Journal of Exposure Analysis and Environmental Epidemiology (2003) 13, 276–282 r 2003 Nature Publishing Group All rights reserved 1053-4245/03/$25.00

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Urinary and serum concentrations of seven in a human reference population subset

LIZA VALENTI´ N-BLASINI, BENJAMIN C. BLOUNT, SAMUEL P. CAUDILL, AND LARRY L. NEEDHAM

National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA

Diets rich in naturally occurring (phytoestrogens) are strongly associated with a decreased risk for cancer and heart disease in humans. Phytoestrogens have estrogenic and, in some cases, antiestrogenic and antiandrogenic properties, and may contribute to the protective effect of some diets. However, little information is available about the levels of these phytoestrogens in the general US population. Therefore, levels of phytoestrogenswere determined in urine (N ¼ 199) and serum (N ¼ 208) samples taken from a nonrepresentative subset of adults who participated in NHANES III, 1988– 1994. The phytoestrogens quantified were the (, , ); the isoflavones (, , , O- desmethylangolensin); and (urine only). Phytoestrogens with the highest mean urinary levels were enterolactone (512 ng/ml), daidzein(317 ng/ ml), and genistein (129 ng/ml). In serum, the concentrations were much less and the relative order was reversed, with genistein having the highest mean level (4.7 ng/ml), followed by daidzein (3.9 ng/ml) and enterolactone (3.6 ng/ml). Highly significant correlations of levels in urineand serum samples from the same persons were observed for enterolactone, enterodiol, genistein, and daidzein. Determination of phytoestrogen concentrations in large study populations will give a better insight into the actual dietary exposure to these biologically active compounds in the US population. Journal of Exposure Analysis and Environmental Epidemiology (2003) 13, 276–282. doi:10.1038/sj.jea.7500278

Keywords: phytoestrogens, lignans, isoflavones, human, exposure, urine, serum.

