARY ISOFLAVONE EXCRETION RATES Table 4 shows the percent differences between the means and medians of the weekly and single day measurements to the monthly samples for all isoflavones combined and each individual isoflavone. Correlation coefficients (r) and P-values from a two-tailed z-test of the natural log values of all the isoflavones from the various time collection periods are also shown. The percent differences of weekly to the monthly medians tended to be greater than the percent differences of the means. The percent differences in the means for total isoflavones of weeks 1, 2, 3, and all weeks combined compared to the monthly means were 8.64,12.66,6.30, and 7.25, respectively. The percent differences for the medians for weeks 1, 2, 3, and all weeks combined were 5.47,22.11,9.81, and 10.66, respectively. The correlations between the weekly and monthly assessments of the individual isoflavones were high (r= ). The correlations for the total isoflavones of all weeks combined were better correlated to the monthly samples than the individual weeks. Week 1 had the highest correlation (r = 0.92) and correlation tended to decrease with each subsequent week (week 2 r = 0.87, week 3 r = 0.78). For the individual isoflavones, the correlations of all weeks with the monthly samples were as high or higher than the correlations of the individual weeks. Also, the correlations of daidzein, genistein, and ODMA of each week and all weeks combined were similar to one another.

48 Table 4. Percent differences in means and medians, and correlation> comparisons between weekly and single day urinary isoflavone excretion rates (UIER) to monthly UIER 41 % Differences: Correlations: Mean Median r P Totallsoflavones Week < Week < Week < All Weeks < Single < Oaidzein Week < Week < Week < All Weeks < Single < Genistein Week < Week < Week < All Weeks < Single < Glycitein Week < Week < Week <.01 All Weeks < Single OOMA Week < Week < Week < All Weeks < Single < Equol Week <.01 Week Week All Weeks <.01 Single Calculated from natural log of values

49 A BO 60 ffi 40 t ~ &......~.".. /. r= 0.91 P< Log r= MonIIfyUER B 80 r = P< ~ " 60 Log r= 0.87 ffi ~ 40 t ~ i " 0 0 '" Monthly IIER C D BO r = 0.92 " P<0.001, Log r = 0.78 ffi 40 t ~ 20 ~ 0 0 ~ '" Mon-..,.UER 80 r= , P< /" moo.. ~' Log r=o.96 f" 120 ~,,...f4'i-;;;:;.:...;--.. " " _um Figure 5. Correlations of total (sum of all isoflavones) urinary isoflavone excretion rates (UIER) from week 1 (A), week 2 (B), week 3 (C), and mean of all weeks combined (D) to total monthly UIER, expressed as nmollmg creatinine. Data presented are from non-togged values. The dotted line represents perfect correlation to the total monthly UIER. The solid line is the best-fit line for the correlation ofthe weekly to the monthly UIER.

50 43 Figure 5 shows the scatter plots of the correlations between the weekly UIERs to the monthly UIER for total isoflavones. There was a wide range of values with 1 to 2 women having high UIERs above 50 nmol/mg creatinine and a few women with U1ERs below 10 nmojlmg creatinine. SINGLE DAY URINARY ISOFLAVONE EXCRETION RATES COMPARED TO MONTHLY URINARY ISOFLAVONE EXCRETION RATES The mean UIER for total isotlavones of the single day samples was nmojlmg creatinine and ranged from 1.74 to nmojlmg creatinine (Table 3). The single day mean UIER for daidzein, genistein, glycitein, OOMA, and equol were 9.59, 5.17, 1.57, 3.45, and 6.71 nmojlmg creatinine, respectively. The percent difference in mean for the total isotlavones of the single day UIER to the monthly UIER was (Table 4). The percent difference in the medians was The percent differences in the medians were higher than the means for most of the single day UIERs of the total isotlavones and each isoflavone. Also, the correlations of the single day U1ERs to the monthly U1ERs for total isotlavones and each isoflavone were high (r = ) and similar to one another, except for glycitein (r = 0.48) (Table 4). Figure 6a shows the correlation of the single day rates to the monthly rates in graphic format. Similarly to the correlations of the weekly to the monthly UIERs, a few women had high UIERs above 50 nmojlmg creatinine, while a few others had low UIERs below 10 nmol/mg creatinine.

