Poster Presentation Abstracts

Poster Presentation Abstracts

Isoflavone quantification

Sensitive HPLC Method for Profiling Plasma Phytoestro-gens. Tarja Nurmi and Herman Adlercreutz. Institute forPreventive Medicine Nutrition and Cancer, Folkhalsan Re-search Center, Clinical Chemistry Division, University ofHelsinki, Helsinki, Finland.

Phytoestrogens have several beneficial effects on humanhealth, i.e., they lower the risk of hormone-dependent cancers,reduce menopausal symptoms and are known to prevent os-teoporosis and coronary heart disease. A Western diet con-tains low amounts of phytoestrogens; therefore, sensitive an-alytical methods are required to determine the phytoestrogenprofiles in nonsupplemented samples, especially when metab-olites are also of interest. Traditional analyses are performed byusing gas chromatography–mass spectrometry, which is sensi-tive and selective but also expensive because of multistagesample pretreatment and a high initial investment. HPLCmethods using UV-detection are simpler to use and the de-vices are usually less expensive; however, the sensitivity is nothigh enough for reliable analysis of nonsupplemented humanplasma samples. Therefore, we developed an HPLC methodusing coulometric electrode array detection for analyzingplasma phytoestrogens (daidzein, genistein, O-desmethylango-lensin, equol, dihydrodaidzein, dihydrogenistein, secoislaricir-esinol, matairesinol, enterolactone and enterodiol). Themethod is especially useful for analyzing nonsupplementedplasma samples from Western populations. The method wasevaluated by determining intra- and interassay precision, res-olution, detection limits, linearities, retention time and detec-tor response repeatability. The simple pretreatment requiredcombined with high sensitivity makes the method a valuabletool in clinical and epidemiologic studies.

Development of Rapid Methods for Measuring Isoflavones inHuman Urine. Chunyang Wang and Regina Wixon. SouthDakota State University, Department of Nutrition and FoodScience, Brookings, SD.

UV-visible spectrophotometry was a very successful techniquein measuring isoflavones in soybeans and processed soy proteiningredients. The same approach was used in the developmentof a rapid method for determining isoflavone concentrations inurine samples. Urine samples (n 5 30) with different levels ofisoflavones were collected from a dietary intervention study.The original samples were scanned, and mathematical modelswere developed to predicate isoflavone concentrations on thebasis of spectrophotometric properties. The method was foundto be ineffective. Various simple sample treatments were con-ducted before the scanning. They included the following:sample dilution, extraction using tetra-butyl methyl ether andsolid-phase extraction. The effectiveness of the UV-visiblemethod was improved, as shown by higher correlation coeffi-

cients (R2 5 0.3–0.6). Solid-phase extraction was found to bethe most effective sample treatment before scanning. Predi-cating models were developed for genistein, daidzein, glyciteinand equol. Other developmental efforts will also be reported.This development effort has great potential to be used inscanning a large number of samples. [Supported by the SouthDakota Soybean Research and Promotion Council.]

Liquid Chromatography–Mass Spectrometry Analysis ofIsoflavones in Naturally Brewed and Chemically HydrolyzedSoy Sauce. Amanda Murray, Michelle Smith,* Marion Kirk*and Stephen Barnes.* Department of Pharmacology and Tox-icology, Altamont School, Birmingham, AL and *Universityof Alabama at Birmingham, Birmingham, AL.

Previous reports showed that soy sauce contains very lowamounts of isoflavones. However, the method of preparation ofsoy sauce was not taken into consideration. In this study, wecompared the isoflavone composition and concentration ofnaturally fermented and chemically hydrolyzed soy sauces.Commercially available soy sauces (fermented, Kikkoman,Kikkoman Lite, Kimlan Ponlai; and chemically prepared, An-gostura, Chun King) were analyzed by reversed-phase HPLC at262 nm with a diode array detector and by HPLC–electrosprayionization–mass spectrometry (LC-ES-MS) using a triplequadrupole mass spectrometer. Isoflavones were recoveredfrom the soy sauces either after precipitation with 4 vol ofmethanol or by C8 Sep-Pak cartridge extraction with andwithout preliminary treatment with b-glucosidase. The ex-tracts were taken to dryness, redissolved in 80% methanol,centrifuged and analyzed by HPLC and LC-ES-MS. Initialexperiments using HPLC-UV analysis revealed that fermentedsoy sauce extracts, unlike the chemically prepared soy sauces,had a large number of UV-absorbing compounds. However,the complexity of the profiles prevented unequivocal identifi-cation of individual peaks as possible isoflavones and theirb-glucoside conjugates. Analysis of extracts by LC-ES-MSscanning over the m/z range 240–650 allowed the develop-ment of reconstructed ion chromatograms for each of theisoflavones in soy (m/z 253 for daidzein, m/z 269 for genisteinand m/z 283 for glycitein). Two suspected isoflavone peaks(m/z 285 and m/z 301) corresponding to the addition of twohydroxyl groups to daidzein and genistein were also detected.No evidence was obtained for the presence of isoflavone b-glu-coside conjugates. Confirmation of the identity of each pri-mary isoflavone was made by carrying out MS–MS experi-ments to obtain daughter ion mass spectra of [M–H]- parentmolecular ions. This LC-ES-MS approach clearly establishedthat fermented soy sauces contain each of the primary isofla-vones (in the unconjugated form) and in 5–10 times greaterconcentrations (42–79 mg/mL for total isoflavone concentra-tion) than in the chemically prepared soy sauces. Only minordifferences in composition or concentrations of isoflavoneswere observed between regular and ‘lite’ forms of one of the soy

0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences. J. Nutr. 130: 680S–711S, 2000.