1. Introduction oxidative balance (Phipps et al., 1993; Wei et al., 1995). Although the physiologic relevance of some of these proper- Decreased risks for heart disease and cancer are associated ties is questionable, consumptionof foods rich inphytoestro- with certaindiets. This protective effect may result from gens is associated with significantly reduced risk for cancer, dietary micronutrients, macronutrients, biologically active osteoporosis, cardiovascular disease, and severity of meno- non-, or a combination of these and other factors. pause (Adlercreutz and Mazur, 1997; Kurzer and Xu, 1997). The human diet contains a complex array of naturally Additionally, certain lignans (Saarinen et al., 2001) and occurring bioactive compounds called . isoflavones reduce carcinogen-induced cancer in laboratory Among these phytochemicals are phytoestrogens, a broad animals (Lamartiniere et al., 1995a, b). group of plant-derived compounds of structure Although considerable health benefits are associated with that canmimic .The largest dietary dosage of diets rich inphytoestrogens, little informationis available phytoestrogenic activity results from two classes of com- about the actual phytoestrogen concentrations in the general pounds: lignans and isoflavones. These groups modulate US population.Phytoestrogenexcretioninurinevaries with many biologically relevant processes, including the type of diet (Adlercreutz et al., 1986; Hutchins et al., productionandmetabolism (Adlercreutz et al., 1987, 1988), 1995a, b; Horn-Ross et al., 1997). Sources for lignans intracellular enzyme levels (Adlercreutz et al., 1993), protein includewholegrains,flax,andsomefruitsandvegetables. synthesis, malignant cell proliferation, cellular differentiation Isoflavone diets consist primarily of , such as (Adlercreutz and Mazur, 1997; Kurzer and Xu, 1997), and and soy-based products (Adlercreutz et al., 1991; Kirkmanet al., 1995; Hutchinset al., 1995a, b). The phytoestrogens are ingested in their natural beta-glycosidic 1. Address all correspondence to: Dr Liza Valentı´ n-Blasini, National forms, which are hydrolyzed to their aglycones in the Center for Environmental Health, Centers for Disease Control and intestine, absorbed, and then glucuronidated in the intestinal Prevention, 4770 Buford Highway, MS F-17, Atlanta, GA 30341, USA. Tel.: +1-770-488-7902; fax: +1-770-488-4609. wall. The major circulating forms of the isoflavones are the E-mail: [email protected] glucuronidated metabolites, and the glucuronidated forms Received 5 November 2002; accepted 3 March 2003 also predominate in the urine (Setchell et al., 2001). The half- Urinary and serum phytoestrogens levels in reference populations Valentı´n-Blasini et al. lives of the phytoestrogens are reported to be in the range of deconjugation internal standards (4-methylumbelliferone 3–10 h (Setchell et al., 2001). Previous studies have reported glucuronide and 4-methylumbelliferone sulfate, Sigma Che- urinary phytoestrogen levels in various populations, but they mical, St. Louis, MO, USA). Serum was diluted and only examined a very few people in each population buffered with ammonium acetate (1 ml of 250 mM, pH 5). (Adlercreutz et al., 1995). The largest reported phytoestrogen Conjugated analytes were hydrolyzed by the addition of b– exposure assessment study measured urinary levels of glucuronidase/sulfatase (Helix pomatia, H-1, Sigma Chemi- phytoestrogens in an ethnically diverse population of 50 cal)andincubatedovernightat371C. Serum deconjugated youngwomeninonegeographic area, the SanFrancisco Bay samples were extracted with Oasis HLB SPE (60 mg HLB, Area (Horn-Ross et al., 1997). We measured the serum and 3 ml, Waters Scientific, Beverly, MA), and urine samples urinary levels of the lignans (matairesinol, enterolactone, and were extracted using C18 SPE column (Varian 500 mg, 3 ml, enterodiol); isoflavones (genistein, daidzein, O-desmethylan- 120 mm pore size); the concentrated extract was resuspended golensin (O-DMA), and equol); and coumestrol (in urine inmobile phase. The phytoestrogensinthe extracted samples only) in