51 44 A III 5... tl 40.. ~ II) 20 O+-...= r------, , o Monthly UIER 60 r= 0.75 P< Log r = B III tl 40 e;, = iii =----, o Corresponding Weekly UIER 60 r = 0.80 P< Log r = Figure 6. Correlation of total urinary isoflavone excretion rates (UIER) from single day samples to total monthly UIER (A), and to the week the single sample was obtained from (8), expressed as nmovmg creatinine. Data presented are from non-logged values. The dotted line represents perfect correlation to (A) the total monthly UIER, (8) the corresponding weekly UIER. The solid line is the best-fit line for the correlation of the single day samples to the (A) monthly UIER and (8) corresponding weekly UIER.

52 45 SINGLE DAY URINARY ISOFLAVONE EXCRETION RATES COMPARED TO WEEKLY URINARY ISOFLAVONE EXCRETION RATES Table 5 shows the correlations of logged values of the single day UIERs to the weekly UIERs and the UIERs of the week from which the single day sample was taken. Single day samples were highly correlated to the corresponding week from which they were derived (r = 0.87, P < 0.001) and to week 1 (r = 0.91, P < 0.001). Correlation also tended to decrease with each subsequent week (week 2 r =0.76, week 3 r =0.67). Table 5. Correlation" comparisons of total urinary isoflavone excretion rates (UIER) from single day samples to the UIER of the week they were obtained from and weekly UIER r P Week < Week < Week < 0.01 All Weeks 0.71 < Week of 0 87 < Single Sample. " Calculated from natural log of values Figure 6b shows the scatter plot of the correlation of the single day UIER to the UIER of the week from which the single sample was taken. This also shows the high UIER for one of the women and low UIERs for a few other women. Figure 7 shows the correlations between the single day UIERs to the individual weekly UIERs and the correlation of the single day UIERs to the UIERs

53 of the mean of all weeks combined. Correlation to week 2 was the lowest, while week 1 was the highest, with a wide range of values for each week. 46

54 A m 60 E'0 i 20 4~ r = 0.80 P< Log r = o_=--~---~--~--~ o W...k1UER '" B ; i 40 r = 0.66 P< 0.01 Log r = c 80 o +-'>=-----=:...---~---~-. o 20 '0 W..k!IIER ,. r = 0.71 P< 0.01 Log r= ~. o+-=---+--~---~--~ o '0 o 80 ~ /... r= 0.64 P< 0.01 Log r = '0...nWMtlll't.lER Figure 7. Correlations between total urinary isoflavone excretion rates (UIER) from single day samples and total UIER from week 1 (A), week 2 (B), week 3 (C), and mean of all weeks combined (D), expressed as nmollmg creatinine. Values presented are from non-logged data. The dotted line represents perfect correlation to the weekly samples. The solid line represents the best-fit line for the correlation of the single day UIER to the weekly UIER.

55 48 STRATIFICATIONS OF URINARY ISOFLAVONE EXCRETION RATE COMPARISONS Stratification by Ethnicitv Table 6 shows the means, standard deviations, and ranges of the UIERs for the total isoflavones stratified by ethnicity, 8MI, and equol excretor status. Table 7 shows the percent differences between the means and medians of the weekly and single day samples as compared to the monthly samples stratified by ethnicity, 8MI, and equol excretor status. Also, correlation coefficients (r) and P-values from a two-tailed z-test of the natural log of values are shown. The mean monthly UIER for Asian and non-asian women were and nmollmg creatinine, respectively (Table 6). The mean monthly UIERs of Asian women ranged from 6.71 to nmollmg creatinine, while those of non- Asians ranged from 6.91 to nmollmg creatinine. According to ethnicity, Asian women had a slightly overall greater percent difference between the median values of all the collection time periods and monthly median values (Table 7). The percent differences between the means of the non-asians and their monthly means were overall slightly greater. Asian and non-asian women had similar correlations to the monthly samples for all collection periods.