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sauces. The results suggest that natural fermentation, unlikechemical hydrolysis, produces soy sauces that contain uncon-jugated isoflavones, some of which have undergone additionalmetabolism.

Deuteration of Isoflavone Metabolites, Dihydrodaidzein andDihydrogenistein. Kristiina Wahala and Sirpa Rasku. Uni-versity of Helsinki, Department of Chemistry, Laboratory ofOrganic Chemistry, University of Helsinki, Helsinki, Finland.

To study the metabolism and biological and physiologic effectsof phytoestrogens, a sensitive and specific quantitative methodis required. The isotope dilution–gas chromatography–massspectrometry–selective ion monitoring (ID-GC-MS-SIM)technique using synthesized deuterated internal standards forthe correction of losses during the procedure is used to quan-titate phytoestrogens from food and biological fluids. An in-ternal standard for quantitative MS must have no unlabeledspecies, and the isotope labels must remain stable under theanalytical conditions used. For polyhydroxy aromatics, it isdesirable that the reference compound contain three to fivestable deuterium atoms because the unlabeled compound, de-rivatized with trimethylsilyl for GC, will show fairly intensem11 and m12 ions in its mass spectrum. Finally, the standardmust be isomerically and isotopically pure. The synthesis ofnew stable [6,8,39,59-D4]-dihydrodaidzein, [3,6,8,39,59-D5]-di-hydrodaidzein and [3,29,39,59,69-D5]-dihydrogenistein involveshydrogen-deuterium exchange of aromatic protons usingD3PO4zBF3/D2O as a deuteration reagent and deprotonation oflabile deuteriums from fully deuterated dihydrogenistein. Thesites of deuteration were determined from the 1H and 13Cnuclear magnetic resonance (NMR) spectra by comparisonwith those of undeuterated compound. Deuterium-carryingcarbon atoms appear as low intensity triplets in the protonnoise-decoupled spectra compared with the intensive singletsof undeuterated compounds. The exchange order of hydrogenswas determined from different deuteration and dedeuterationexperiments by following the progress of the reaction by NMR.The isotopic purity of the product is determined from the massspectra of trimethylsilylated product to avoid M-1 losses fromphenolic hydroxyls. The new deuterium-labeled dihydrodaid-zein and dihydrogenistein can be reliably used as referencecompounds and introduced at the beginning of the analyticalprocedure because the deuterium labels are securely bound andwill survive the various isolation, purification and derivatiza-tion steps. The ID/GC/MS/SIM method has now been used forthe quantitation of these isoflavone metabolites in humanurine.

Phytoestrogen Content of Various Natural Products. TarjaNurmi and Herman Adlercreutz. Institute for PreventiveMedicine Nutrition and Cancer, Folkhalsan Research Center,Clinical Chemistry Division, University of Helsinki, Helsinki,Finland.

Phytoestrogens, shown to possess several beneficial effects onhuman health, are of particular interest for the pharmaceuticalindustry. Phytoestrogens seem to lower the risk of hormone-dependent cancers and reduce menopausal symptoms; they areknown to prevent osteoporosis and coronary heart disease inexperimental studies. A Western diet contains amounts ofphytoestrogens that are too low to allow their levels in plasmato become high enough for biological activity. Instead ofchanging the traditional diet, it is now possible to supplementit with isoflavone-containing concentrated natural products.

Positive health effects may occur with moderate amounts ofphytoestrogens and high amounts may not have any effects.There is a difficult dilemma, i.e., what is a moderate amountand what is a sufficient amount? In cancer treatment, on theother hand, very high amounts may be needed. The phy-toestrogen content of natural products varies significantly, andthe amounts recommended to be consumed are not alwaysspecified. Ten commercial products were analyzed by usingHPLC with a coulometric electrode array detector. The ana-lytical method was carefully standardized and optimized toseparate the following isoflavones: daidzein, genistein, gly-citein, formononetin, biochanin A, and daidzein- andgenistein-7-O-glucosides and their malonyl-glucosides. Thesamples were analyzed before and after the hydrolysis to quan-tify the conjugate forms of the phytoestrogens occurring in theproducts. Phytoestrogen content of the concentrated naturalproducts was slightly or moderately lower than the amountsclaimed by manufacturers. However, in one preparation, thecontent of phytoestrogens was minimal.

HPLC: Electrochemical Detection of Isoflavones and Lig-nans in Human Plasma. Paul H. Gamache and Ian N. Ac-worth. ESA Inc., Chelmsford, MA.