a larger and more geographically diverse US were separated by reversed-phase HPLC (50 mm Prism population of adults of different race/ethnicity and both column, Keystone Scientific, Bellafonte, PA, USA) and genders with different dietary habits. measured by tandem mass spectrometry (Sciex API III, Perkin-Elmer Sciex Instruments, Wellesley, MA, USA) using heated nebulizer atmospheric pressure chemical ionization in Materials and methods thenegativeionmode. Urinary concentrations of the phytoestrogens are reported Study subjects as nanograms of phytoestrogen per milliliter of urine (ng/ml) Urine and serum samples were obtained from a subset of and as micrograms of phytoestrogens per gram of urinary samples from adult participants of the Third National Health creatinine (mg/g); serum concentrations are reported as ng/ and Nutrition Examination Survey (NHANES III), 1988– ml. Quality control samples (spiked serum or urine) and 1994. This subset was chosenat random from individuals standards were analyzed along with unknown samples. To who volunteered to give an extra blood and urine sample. diminish the high degree of skewness of the data, the The samples were takenat various times of the day anddo concentrations (mg/g creatinine) were graphed using a not represent fasting samples. This population subset was not logarithmic scale. Geometric means were determined with designed to be representative of the US population but rather the values corrected by urinary creatinine concentrations. to serve as a reference range group of civilian, noninstitutio- For statistical purposes, nondetectable values were treated as nalized adults, age 20–58 years (mean of 36.7710.2 years). the limit of detectiondivided by two (LOD/2). Detection The gender distribution was 61% female. Race/ethnicity of limits were inthe low ng/ml (Valentı ´ n-Blasini et al., 2000). these samples was as follows: non-Hispanic white 29%; non- Statistical analysis of the correlation of each of the Hispanic black 43%; Mexican American 23%; and other phytoestrogen concentrations in urine and serum from the 5%. The paired urine and serum samples from 66 people same individuals was performed using a Spearman’s rank were simply based onthe availability of samples for which we correlation analysis. Multivariate statistical analysis (annual had adequate volume of both matrices. income, education, age, ethnicity, rural or urban residence, and gender) was accomplished using PROC GLM in SAS Method (SAS User’s Guide: Statistics, 1982 Edition. SAS Institute, Randomly selected urine (N ¼ 199) and serum (N ¼ 208) Inc., Cary, NC, USA). samples were analyzed for phytoestrogens using solid-phase extraction (SPE) followed by HPLC and tandem mass spectrometry, as previously described (Valentı´ n-Blasini et al., Results 2000). All of these samples had beenstored at À701Csince collection. Genistein, coumestrol, and daidzein were pur- Urinary Levels of Phytoestrogens chased from Indofine Chemical (Somerville, NJ, USA). The Urinary levels (in ng/ml) of phytoestrogens in our reference remaining phytoestrogens and the labeled phytoestrogens, group are summarized inTable 1. Sincecoumestrol was except for daidzein, were purchased from Professor Kristina detected in only nine urine samples, its data are not Wa¨ ha¨ la¨ of the University of Helsinki, Finland; daidzein was presented. The highest mean level (512 ng/ml) was observed purchased from Cambridge Isotope Laboratories (Andover, for enterolactone, a , followed by the isoflavanoids, MA, USA). All samples were spiked with a mixture of stable daidzein (317 ng/ml) and genistein (129 ng/ml). The lowest 2 isotope labeled internal standards, isotopic purity: ([ H6]- mean level was found for the lignan, matairesinol (15 ng/ml), 2 2 enterolactone, 490%; [ H6]-enterodiol, 490%; [ H6]- which metabolizes to enterolactone. Figure 1 presents the 2 2 matairesinol, 95%; [ H4]-equol, 90%; [ H5]-O-DMA, distributionof the phytoestrogenlevels ona creatinine- 2 2 80%; [ H3]-daidzein, 97%; and [ H4]-genistein, 90%), and adjusted basis in this nonrepresentative, but multiethnic,