56 49 Stratification bv BMI The mean monthly UIERs of women with BM and BMI > 25 were and nmollmg creatinine, respectively (Table 6). The mean monthly UIERs of those with a BMI ranged from 6.71 to nmol/mg creatinine, while those with a BMI > 25 ranged from t nmollmg creatinine. The percent differences in means for both groups of women were similar (Table 7). The percent differences in the medians for women with a BMI between 19 and 25 were greater for most time periods than those with a BMI greater than 25. Correlations according to BMI were also similar for both subgroups for all weeks and the single day samples. Stratification by Equal Excretor Status The mean monthly UIER of equal excretors and non-equal excretors were and nmol/mg creatinine, respectively (Table 6). The mean monthly UIERs of equal excretors ranged from to nmollrng creatinine, while non-equal excretors ranged from 6.71 to nmol/mg creatinine. Non-equal excretors had higher percent differences of their mean UIERs for all time collection periods than equal excretors except for week 2 and all weeks combined (Table 7). Non-equol excretors also tended to have higher percent differences of their median UIERs for most of the time collection periods. Women who were non-equal excretors had similar correlations to equal excretors for all time collection periods, except for week 2 of the equal excretors (r =0.55).

57 50 Of the 7 (37%) of the women who excreted equol, equol excretion rates tended to be higher than their OOMA excretion rate, while non-equol excretors displayed the opposite pattern (Table 8). Also, equol excretors had lower UIERs for OOMA and daidzein.

58 Table 6. Urinary isoflavone excretion rates for total isoflavones stratified byethnicity, 8MI, and equol excretor status 51 Mean SO Range Asian Monthly Week Week Week All Weeks Single Non-Asian Monthly Week Week Week All Weeks Single BM Monthly Week Week Week All Weeks Single MI > 25 Monthly Week Week Week All Weeks Single Equol Monthly Excretor Week Week Week All Weeks Single Non-Equol Monthly Excretor Week Week Week All Weeks Single 'Values in nrnola11g creatinine

59 Table 7. Percent differences in means and medians, and correlation" comparisons between weekly and single day to monthly urinary isoflavone excretion rates stratified by ethnicity, BMI, and equol excretor status % Differences: Correlations: Mean Median r P Asian Week < 0.01 Week < Week All Weeks < Single Non-Asian Week < 0.01 Week < 0.01 Week < All Weeks < Single < 0.01 BM Week < Week < Week < 0.01 All Weeks < Single < BMI > 25 Week Week Week All Weeks < 0.01 Single Equol Excretor Week < 0.01 Week Week All Weeks < Single < 0.01 Non-Equol Excretor Week < Week < Week < 0.01 All Weeks < Single < Calculated from natural log of values 52

60 Table 8. Mean monthly daidzein, OOMA, and equol excretion rates' of equol and nonequol excretors 53 Non-Equol Equol Excreters Excretors Oaidzein OOMA Equol Values in nmol/mg creatinine

61 54 CHAPTER 4: DISCUSSION SUMMARY According to this study, obtaining weekly samples showed the highest correlation with monthly UIER in determining soy intake and compliance. Measuring a single day sample for each week also showed a high correlation to the week it was measured from. The mean monthly UIERs for the total parent isoflavones were found to be highest for daidzein, followed by genistein and glycitein. We found interindividual variability in the mean monthly UIERs among the women. Equol was excreted in approximately 37% of women, who also had lower OOMA and daidzein excretion rates than non-equol excretors. No large differences in correlations were seen when SUbjects were stratified by ethnicity, 8MI, or equol excretor status. Measuring UIERs from different weeks did not differ greatly in relation to the monthly sample, but the week 1 UJERs showed the highest correlation. Oaidzein, genistein, and OOMA had similar correlations ofweekly and single UIERs with monthly U1ERs. We also found the single day UIERs of the total isoflavones to be significantly correlated to week 1 and to the week they were taken from, but not as well correlated with weeks 2 and 3.