Interest in potential health benefits of phytoestrogens hascreated the need for simple and reliable techniques for theirmeasurement. A highly sensitive method that uses HPLC withcoulometric array detection was developed for the determina-tion of plasma phytoestrogens. Analytes were separated in 25min by reversed-phase (C18) isocratic elution using a water/methanol/acetonitrile, 68:25:7 (v/v/v) mobile phase contain-ing sodium acetate buffer (0.2 mol/L, pH 4.8) at a flow rate of0.6 mL/min and column temperature of 42°C. Eight serialelectrochemical sensors were used at potentials of 340, 470,500, 530, 560, 620, 680 and 760 (mV vs. Pd) to generatevoltametric response relationships for each analyte. After en-zymatic hydrolysis, plasma samples were acidified, washed withhexane and extracted with diethyl ether. Combined extractswere evaporated and the residue was dissolved in 50% (by vol)aqueous methanol before HPLC–electrochemical detection(ECD) analysis. Lower limits of quantification (15% relativestandard deviation) estimated from plasma extracts were 0.8,1.2, 0.8 and 1.4 ng/mL of daidzein, enterolactone, equol andgenistein, respectively. By least-squares regression, the re-sponse was linear with concentration for all analytes (r 0.99,six levels, 10.0–2500 ng/mL, two replicate extractions at eachlevel on 2 d). Intra- and interassay variability for plasmaaugmented with 50 ng/mL of standards was ,5.5% RSD (n5 10) and ,7.7% RSD (4 d), respectively. Plasma levels(mean 6 SD) obtained from 15 adult volunteers were 2.76 3.9, 4.2 6 3.4, ,1.2 and 2.7 6 2.1 ng/mL of daidzein,enterolactone, equol and genistein, respectively. The de-scribed method is suitable for measuring plasma phytoestrogenin human subjects at basal levels and after supplementation.

The Synthesis of 13C-Labeled Phytoestrogens. Tara Fryatt,Nigel P. Botting and Mark F. Oldfield. University of St.Andrews, St. Andrews, Scotland.

Accurate analysis of the biological effects of phytoestrogens onhuman health has been hindered by the lack of stable internalstandards for gas chromatography–mass spectrometry analysisand for metabolic studies. The primary objective of this workwas to develop efficient syntheses of labeled phytoestrogens foruse in studies such as these. The initial targets were 13C-

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labeled daidzein and genistein, compounds that are recognizedas protective factors against the development of hormone-dependent cancers. Synthetic procedures for the preparationof the isoflavonoid phytoestrogens formononetin, biochaninA, daidzein and genistein, incorporating a single 13C label atthe 4-position were developed. These procedures use adapta-tions of existing methodologies and suitable labeled precursors.Current work is focusing on multiply labeled 13C-isoflavonoidderivatives. Work is also being carried out on developingmethods for the synthesis of 13C-labeled lignans, for example,secoisolariciresinol and matairesinol.

Isoflavone absorption and metabolism

Transport and Metabolism of Soy Isoflavones (Glycosides vs.Aglycones) in Caco-2 Cells and in Perfused Gut Segments.Aukje Steensma,*† Hub P.J.M. Noteborn,* Marcel J. B.Mengelers,* Harry A. Kuiper*. *State Institute for QualityControl of Agricultural Products (RIKILT), Wageningen, TheNetherlands and †Wageningen Agriculture University, De-partment of Food Technology and Nutritional Sciences, SubDepartment of Toxicology, Wageningen, The Netherlands.

Genistein and daidzein are present in minor amounts in soy-beans and soy-derived foods, whereas their sugar-conjugatedderivatives are present in relatively high amounts (1–3 mg/gproduct). Data on the bioavailability of isoflavones are scarce.In this study, Caco-2 cells derived from a human colon ade-noma carcinoma were used as a model for intestinal absorp-tion. Information was obtained on the transport, metabolismand mechanism of action of genistein, daidzein and theirglycosides by growing the cells on semipermeable filters. Thesein vitro transport and metabolism data were compared withthose from intestinal perfused segments of the rat. In Caco-2cells, a significant difference in the transport and metabolismbetween the aglycones and their glycosides was observed.Genistein and daidzein added at the apical side were trans-ported to the basolateral side, whereas their glycosides werehardly transported in this direction. In perfused gut segments,the transport of genistein was also higher than its glycoside.Furthermore, the transport of genistein was highest in the ilealsegment, whereas there were no differences in transport of theglycoside in various other segments tested. In both modelsystems, the glycosides were metabolized to their respectiveaglycones. Genistein was metabolized mainly to sulfates andglucuronides in the Caco-2 cells and to glucuronides in theperfused gut segments. Our data also indicated that Caco-2cells and rat segments contained exogenous or endogenousglucosidase activity (or both) because only aglycones could bedetected at the basolateral side of the Caco-2 cells and in theresorbate of perfused gut segments.

Absorption of Isoflavone Aglycones and Glycosides in Post-menopausal Women. M. Richelle, S. Pridmore-Merten, S.Bodenstab,* I. Tavazzi, S. Jecklin* and E.A. Offord. NestleResearch Center, Lausanne, Switzerland and *Nestle ProductTechnology Centre, Konolfingen, Konolfingen, Switzerland.

Isoflavones naturally present in soybeans and soy-based foodsare mainly in the glucoside form. It is believed that removal ofthe sugar moiety of the glucoside (by b-glucosidase activity) isrequired for its absorption through the intestinal wall. Wecompared the bioavailability of isoflavone aglycones and glu-cosides from an isoflavone-rich extract consumed as a drink.The isoflavone-rich extract was hydrolyzed enzymatically by

commercial b-glucosidases to produce aglycones. Drinks wereconstituted by mixing the isoflavone-rich extracts with water,sugar and orange flavor. The pharmacokinetics of isoflavonesin plasma were determined over 34 h in six postmenopausalwomen. After the ingestion of both soy drinks (aglycone orglucoside), plasma total isoflavone increased rapidly, reachinga maximal concentration of 4 mmol/L between 5 and 7 h.Thereafter, plasma isoflavones decreased slowly, leading toplasma concentrations that were still elevated at 34 h postab-sorption. The ratio of plasma daidzein to genistein mimickedthe ratio found in the soy drink. A low level of dihydroxy-daidzein (0.2 mmol/L), a metabolite of daidzein, appeared inplasma after 4 h. The pharmacokinetics of plasma isoflavoneswere similar with both products. In conclusion, hydrolysis ofglucoside before ingestion does not improve the bioavailabilityof isoflavones from isoflavone-enriched extracts.