Journal of Exposure Analysis and Environmental Epidemiology (2003) 13(4) 277 Valent ı´n-Blasini et al. Urinary and serum phytoestrogens levels in reference populations

Ta bl e 1 . Urinary phytoestrogen concentrations from nonrepresentative subset of the NHANES III population.

PhytoestrogenN MeanMinimum Percentiles Maximum GM

25th 50th 75th

Isoflavones Daidzein199 317 5 22 74 213 15900 58 Equol 199 36 1.5 3.3 8.9 18 4140 6.5 Genistein 199 129 1.5 13 35 93 7290 28 O-DMA 199 50 2.1 2.1 4.6 25.6 1840 5.9

Lignans Enterodiol 199 63 1.5 13 27 70 2510 23 Enterolactone 199 512 5 67 209 553 5580 144 Matairesinol 199 15 0.4 3.2 8.6 18 92 6.6

Mean, minimum, maximum and percentiles expressed as ng/ml. Geometric mean (GM) expressed in mg of phytoestrogen/g of creatinine.

100000.00 Data analysis regarding urinary levels of lignans revealed several statistically significant findings. The lignans present in 10000.00 highest urinary concentrations, enterolactone, and entero- diol, showed significant interaction with income, gender, and 1000.00 age. For enterolactone, data analysis showed a significant interaction (p ¼ 0.046) between gender and income, and a 100.00 nominally significant interaction (p ¼ 0.0646) betweeneduca- tionandincome.Formaleswith12orfeweryearsof 10.00 education, enterolactone urinary levels decreased proportion- µg/g creatinine ally with increasing income; conversely, for females with 12 1.00 or fewer years of education, enterolactone levels increased

0.10 proportionally with increasing income. For males with more than 12 years of education, enterolactone levels increased