62 55 COMPARISON WITH PAST RESEARCH One study to date has investigated different urine collection times to determine the accuracy of urinary isoflavones in assessing self-reported soy intake (140). They reported isoflavone excretion rates from one overnight sample to be significantly correlated to a second sample collected 48 hours later. This suggests that collecting one urine sample may provide adequate information for recent soy intake. Our study went further to investigate how often urine should be analyzed to obtain the most accurate reflection ofmonthly UIERs. At the same time, the study design has to be feasible to subjects and staff. The variability of UIERs seen among the subjects in our study has been noted in other studies (25, 111, 115, 126, 135). The order of decreasing UIER of the parent isoflavones found in this study is also in agreement with past studies (70,111,133,135,141). Equol was seen in approximately 30% of subjects in other studies (115, 122), which is similar to our results. However, those who were classified as nonequol excretors may have excreted equol in undetectable amounts because HPLC may not be sensitive enough to detect lower levels of equol and equol may have poor UV absorption characteristics (114). Additionally, the higher equol excretion rates compared to OOMA in equol excretors and the opposite trend seen in non-equol excretors was also seen in several studies (115, 123, 135). This was expected since daidzein is the precursor for equol, thus, presumably less daidzein would be available for OOMA formation.

63 56 Analyzing excretion rates according to ethnicity, 8MI, and equol excretor status did not show large differences between the subgroups. Past studies have observed Asians to have higher UIERs (133,140), but this was due to higher soy intake, which would not be expected to be seen in our study as all women are presumably consuming equal amounts of isoflavones. STRENGTHS OF THIS STUDY One of the strengths of this study is the fact that we were using subjects who were already on a long-term soy diet. This eased subject recruitment and decreased the length and cost of the study. Also, analyzing urinary isoflavones by HPLC provided data in a timely matter. Using a small volume of urine required little storage space and decreased participant burden in collecting urine. Compliance was high as we succeeded in having subjects collect 25 overnight samples. Furthermore, the presence of equol excretors also allowed us to compare isoflavone excretion with non-equol excretors and determine the variability between them. LIMITATIONS TO THIS STUDY There were also limitations in this study. Analyzing daily UIERs over three weeks was costly. The single day samples were not obtained from the same week for every subject. In order to standardize the comparison ofthe single day samples to the monthly and weekly samples, the single day samples should be

64 57 obtained from the same week for every subject. We could have also have analyzed a single day sample from a day within a weekend to determine if variations exist between sampling a single day during the weekdays or weekends. Also, the HPLC method may not have been sensitive enough to detect low levels of equol in other women who did not excrete equol in their urine. Even though we were able to sample various ethnicities in this study, it was a poor representation of the ethnic distribution in Hawaii. Hawaii's population consists of approximately 24% Caucasians, 17% Japanese, 14% Filipino, 9% Native Hawaiian, 5% Chinese, 2% Black/African-American, 2% Korean, and 27% other ethnic groups (147). The main ethnicities in this study were Caucasians (37%), with Japanese comprising 16%, and Filipino comprising only 5%. 32% were of mixed race. It would have been better to increase the sample size and to include women with other ethnicities to allow for a wider range of comparison between ethnicities and to observe more variability. IMPLICATIONS Collecting monthly urines is the gold standard for assessing dietary compliance. In viewing our data, the extra effort and inconvenience to the SUbjects cannot justify the small degree of additional accuracy in collecting monthly urines when compared to collecting weekly urines. Our results show that collecting monthly urine samples would only detect an additional 1 to 3