Kinetic Models Describing In Vitro Transport and Metabo-lism of Isoflavones and Their Glycosides in Human Caco-2Cells. Aukje Steensma,*† Marcel J. B. Mengelers,* HubP.J.M. Noteborn* and Harry A. Kuiper.* *State Institute forQuality Control of Agriculture Products (RIKILT-DLO),Wageningen, The Netherlands and †Wageningen AgricultureUniversity, Department of Food Technology and NutritionalSciences, Sub Department of Toxicology, Wageningen, TheNetherlands.

Kinetic models can be applied to describe the in vitro trans-port and metabolism of isoflavones by using intestinal epithe-lial cells (Caco-2). These models may enable a quantitativecomparison of in vitro and in vivo bioavailability parameters.An extended kinetic model was developed in Caco-2 cellsgrown on semipermeable filters that described the transport ofisoflavones from the apical to basolateral side and vice versa.The model also included the metabolic activity of the cells.Transport of the isoflavones across the intestinal cells wascontrolled by diffusion. However, the transport rate of theglycosides across Caco-2 cells was too low to enable a kineticmodeling. Additional metabolic studies were carried out toincorporate metabolic rates into the kinetic model. The met-abolic rates obtained from the metabolism studies could beincorporated into the model used for describing the transportexperiments without alterations.

Absorption and Deglycosylation of Isoflavone Glycosides inthe Small Intestine. A. P. Wilkinson, J. M. Gee, A. J. Day,M. S. DuPont, P. W. Needs, G. W. Plumb, I. T. Johnson,M. R. A. Morgan* and G. Williamson. Institute of FoodResearch, Norwich Research Park, Norwich, UK and *ProctorDepartment of Food Science, University of Leeds, Leeds, UK.

The predominant isoflavones in soy are the glycosides, al-though low levels of the aglycones are also present. Isoflavonesappear in plasma within 30 min of ingesting soy, indicatingsome absorption from the small intestine. The aim of thisstudy was to investigate whether isoflavone aglycones areabsorbed preferentially in the small intestine compared withtheir glycosides. Daidzin and daidzein absorption was studiedwith the use of an in vitro rat everted-gut model. Everted sacsof rat proximal jejunum were filled with physiologic saline andsuspended for 15 min at 37°C in oxygenated (95% CO2/5%O2) Krebs buffer (pH, 7.2–7.4) containing either daidzein ( 0,10, 100 or 1000 mmol/L) or daidzin ( 0, 1, 10 or 100 mmol/L).After the incubation, the serosal fluids were collected andstored. An ELISA for daidzein and HPLC were used to both

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quantitate amounts of daidzin or daidzein transported acrossthe rat small intestine and investigate the chemical formspresent in the serosal fluid. A similar degree of absorption ofdaidzin and daidzein was observed. Absorbed daidzin anddaidzein were rapidly metabolized within the small intestinalenterocytes. The principal metabolite of both daidzin anddaidzein found in the serosal fluid was daidzein-O7-glucuro-nide. The mechanisms involved in the absorption and metab-olism of daidzein and daidzin may be as follows. Daidzeindiffuses passively into the enterocyte where it is conjugatedwith glucuronic acid, a reaction mediated by UDP-glucuronyltransferase, forming daidzein-O7-glucuronide, which is subse-quently transferred across the basolateral membrane into theserosal fluid. Two mechanisms may account for daidzin uptakeand metabolism. It may enter the enterocyte by interactionwith an active sugar transport mechanism. Once inside thecell, daidzin can be hydrolyzed by an intracellular cytosolicb-glucosidase recently shown to be present in the human smallintestine (Day et al. 1998). This enzyme can effectively hy-drolyze daidzin and genistin. After daidzin hydrolysis, theliberated daidzein is conjugated with glucuronic acid as de-scribed above. Alternatively, daidzin may be hydrolyzed at themucosal brush border membrane by another enzyme (lactasephlorizin hydrolase). Our recent studies have shown that thisenzyme can hydrolyze isoflavone glycosides. Thus, at the mu-cosal brush border, daidzin may be hydrolyzed by lactase phlo-rizin hydrolase and the daidzein formed then diffuses into theenterocyte to be metabolized as described above. It is fre-quently inferred that absorption of isoflavone glycosides occursonly after their hydrolysis by b-glucosidases associated withthe large intestinal microflora. The results of this study showthat both daidzin and daidzein can be absorbed from the smallintestine and that there is little difference between the two intheir rate of uptake.Day, A. J. , DuPont, M. S. , Ridley, S, Rhodes, M., Rhodes M.J.C., Morgan, M.R.A.

& Willamson, G. (1998) Deglycosylation of flavonoid and isoflavonoid gly-cosides by human small intestine and liver b-glucosidase activity. FEBS Lett.436: 71–75.