0.01 proportionally with increasing income, but at half the rate of O-DMA GEN EQ ENTD DAID MAT ENTL females with more than12 years of education.Data analysis Phytoestrogen for enterodiol showed that the levels of enterodiol increase as Figure 1. Urinary distribution of phytoestrogens in nonrepresentative income increases. Similar to the enterolactone data, the NHANES III population subset using a Box and Whiskers plot. Concentration is expressed in mg/g creatinine in a logarithmic scale. increase is much larger for females than for males and also genistein (GEN), O-desmethylangolensin (O-DMA), matairesinol much larger for persons living in urban areas, compared to (MAT), equol (EQ), enterodiol (ENTD), daidzein (DAID), and those living in rural areas. The enterodiol levels increase coumestrol (COUM). for persons with more than 12 years of education but not forpersonswith12orfeweryearsofeducation.Thereis a significant interaction between gender and residence populationsubset. For comparisonsamongthe various (p ¼ 0.0150) for enterodiol, with urban males having lower demographic factors, the urinary creatinine-adjusted data levels thanrural males, but urbanfemales havinghigher were log transformed because the data were not normally levels than rural females. Matairesinol showed significant distributed, but were right-skewed. interactionbetweeneducationandresidence. Onaverage, Within our reference range population, we observed the urbansubjects with 12 or fewer years of educationhad higher highest levels of phytoestrogens in non-Hispanic whites, with levels thanurbansubjects with more education,but the the exception of matairesinol being found highest in non- reverse was true for rural residents. Hispanic blacks. Statistically significant differences among Data analysis on isoflavone levels provided far fewer ethnic groups were observed only for equol (p ¼ 0.0001) and significant findings. In fact, the major isoflavones, daidzein matairesinol (p ¼ 0.019). For equol, the levels were signifi- and genistein, showed no significant correlations with any of cantly higher in non-Hispanic whites followed by non- the demographic variables examined. O-Desmethylangolen- Hispanic blacks (p ¼ 0.0002). For matairesinol, the levels sin showed significant interaction among income, gender, and were significantly higher in non-Hispanic blacks than in age. For the younger group of adults, O-DMA urinary levels MexicanAmericans( p ¼ 0.0035). decreased proportionally with increasing income, while for

278 Journal of Exposure Analysis and Environmental Epidemiology (2003) 13(4) Urinary and serum phytoestrogens levels in reference populations Valentı´n-Blasini et al.

Ta bl e 2. Serum phytoestrogen concentrations (ng/ml) from nonrepresen- tative subset of the NHANES III population.

PhytoestrogenRange MeanDetectable (nondetectables)

Isoflavones Daidzeinn.d.–1623.9 179 (29) Equol n.d.–8.2 oLOD 2 (206) Genistein n.d.–203 4.7 116 (92) O-DMA n.d.–29 1.0 138 (70)

Lignans Enterolactone n.d.–112 3.6 189 (19) Enterodiol n.d.–19 1.8 116 (92) Matairesinol n.d. –3.3 oLOD 3 (205) Figure 2. Scatter plot for the lignan, enterolactone. Centerlines n.d.=nondetectable. representthe error invariables regressionof log 10 [urinary enter- olactone, creatinine corrected (mg/g creatinine)] onto log10 [enterolac- tone serum levels, (ppb)]. Outer lines represent the 95% confidence the older age group O-DMA levels increased proportionally limits on individual points. with increasing income. Equol levels tended to be signifi- cantly higher (p ¼ 0.0242) inrural residents thaninurban Table 3 . Spearmancorrelationcoefficientsfor phytoestrogenlevels in residents. matched urine and serum samples from 66 persons.

Serum Levels of Phytoestrogens PhytoestrogenCorrelation P-value Phytoestrogenlevels were quantified in208 serum samples, Isoflavones and their levels are summarized in Table 2. The serum levels Daidzein0.72 0.0001 of the phytoestrogens are several times less than the Genistein 0.79 0.0001 respective urinary levels. The highest serum mean levels were O-DMA 0.41 0.0007 observed for the isoflavones, genistein (4.7 ng/ml) and Lignans daidzein (3.9 ng/ml). For the lignans, enterolactone was Enterodiol 0.62 0.0001 present in highest amounts (mean of 3.6 ng/ml) followed by Enterolactone 0.84 0.0001 enterodiol (mean of 1.8 ng/ml). Concentrations of equol and matairesinol were below our limit of detection. reported values are from a diverse adult US population, a Correlation between Urinary and Serum Levels broader populationbase thanprevious studies. Of the eight From the total samples analyzed (401), 132 consisted of phytoestrogens we measured, the highest arithmetic mean paired samples (urine and serum samples collected at the urinary levels were observed for enterolactone (512 ng/ml), same setting from the same individual) from 66 individuals. daidzein (317 ng/ml), and genistein (129 ng/ml) (Table 1). Figure 2 presents a scatter plot for enterolactone showing the The finding of enterolactone having the highest levels in this relation between serum levels and creatinine corrected urinary subsample is consistent with a general US diet that is higher levels for these 66 people. Table 3 summarizes the Spearman inwhole grainsor cereals thaninsoybeansources of coefficients obtained for the concentrations of different phytoestrogens. The large difference in the concentrations of phytoestrogens in urine and serum samples taken from the these phytoestrogens between the 75th percentile and the same 66 individuals. maximum levels is probably due to a relatively small number of people taking food supplements containing these phytoes- trogens or having a dietary intake with very high concentra- Discussion tions of them. Owing to the skewness of the data, it may be more important to estimate the general population by We report urinary and serum concentrations of selected examining the geometric means or medians (50th percentile) phytoestrogens in this NHANES III subset of adults living in (Table 1), which both decrease inthe same order as the the US. Previous studies have reported urinary and serum means.Inthecaseofcoumestrol,onlynineurinesamples phytoestrogens levels for various populations. Most of these had detectable amounts suggesting a very low dietary studies focused on populations of specific ethnic groups, exposure to this phytoestrogen. dietary habits and/or gender, and with a low number of To compare our urinary phytoestrogen results with those subjects (Hermanet al., 1995). Eventhough our results do of previous studies, we transformed the geometric mean not represent the composition of the general population, our values (mg/g creatinine) to similar units used in these studies