65 58 subjects who are non-compliant compared to the weekly samples. In large studies, a difference of a few subjects would be insignificant for determining overall dietary compliance amongst the subjects. However, in small studies, obtaining monthly urine samples may be worth the extra effort. We observed each week to be similar with one another in their correlations to the monthly rates for total isoflavones and each isoflavone except equol. This indicates collecting urine from one week represents all 3 weeks. Also, week 1 was the most highly correlated to the monthly UIER and correlation tended to decrease slightly with each subsequent week. This suggests sampling week 1 may be the most accurate week of the 3 weeks in assessing soy intake. However, this trend could be due to chance, higher soy intake, or menstrual cycle characteristics, therefore, a future study with larger sample size should be done to determine the consistency of the trend. The UIER from all weeks combined had a higher correlation with the monthly UIER as compared to the single day UIER. Therefore, collecting weekly urine samples for analyzing urinary isoflavones is the most accurate method next to collecting monthly samples. Also, the similarities in correlations between daidzein, genistein, and OOMA suggests the possibility that analyzing one of the three isoflavones could represent the correlations for all three isoflavones. The single day samples were not as well correlated with the individual weeks, which may be due to day to day variation in soy consumption. This indicates a single day sample is not sufficient for assessing soy intake very

66 59 accurately. Our observation of the single day UIERs of total isoflavones to be significantly correlated to the week they were derived from suggests that collecting a single day sample within a one-week period may be sufficient and accurate in representing that week's soy intake. Inter-individual variability noted in this study may be due to some of the women consuming their required daily servings all in the morning or all in the evening, resulting in a wide range of time between their last soy intake and the urine collection the following morning. Since the half-life of isoflavones is approximately 5-8 hours, timing of soy intake is likely a factor in the variability. Also, the variability may be due inter-individual differences in gut microflora, different sources of soy intake, non-compliance, or other dietary factors. Additionally, the lack of variability across the subgroups may be due to an insufficient sample size in each subgroup. The high excretion rates seen by two subjects are possibly due to factors such as, inconsistency of intake with self-reported intake and/or a high consumption of fermented foods. Unfermented soy foods consist of primarily conjugated forms and high amounts of intake may overwhelm the bacterial deconjugating enzymes in the intestine, resulting in less isoflavones to be absorbed (130,131). Fermented foods contain primarily aglycones, which do not require the rate-limiting step of de-conjugation and are, therefore, efficiently absorbed. Also, the high excretion rates could be explained by an unreported additional intake of soy foods or soy supplements.

67 60 The low excretion rates seen by several of the subjects may be possibly due to non-compliance to the soy diet protocol and/or limited absorption of the isoflavones into the enterocyte. The lack of absorption may be due to consuming insoluble fiber concurrently with the soy food, which increases fecal bulk and transit time. This may result in decreased absorption ofisoflavones due to less exposure ofisoflavones to the gut microflora and less time for isoflavones to be absorbed. Also, absorption may be affected by consuming other foods concurrently with the soy food. These foods may require metabolism and absorption. This may consequently interfere with efficient isoflavone absorption as other foods may require degradation by the same bacterial enzymes, therefore, less of the enzymes would be available for isoflavone metabolism. Additionally, the women who had low UIERs may have insufficient intestinal microflora to metabolize the conjugated forms to aglycones for absorption. This could be due to antibiotic intake which diminishes gut microflora or environmental factors. Thus, consuming soy daily may overwhelm the insufficient bacterial enzymes and a portion of the isoflavones may escape hydrolysis and absorption and pass into the colon. APPLICATIONS Measuring UIERs can be used in addition to the traditional methods of any diet assessment, such as 24-hour recalls or food frequency questionnaires, to