Review of Glycitein Bioavailability and Biological Effects.Suzanne Hendrich, Tong T. Song, Yan Zhang, Gui-JuanWang and Patricia A. Murphy. Food Science and HumanNutrition, Iowa State University, Ames, IA.

Glycitein (49,7-dihydroxy-6-methoxyisoflavone) constitutes5–10% of total isoflavones in most soybean foods and ;40% oftotal isoflavones in soy germ (SoyLife). Glycitein dispositionand bioavailability were assessed in humans and its estroge-nicity was assessed in mice. Total isoflavones of 4.5 mmol/kgbody weight were fed to seven men and seven women fromsoymilk or SoyLife in a single meal in a randomized crossoverdesign. Urinary excretion and plasma content of isoflavoneswere analyzed by HPLC-UV. Interindividual variation inisoflavone disposition was controlled by selecting subjects ofthe moderate fecal isoflavone degradation phenotype (averagein vitro fecal genistein half-life of 8.9 6 4.3 h). Urinaryexcretion as a percentage of ingested dose differed significantlyamong isoflavones as follows: glycitein, 55%; daidzein, 46%;and genistein, 29% (P , 0.05). Plasma isoflavone contentsafter soymilk feeding paralleled soymilk isoflavone contents(genistein daidzein glycitein; P , 0.05) in both sexes. Plasmaisoflavones paralleled SoyLife isoflavone contents in men(daidzein glycitein genistein), but at 6 h after dosing, plasmaglycitein and genistein did not differ in women. The sexdifference may be due to isoflavone biotransformation differ-ences and may require further study. Weanling female B6D2F1

mice were dosed with glycitein or genistein (3.0 mg/d for 4 d)or diethylstilbestrol (0.03 mg/kg for 4 d). Uterine weight wasincreased by 150% by glycitein, 50% by genistein and 60% bydiethylstilbestrol, all significantly greater than the control.Glycitein was threefold more estrogenic than genistein (P, 0.05), suggesting that although glycitein is present in lesseramounts in most soy ingredients and foods, it may be respon-sible in part for certain biological effects of soy-containingfoods.

Metabolites of Dietary Phytoestrogens Daidzein, Genisteinand Glycitein. S. Heinonen, K. Wahala* and H. Adlercreutz.Folkhalsan Institute for Preventive Medicine, Nutrition andCancer, and Department of Clinical Chemistry, University ofHelsinki, Finland and *Laboratory of Organic Chemistry, De-partment of Chemistry, University of Helsinki, Finland.

Soy and soy-based foods are rich sources of the isoflavonesdaidzein, genistein and glycitein. The beneficial properties ofthese dietary phytoestrogens include prevention of hormone-dependent diseases, such as breast and prostate cancers, osteo-porosis and cardiovascular disease. In this investigation, themetabolites of soy isoflavones were studied in humans after soywas consumed. The isolation and the characterization of theurinary metabolites were carried out with absorption chroma-tography on Sephadex LH-20 and gas chromatography–massspectrometry (GC–MS). The structures of the isolated isofla-vones were confirmed by using authentic reference com-pounds. We identified dihydrogenistein, 69-OH-O-desmethyl-angolensin, and cis-4-OH-equol in addition to the knownisoflavonoids, daidzein, genistein and glycitein, and the knownmetabolites, equol, O-desmethylangolensin and dihydrodaid-zein, by comparing the retention times and the spectra of theurinary compounds with those of reference standards. For thefirst time, the metabolites of glycitein were investigated, andnew compounds such as 59-OH-O-desmethylangolensin and59-methoxy-O-desmethylangolensin were isolated and identi-fied tentatively by GC–MS. The metabolic pathways for daid-zein, genistein and glycitein are presented on the basis of theisolation and identification of these isoflavonoids from humanurine.

Plasma Isoflavone Concentrations in American Men andWomen Consuming Different Levels of Isolated Soy Proteinfor up to 6 Months. S. Teixeira, S. M. Potter* and J. W.Erdman, Jr. Division of Nutritional Sciences, University ofIllinois at Urbana-Champaign, Urbana, IL and *Protein Tech-nologies International, St. Louis, MO.

Isoflavones are plant compounds with mild estrogenic activity.These compounds are found in high concentrations in soy-beans and in many soy foods. It has been known for some timethat Asian populations with high intakes of isoflavone-richproducts have high concentrations of these compounds inblood and concomitantly a lower incidence of cardiovasculardisease, several types of cancer, osteoporosis and menopausalsymptoms. As part of two larger studies (a men’s and a wom-en’s study) on the effects of soy protein consumption on bloodlipid concentrations, we measured plasma concentrations ofseveral isoflavones by HPLC coulometric array detection(eight-channel detector) and HPLC mass spectrometry. Theplasma levels of daidzein, dihydrodaidzein, O-desmethylango-lensin, equol, genistein, p-ethyl phenol, glycitein and totalisoflavones were analyzed in 81 men and 66 postmenopausalwomen. In both studies before the soy feeding, the subjects