Journal of Exposure Analysis and Environmental Epidemiology (2003) 13(4) 279 Valent ı´n-Blasini et al. Urinary and serum phytoestrogens levels in reference populations

(mmol/day). Assuming an average excretion amount per day US, the SFBA study, the following is observed. In our study, for creatinine of 2.145 g, the values were converted to mmol/ the levels of the isoflavones found in non-Hispanic whites day (Diem and Geigy, 1962). Levels of the lignan (n ¼ 55) were two to five times higher, while the levels of the enterolactone (1.04mmol/day) were similar to the published lignans were three times lower than in the SFBA population levels for Latina women in the San Francisco Bay Area group (n ¼ 15). For non-Hispanic blacks (n ¼ 83), the (SFBA) study (Horn-Ross et al., 1997) and for Japanese isoflavones in our subset were two to five times higher while menandwomen(0.9 mmol/day) (Adlercreutz et al., 1995). the lignans were two times lower. For Mexican Americans Onthe other hand, they were approximately two times lower (n ¼ 45), the reverse is observed: the lignans were one to two than published levels reported in omnivorous US and times higher inour group, but the isoflavones, genisteinand Finnish women. Levels of enterodiol (0.16mmol/day) were daidzein, were two to three times lower. O-Desmethylango- similar to those found in post-menopausal omnivorous and lensin was consistently higher in our population subset; in lacto-ovovegetarianUS womenbut were two to three times particular, MexicanAmericanshad three times higher levels lower than for Japanese men and women. The enterodiol thanthe Latinagroup ( n ¼ 15) inthe SFBA study. levels were two times higher thaninLatinawomeninthe The geometric mean concentrations in our study were SFBA study but four times lower thaninwhite SFBA obtained from an ethnically diverse population, representing women. The levels were similar to the levels for persons a broad distributionof dietary habits, ages, incomes, and consuming a soy experimental diet that was high in residences that could explain possible differences between the isoflavones and low in lignans (Kirkman et al., 1995). values. Most of the differenceswithinpopulationcould be For the isoflavone, genistein (0.22mmol/day), the levels attributed to different dietary habits. Other factors could were two times higher thaninpersonsexposed to a contribute to the observed differences, such as basal diet carotenoid or cruciferous experimental diet and six times composition, phytoestrogen content of plant foods or food lower than those eating a soy diet. The carotenoid diet and products consumed by residents of different geographic cruciferous diet correspond to a diet with a higher intake of areas, as well as cultural differences in food composition, lignans and minimum amount of isoflavones. The levels preparationandconsumption.Factors other thandietary obtained inour populationreflect a diet with a low isoflavone intake, such as by intestinal , endogenous exposure. Whencompared with the SFBA study, the levels hormones, and antibiotic use also influence phytoestrogens’ were similar to white US womenbut three times lower than excretion(Adlercreutz et al., 1986). Gut absorptionof inLatinaandfour times higher thaninAfrican-American phytoestrogens is influenced by the composition of gut women. For daidzein (0.49 mmol/day), similar values have microflora, which is influenced by dietary habits, and it could beenobserved for vegetarianFinnishwomenandSFBA differ between two persons eating the same diet (Knight and Latina women. The levels were higher than levels reported Eden, 1996). For example, some persons cannot metabolize for a carotenoid experimental diet but lower than a daidzeinto equol, resultingina low equol excretion. cruciferous diet. The levels were dramatically lower than Daidzein metabolism to equol varies considerably among reported for a soy diet. For example, Japanese women people and is influenced by other dietary components, such consuming approximately 16 and 30 mg/day of daidzein, as carbohydrate intake. High carbohydrate intake leads to equol, and genistein excreted approximately 3000–5000mg/l higher productionof equol thandoes low carbohydrate of each of these isoflavones in their urine (Arai et al., 2000). intake (Setchell and Cassidy, 1999; Bennetau-Pelissero et al., For O-DMA (0.049 mmol/day) the levels were similar to 2000). Inthe NHANES III population,variables like this Japaneseinthe SFBA; two times higher thaninwhite in could not be assessed, which could contribute additional SFBA, Helsinki, and Boston omnivorous women; two times information to the interpretation of differences between lower than in vegetarians in Boston; and nine times lower different populations and possibly add insight in the broad thanmacrobiotics inthe same study. Compared with a soy variability observed. However, we did observe that the ratio experimental diet, the levels of O-DMA in our population of daidzeinto equol urinarylevels was less inMexican were seventimes lower. The relationbetweenthe dose and Americans (5.7) and non-Hispanic whites (6.4) compared to urinary excretion is linear for many of the phytoestrogens non-Hispanic blacks (13.2), suggesting lesser conversion of measured, except for equol (Karr et al., 1997). Since daidzeinto equol inthe latter population.We also observed excretory half-lives are reported to be inthe rangeof 3– that for all ethnic groups, the ratios of enterodiol to 10 h (Setchell et al., 2001), urinary levels reflect recent enterolactone, which can interconvert, were less than 1. This consumption. finding of higher excretion amounts of enterolactone is in In general, in our study the concentrations for lignans agreement with other human studies (Adlercreutz et al., correlate with levels reported for other populations, while the 1986; Kirkmanet al., 1995). Females tended to have isoflavone concentrations in our study reflect a diet low in marginally higher levels of enterodiol and lower levels of isoflavones. If we compare our population subset in terms of enterolactone, while the opposite was true for males. the different ethnic groups with a study also conducted in the Differences might be related to variations in the composition