68 61 determine dietary compliance of study subjects. This combination would be beneficial in determining if other dietary factors are affecting UIERs. Urinary isoflavones can also be used to validate food frequency questionnaires. As was used in this study, collecting weekly urine samples, two weekday overnight collections, 24 hours apart, and one weekend overnight collection is recommended. This would represent a one-week time period, while maintaining compliance. Due to inter-individual variability in urinary isoflavone excretion, it cannot be determined if a participant is compliant to the diet protocol by only measuring urinary isoflavones without a baseline study. Each subject's excretion pattern needs to be assessed prior to entering a clinical trial. This will provide researchers a basis to compare each subject's isoflavone excretion patterns during the actual study with the baseline value. This should be done for both control and intervention groups. Dietary intakes of control groups also need to be assessed to ensure they are complying with a soy-free diet. Also, randomly alternating urine collection days may help determine compliance as the subject will not know which days to collect urine and it may prevent subjects from consuming soy only on the days prior to each collection. However, randomly alternating urine collections is not as practical and could become inconvenient to the subjects and decrease compliance in the study.

69 62 These results cannot necessarily be applied to populations who do not consume soy on a daily basis. These results are most applicable to studies requiring daily soy intake. Our correlations would not be expected to be as high for individuals who do not consume soy on a daily basis because urinary isoflavones reflect mostly the intake of soy within the past 48 hours due to the short half-life of the isoflavones. Single day samples cannot be used for those not on a daily soy diet due to day to day variation in soy consumption. It is uncertain if these results can be applied to men. Gender differences have not been seen in metabolism and excretion of urinary isoflavones in two studies (68, 148). One study found gender differences in excretion during one month of daily soymilk intake, but the amount of isoflavone intake was high ( mg), which may have affected metabolism and excretion (150). Future studies should be done to determine if excretion rates in men differ over a onemonth time period with a lower daily isoflavone intake (45 mg) such as that used in this study. We can also not be sure if these results can be applied to postmenopausal women and individuals ofvarious ages. No differences have been reported to be seen between menopausal status and different age groups of women in a short term study (131). But it is not known if their excretion rates differ over a one-month time period. It is also unknown if these results would apply to younger men and women.

70 63 Due to the small sample size of this study, it is unknown if our findings would apply to all ethnicities, subjects with high or low 8Mls, and those who are considered equol excretors. Our findings showed there were no large differences between these subgroups in this study. However, our results could be due to chance and future studies with larger sample sizes can determine if differences actually exist. Analyzing urinary isoflavone excretion as a measure of compliance is not necessary for studies that place subjects under monitored diets unless it is being used to investigate other factors. Compliance is observed by staff, therefore, the extra time and effort to measure urinary isoflavones would be unnecessary. CONCLUSION Measuring urinary isoflavone excretion everyday is the gold standard for dietary assessment in clinical and epidemiological studies requiring compliance with a soy diet. However, requiring SUbjects to collect urine everyday is impractical and it may result in difficulties with subject recruitment and compliance. Other studies requiring compliance to a soy diet have used selfreported intakes and/or one or multiple overnight to 24-hour urine collections, implemented strict diets, or monitored food consumption to assess dietary intake. No other studies have investigated other urine collection times in assessing soy intake.

71 64 Therefore, this study investigated the accuracy of single day and weekly urinary isoflavone collections in comparison to monthly collections for assessing self-reported soy intake and compliance. From our results, it was seen that all time collection periods were highly correlated with the monthly UIERs. Analyzing urinary isoflavones on a weekly basis were highly correlated with the monthly UIERs. Overall, measuring UIERs on a weekly basis had the highest correlation with the gold standard of measuring isoflavones everyday. When compared to monthly UIERs, the additional degree of accuracy in finding non-compliant subjects using weekly samples was small and insignificant when determining overall subject non-compliance. Being able to find only a few additional noncompliers would not be worth the extra effort and time required by the subjects and staff to collect urine samples everyday. Therefore, collecting weekly samples would be the most accurate and most feasible method for determining dietary compliance. These results are most applicable to future studies requiring compliance to a daily soy diet, and can facilitate subject recruitment and compliance.

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