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consumed a National Cholesterol Education Program Step Idiet for 3 wk (men) or 2 wk (women). In the men’s study, 92subjects, divided into five groups, were fed 50 g/d of a soyprotein–casein mixture in different proportions (0:50, 20:30,30:20, 40:10 and 50:0 g/d) for 6 wk, with corresponding levelsof isoflavones (0, 29.1, 44.8, 62 and 95.1 mg aglycones/d).Blood isoflavones were measured at 0, 3 and 6 wk. In thewomen’s study, three groups (n 5 81 subjects) were fed 40 g/dof soy protein with no isoflavones (casein only) or a moderate(55.6 mg aglycones/d) or high level (90 mg aglycones/d) ofisoflavones. The subjects received the study protein for 6 mo.Blood isoflavones were measured at 0 and 24 wk. The effects ofchronic consumption of different amounts of isoflavones onblood isoflavone concentrations were analyzed by multiplelinear regression. The outcome measured was the isoflavoneconcentration for each subject, with treatment effects repre-sented as each group contrasted with the control group (ca-sein). In the men’s study, soy feeding resulted in higher plasmaconcentrations of all isoflavones except for O-desmethylango-lensin and glycitein. In the women’s study, soy feeding resultedin higher plasma concentrations of all isoflavones analyzed.Plasma isoflavone concentrations after soy feeding, except forequol, were higher in women than in men. At baseline, plasmaisoflavone concentrations were higher in men. [Supported inpart by the Illinois Soybean Program Operating Board, theIllinois Council for Food and Agriculture Research, and Pro-tein Technologies International.]

Novel Chlorinated and Nitrated Derivatives of Soy Isofla-vones Formed by Proinflammatory Oxidants. Brenda J.Boersma, Rakesh P. Patel, Marion Kirk, Victor M. Darley-Usmar and Stephen Barnes. University of Alabama at Bir-mingham, Department of Pharmacology and Toxicology, Bir-mingham, AL.

Several chronic inflammatory diseases including atherosclero-sis and many types of cancer are associated with the produc-tion of the proinflammatory oxidants hypochlorous acid(HOCl) and peroxynitrite (ONOO2). Because the soy isofla-vones have structural similarities with tyrosine, a biologicaltarget of HOCl and ONOO2, we hypothesized that isofla-vones react with HOCl and ONOO2, thereby modulating thebiological responses to these proinflammatory oxidants. Ininitial studies carried out in vitro using HPLC and liquidchromatography–mass spectrometry (LC–MS), we demon-strated that the isoflavones genistein and biochanin A areconverted to their mono- and dichlorinated derivatives byHOCl, whereas daidzein forms a monochlorinated derivative.In the case of ONOO2, mononitrated products of genisteinand daidzein, but not biochanin A, were formed. Treatment ofgenistein with HOCl and sodium nitrite led to the formationof a doubly substituted chloro-nitro derivative. In biologicalexperiments, we used polymorphonuclear neutrophils (PMN)that were activated with phorbol 12-myristate 13-acetate(PMA) to simulate a respiratory burst that generates severalreactive oxygen species, including HOCl. When genistein,daidzein or biochanin A was added to this cellular system,monochlorinated derivatives identical to those found in our invitro studies were detected by LC–MS. In addition, humanleukemia (HL-60) cells, which can be differentiated withdimethylsulfoxide to form PMN-like cells, are activated withPMA to produce reactive oxygen species. Addition of each ofthe isoflavones to these cells also led to the formation ofsimilar amounts of the monochlorinated products. These re-sults indicate that the chlorinated derivatives of the isofla-

vones could be formed under pathologic conditions whenreactive oxygen species are generated. By using the HL-60cells, we have a model system in which we can investigate theformation and properties of these novel isoflavone metabolitesin a renewable biological system.

Flavones and Isoflavones: Synthesis and Isotopic Labeling.Matthew R. Benton and Nigel P. Botting. School of Chem-istry, University of St. Andrews, Andrews, Fife, UK.

At present, we are interested in the synthesis of isoflavonesand flavones. Both of these classes of compounds are elicitingconsiderable interest because of their biological effects. Inparticular, they show anticancer activity and are being inves-tigated as potential chemopreventive agents. The poster de-scribes our studies on the synthesis of novel isoflavones and thedevelopment of improved routes for the synthesis of isoflavonemetabolites (e.g., glucosides or glucuronides). Recently, wehave also been developing methods for the synthesis of isofla-vones containing multiple 13C atoms. These derivatives can beused as internal standards for analysis and also in metabolicstudies. This work is now being extended to examine thesynthesis of 13C-labeled flavones. Two of the initial targets areapigenin and naringenin labeled with three 13C atoms.

Maternal and Cord Blood Phytoestrogen Levels in Indone-sian Women. Fabien S. Dalais, Andreanyta Meliala andMark L Wahlqvist. International Health and DevelopmentUnit, Monash University, Alfred Hospital, Prahran, VIC,Australia.

Epidemiologic, cell, animal and human studies have suggesteda potential beneficial link between phytoestrogens from soyand cancer, cardiovascular disease, bone metabolism andmenopausal symptoms. Japanese individuals have among thelowest rates of hormone-dependent cancer and low rates ofcardiovascular disease and menopausal symptoms. It has beenshown that Japanese mothers and their infants have highlevels of isoflavones in their plasma and cord blood. Indone-sian individuals also consume high levels of soybean in theform of tempeh and tofu. The aim of this study was to deter-mine whether Indonesian mothers and their infants wereexposed to levels of isoflavones similar to those of the Japaneseindividuals and possibly received similar health benefits. Bloodand cord blood samples were collected from 30 women givingbirth at Bethesda Hospital in Yogyakarta, Indonesia. Thematernal levels of the isoflavones genistein and daidzein(mean 6 SEM) were 83.1 6 11.7 and 28.9 6 6.49 nmol/L,respectively, and the cord blood levels were 91.7 6 12.5 and33.9 6 5.62 nmol/L, respectively. On average, these levels aremarginally lower than those published for Japanese individu-als. Because these individuals are exposed to isoflavones at anearly stage of life, these compounds may modify hormonalmetabolism and in turn alter disease profiles later in life.