280 Journal of Exposure Analysis and Environmental Epidemiology (2003) 13(4) Urinary and serum phytoestrogens levels in reference populations Valentı´n-Blasini et al. of the colonic bacteria affecting the further oxidation of populations will help elucidate the potential role of enterodiol to enterolactone or to a higher intake of phytoestrogens in protecting against hormone-dependent enterodiol. Similar results were observed in a study of lignan diseases. This study increases insight into the actual dietary andisoflavonoidurinaryexcretioninmenandwomen exposure to these biologically active compounds in the US consuming the same diets (Kirkman et al., 1995). population. The concentrations of the phytoestrogens were much lower inserum thaninurine(Tables 1 and2), which pointsto the fast clearance of these chemicals from the body. In addition, Acknowledgments a higher number of samples had nondetectable concentra- We acknowledge personnel at the National Center for Health tions in serum than urine. For matairesinol and equol, Statistics of the Centers for Disease Control and Prevention nondetectable concentrations were obtained for most of the for conducting NHANES, for providing results of creatinine serum samples. Detectable levels of equol inserum have been measurements, and for assistance with demographic informa- reported inprevious studies, but the reported values were tion. below our method’s limit of detection(Adlercreutz et al., 1993; WhittenandPatisaul, 2001). Time of collection of the blood sample is animportantvariable for the References determination of phytoestrogens because of their short half- lives (King and Bursill, 1998). NHANES III obtained Adlercreutz H., Fotsis T., Bannwart C., Wahala K., Makela T., Brunow samples from the subjects without consideration of elapsed G., and Hase T. Determination of urinary lignans and phytoestrogen time after meals. metabolites, potential and anticarcinogens, in urine The range of levels in serum for the multiethnic population of womenonvarious habitual diets. J Biochem 1986: 25: 791–797. covers one to three orders of magnitude depending on the Adlercreutz H., Fotsis T., Kurzer M.S., Wahala K., Makela T., Hase T. analyte. The highest mean level of 4.7 ng/ml was observed for Isotope dilutiongas chromatographic–mass spectrometric method genistein, followed by daidzein with 3.9 ng/ml, and enter- for the determination of unconjugated lignans and isoflavonoids in olactone with 3.6 ng/ml. When compared with other humanfeces, with preliminaryresults inomnivorous andvegetarian populations, total isoflavone concentrations, except for women. Anal Biochem 1995: 225: 101–108. Adlercreutz H., Fotsis T., Lampe J., Wahala K., Makela T., Brunow equol, were approximately two to five times higher thanthe G., and Hase T., Quantitative determination of lignans and values reported for omnivorous Finnish women, and two to isoflavonoids in plasma of omnivorous and vegetarian women by three times lower than levels observed for vegetarian Finnish isotope dilutiongas chromatography–mass spectrometry. Scand J women. For lignans in our study, enterolactone was Clin Lab Invest Suppl 1993: 215: 5–18. approximately three times lower, and enterodiol was two Adlercreutz H., Hockerstedt K., Bannwart C., Bloigu S., Hamalainen E., Fotsis T, and Ollus A. Effect of dietary components, including times higher than the values reported for omnivorous Finnish lignans and phytoestrogens, on enterohepatic circulation and liver women(Adlercreutz et al., 1993). metabolism of estrogens and on sex hormone binding globulin Highly significant correlations of the levels in serum and (SHBG). J Steroid Biochem 1987: 27: 1135–1144. urine were observed in the 66 people with matched serum and Adlercreutz H., Hockerstedt K., Bannwart C., Hamalainen E., Fotsis urine samples for daidzein, enterodiol, enterolactone, and T., andBloigu S. Asociationbeweendietary fiber, urinaryexcretion of lignns and isoflavonic phytoestrogens, and plasma non-protein genistein (Table 3). All Spearmancorrelationcoefficients bound sex hormones in relation to . In: Bresciani F, were significantly different from zero (p ¼ 0.0001, except for King RJB, Lippman ME, Raynaud J-P (Eds.) Progess in Cancer O-DMA p ¼ 0.0007). Since the method was not adequately Research and Therapy: Hormones and Cancer Vol. 35, RavenPress: sensitive for detecting equol and matairesinol in serum, these New York, NY 1988, pp. 409–412. levels could not be compared with urinary levels. The high Adlercreutz H., Honjo H., Higashi A., Fotsis T., Hamalainen E., Hasegawa T., and Okada H. Urinary excretion of lignans correlations between urine and serum levels for the other and isoflavonoid phytoestrogens in Japanese men and women measured phytoestrogens suggest that urine levels can be used consuming a traditional Japanese diet. Am J Clin Nutr 1991: 54: to assess phytoestrogenhumanexposure without the needfor 1093–1100. invasive phlebotomy, especially in cases where it is difficult to Adlercreutz H. and Mazur W. Phyto-oestrogens and Western diseases. obtaina blood sample, for example, infantexposure studies. Ann Med 1997: 29: 95–120. Arai Y., Uehara M., Sato Y., Kimira M., Eboshida A., Adlercreutz H., The values reported here provide a reference range for a Watanabe S. Comparison of isoflavones among dietary intake, multiethnic adult US population. The differences in phy- plasma concentration and urinary excretion for accurate estimation toestrogenlevels betweenethnicgroups might be largely of phytoestrogenintake. J Epidemiol 2000: 10: 127–135. influenced by differences in dietary intake and/or metabo- Bennetau-Pelissero C., Le Houerou C., Lamothe V., Le Menn F., lism. The high correlation observed for urinary and serum Babin P., and Bennetau B. Synthesis of haptens and conjugates for ELISAs of phytoestrogens. Development of the immunological tests. levels validates that noninvasive collection techniques, such as J Agric Food Chem 2000: 48: 305–311. those used for urine, can be used to assess phytoestrogen Diem K. (ed.) Documenta Geigy: Scientific Tables 6th ed. Geigy exposure. The assessmentof phytoestrogenexposure inlarge Pharmaceuticals, Ardsley 1962.