Absorption of Soy Isoflavone Aglycone in Humans and ItsAntiatherosclerotic Effect in Rabbits. Toru Izumi, Jun Ya-makoshi, Akio Obata, Koichiro Tobe, Makoto Saito, Shige-hiro Kataoka and Mamoru Kikuchi. Kikkoman Corporation,Research and Development Division, Noda City, Japan.

Isoflavone is one of the biologically active compounds insoybeans. There are two types of soy isoflavones, i.e., glucosideforms (IFG) and aglycone forms (IFA). The absorption of IFGhas been reported but that of IFA has not yet been reported.

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We tested the absorption of IFA in humans and their anti-atherosclerotic effect in rabbits. We measured the isoflavoneconcentration in plasma by HPLC after the intake of IFA andIFG by humans. IFA were absorbed faster and in largeramounts than were IFG from both low (0.11 mmol) and high(1.7 mmol) single intakes. The plasma concentration ofgenistein was higher than that of daidzein after intakes of IFAand IFG. We also tested the antiatherosclerotic effect of IFAand IFG in cholesterol-fed rabbits. IFA significantly inhibitedthe progression of atherosclerosis in a dose-dependent manner(IFA 1.0%, P , 0.01; IFA 0.33%, P , 0.05). In rabbits fed adiet containing 0.55% IFG (equimolar to 0.33% IFA), theprogression was not significantly inhibited. Our data show thatIFG are not directly absorbed from the gut but are convertedto IFA by intestinal bacteria and then are absorbed. Thus, IFAare more effective than IFG for the prevention of atheroscle-rosis.

Osteoporosis

Soy Consumption and Bone Mineral Density in Older Jap-anese American Women in King County, Washington.Madeline M. Rice,* Andrea Z. LaCroix,*† Johanna W.Lampe,† Gerald van Belle,* Mark Kestin,*†** Barbara L.Drinkwater,‡ Amy B. Graves†† and Eric B. Larson*. *Uni-versity of Washington, Seattle, WA; †Fred Hutchinson Can-cer Research Center, Seattle, WA; **Bastyr University, Seat-tle, WA; ‡Pacific Medical Center, Seattle, WA; and††University of South Florida, Tampa, FL.

The purpose of this study was to examine the relation betweensoy consumption and bone mineral density (BMD) in 267older Japanese-American women aged 65–93 y. Soy consump-tion was measured using a 14-item soy food-frequency ques-tionnaire. Soy isoflavone intake was estimated by using pub-lished isoflavone (genistein plus daidzein) content of soy foods.BMD of the hip and spine was measured by using dual-energyX-ray absorptiometry. Least-squares means general linear mod-els were used to estimate mean BMD according to categories ofcurrent and lifetime soy consumption. Current soy isoflavoneintake was grouped by tertiles (low, moderate, high). Lifetimesoy intake was categorized as low (lowest current isoflavonetertile with same or less intake during adolescence, late 20sand late 40s), high (highest two tertiles with same or moreintake during adolescence, late 20s and late 40s) and varied(all other women). All analyses were adjusted for age, weight,language spoken at the interview, age at menarche and post-menopausal years without estrogen. Femoral neck BMD inwomen who consumed high amounts of soy throughout lifewas 0.680 g/cm2 compared with 0.628 g/cm2 in women whoconsumed very little soy throughout their lifetime (P 5 0.03).Among women currently using fiber supplements, lumbarspine BMD was 0.968 g/cm2 in women in the highest tertile ofcurrent isoflavone intake compared with 0.843 g/cm2 inwomen in the lowest tertile (P 5 0.01). No association wasobserved between current isoflavone intake and lumbar spineBMD in women who were not using fiber supplements.Women using postmenopausal estrogen appeared to benefitmost from high soy consumption, although this effect modifi-cation was not significant. Postmenopausal estrogen users whowere high soy consumers had the highest BMD at all sites.

Urinary Isoflavone Levels and Several Factors That Influ-ence Bone Metabolism in Postmenopausal Women. Chung JaSung, Sun Hae Choi and Byoung-Seob Ko.* Department of

Food and Nutrition, Sookmyung Women’s University, Seoul,Korea and *Korea Institute of Oriental Medicine, Seoul, Ko-rea.

Several studies showed that isoflavones in soy protein areresponsible for its bone-sparing effects. Soy products containlarge amounts of isoflavones, which have estrogenic and an-tiestrogenic properties, and may protect against postmeno-pausal osteoporosis. The purposes of this study were to inves-tigate the association between urinary isoflavones and a boneresorption biochemical maker, deoxypyridinoline (DPD), andto investigate the correlation of DPD with several factors,including bone mineral density (BMD) of the spine and fem-oral neck. We interviewed 160 postmenopausal women aged47–85 y and selected 60 women who had a higher frequencyof soy product consumption to answer a questionnaire aboutsoy food consumption. We administered a 24-h dietary recall,made anthropometric measurements and collected spot urinesamples. BMD was assessed by dual-energy X-ray absorption-metry. Urinary DPD and pH values were measured and majorisoflavones (genistein and daidzein) were analyzed by usingHPLC. The concentration of urinary isoflavones was nega-tively correlated with urinary DPD (P , 0.01). DPD level wasnegatively correlated with weight and height. Urinary isofla-vone levels were positively correlated with age. With multipleregression analysis, DPD was negatively associated with uri-nary genistein and femoral BMD; spinal BMD was positivelyassociated with femoral BMD, body mass index and urinarypH; and femoral BMD was negatively associated with DPD.We conclude that urinary isoflavone levels, urinary pH andbody mass index affect urinary DPD and BMD. Therefore,consuming soy food can be one way to protect against boneresorption.