Journal of Exposure Analysis and Environmental Epidemiology (2003) 13(4) 281 Valent ı´n-Blasini et al. Urinary and serum phytoestrogens levels in reference populations

HermanC., Adlercreutz B.R., GoldinB.R., Gorbach S.L., Hockerstedt Lamartiniere C.A., Moore J., Holland M., and Barnes S. Neonatal A.V., Watanabe S., Hardin M.J., Makela T.H., Wahala K.T., Hase genistein chemoprevents mammary cancer. Proc Soc Exp Biol Med T.A., andFotsis T. Soybeanphytoestrogenintake andcancer risk. 1995a: 208: 120–123. JNutr1995: 125: 757S–770S. Lamartiniere C.A., Moore J.B., Brown N.M., Thompson R., Hardin Horn-Ross P.L., Barnes S., Kirk M., Coward L., Parsonnet J., and M.J., and Barnes S. Genistein suppresses mammary cancer in rats. Hiatt R.A. Urinary phytoestrogenlevels inyoungwomenfrom a Carcinogenesis 1995b: 16: 2833–2840. multiethnic population. Cancer Epidemiol Biomarkers Prev 1997: Phipps W.R., Martini M.C., Lampe J.W., Slavin J.L., and Kurzer M.S. 6: 339–345. Effect of flax ingestion on the menstrual cycle. J Clin Endocrinol Hutchins A.M., Lampe J.W., Martini M.C., Campbell D.R., and Metab 1993: 77: 1215–1219. SlavinJ.L. Vegetables, fruits, andlegumes: effect onurinary Saarinen N.M., Huovinen R., Warri A., Makela S.I., Valentı´ n-Blasini isoflavonoid phytoestrogen and lignan excretion. JAmDietAssoc L., Needham L., EckermanC., CollanY.U., andSantti R. Uptake 1995a: 95: 769–774. and metabolism of in relation to its antic- Hutchins A.M., Slavin J.L., and Lampe J.W. Urinary isoflavonoid arcinogenicity in DMBA-induced rat mammary carcinoma model. phytoestrogenandlignanexcretionafter consumptionof fermented Nutr Cancer 2001: 41: 82–90. and unfermented soy products. JAmDietAssoc1995b: 95: Setchell K.D., BrownN.M., Desai P., Zimmer-Nechemias L., Wolfe 545–551. B.E., and Brashear W.T. Bioavailability of pur isoflavones in healthy Karr S.C., Lampe J.W., Hutchins A.M., and Slavin J.L. Urinary humans and analysis of commericial soy isoflavone supplements. isoflavanoid excretion in humans is dose dependent at low to JNutr2001: 131(Suppl. 4): 1362S–1375S. moderate levels of soy-proteinconsumption. Am J Clin Nutr 1997: Setchell K.D., and Cassidy A. Dietary isoflavones: biological effects and 66: 46–51. relevance to human health. JNutr1999: 129: 758S–767S. King R.A., and Bursill D.B. Plasma and urinary kinetics of the Va lent ı ´ n-Blasini L., Blount B.C., Rogers H.S., and Needham L.L. isoflavonesdaidzeinandgenisteinaftera singlesoy meal inhumans. HPLC-MS/MS method for the measurement of seven phytoestro- Am J Clin Nutr 1998: 67: 867–872. gens in human serum and urine. J Expos Anal Environ Epidemiol KirkmanL.M., Lampe J.W., Campbell D.R., MartiniM.C., andSlavin 2000: 10: 799–807. J.L. Urinary lignan and isoflavonoid excretion in men and women Wei H., Bowen R., Cai Q., Barnes S., and Wang Y. and consuming vegetable and soy diets. Nutr Cancer 1995: 24: 1–12. antipromotional effects of the isoflavone genistein. Proc Soc Knight D.C., and Eden J.A. A review of the clinical effects of Exp Biol Med 1995: 208: 124–130. phytoestrogens. Obstet Gynecol 1996: 87: 897–904. Whitten P.L., and Patisaul H.B. Cross-species and interassay compar- Kurzer M.S., and Xu X. Dietary phytoestrogens. Annu Rev Nutr 1997: isons of phytoestrogen action. Environ Health Perspect 2001: 17: 353–381. 109 (Suppl. 1): 5–20.

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