Effects of Soy Isoflavones, Daidzein, Genistein and Gly-citein, on Bone Loss and Lipid Metabolic Pathway in Ovari-ectomized Rats. Hitoshi Ishida, Takehiko Uesugi,* ToshiyaToda* and Kuniro Tsuji. School of Pharmaceutical Science,University of Shizioka, Shizuoka Japan and *Fujicco Co. Ltd.,Kobe, Japan.

It would be helpful to discover a natural dietary substance thatwould minimize the risk of bone loss and normalize lipidmetabolism in postmenopausal women. Recently, some soyproducts containing isoflavones (e.g., daidzein, genistein orglycitein) were shown to have positive effects on bone densityand to reduce abdominal fat in ovariectomized (ovx) rats. Itwas found that ipriflavone, which is structurally related to soyisoflavones, shows a similar effect. Therefore, we hypothesizedthat soy isoflavones might be effective in ameliorating thebone loss and hypercholesterolemia due to ovarian hormonedeficiency. To test our hypothesis, we studied the effects ofdaidzein, genistein and glycitein on bone loss and lipid me-tabolism in ovx Sprague-Dawley rats (age 11 wk). Each com-pound was administered orally to ovx rats for 4 wk. The femursof these rats showed significantly lower density and breakingstrength than did those of sham-operated rats. These changeswere largely prevented in rats that received daidzein, genisteinor glycitein at a dose of 50 mg/(kg z d) and in rats that receivedsubcutaneous estrone [7.5 mg/(kg z d)] as a positive control.Ovariectomy caused atrophy of the uterus and increased theratio of the urinary excretion of pyridinoline and deoxypyr-idinoline to endogenous creatinine. This was prevented byadministration of daidzein, glycitein or estrone but, interest-ingly, not genistein. With respect to food intake and body

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weight, rats in the ovx group had significantly higher finalbody weights and food intake than did rats in the sham-operated group. Daidzein and glycitein, like estrone, preventedthis ovariectomy-induced increase in body weight gain andfood intake, whereas genistein did not. Daidzein and glyciteinreduced abdominal fat and the level of serum total cholesterolin comparison with the control group as did estrone. Theresults indicate that daidzein and glycitein seem to be proestro-genic compounds; genistein appears to have a mechanism andsite of action different from those of these two compounds.

Relationship Between Urinary Isoflavones and Bone Metab-olism in Postmenopausal Japanese Women. Y. Fukui, A.Miura,*† Y. Nara,** T. Uesugi, K. Honda and Y. Yamori.*†

Fujicco Co. Ltd., Kobe, Japan; *WHO Collaborating Centerfor Research on Primary Prevention of Cardiovascular Dis-eases, University of Kyoto, Kyoto, Japan; †Department ofEnvironmental Preservation and Development, GraduateSchool of Human and Environmental Studies, University ofKyoto, Kyoto, Japan; and **Graduate School of IntegratedScience and Art, University of East Asia, Shimonoseki, Japan.

Soy isoflavones are recognized as having beneficial effects onbone health. This study examines the effect of daily isoflavoneintake on bone metabolism. We investigated the association ofurinary isoflavones excretion with bone density or urinarybone resorption markers in postmenopausal Japanese women.Subjects included two populations as follows: middle-agedJapanese women (n 5 39, 54.8 6 3.3 y) living in Japan (JJ)and elderly Japanese women (n 5 48, 74.4 6 3.7 y) living inHawaii (JH). Urine samples were collected for 24 h and wereanalyzed for the urinary isoflavones daidzein and genistein andbone resorption markers pyridinoline and deoxypyridinolineby HPLC. The bone density, given as the stiffness value, wasestimated by an ultrasonic bone densitometer. All subjectswere divided by stiffness values into three groups as follows:the higher stiffness, middle stiffness and lower stiffness groups.In both populations, the higher stiffness group had signifi-cantly higher urinary isoflavone (daidzein and genistein) ex-cretions than did the lower stiffness group (JJ: 20.0 6 12.8 and8.6 6 7.7 mmol/d; JH: 26.6 6 24.5 and 9.7 6 8.9 mmol/d,respectively, for the higher stiffness and lower stiffness groups;P , 0.05). Significant inverse correlations were found betweenurinary isoflavones excretion and bone resorption markerspyridinoline and deoxypyridinoline in JJ (r 5 20.338, P, 0.05 and r 5 20.387, P ,0.05, respectively, for pyridino-line and deoxypyridinoline). Isoflavones taken from daily Jap-anese diets were related inversely to bone resorption markersand positively to bone density in menopausal women. Thus, asufficient daily isoflavone intake is expected to contribute tothe prevention of postmenopausal osteoporosis.

Effects of Soy Isoflavones on Blood Lipids, Blood Pressureand Biochemical Markers of Bone Metabolism in Postmeno-pausal Women. William W. Wong. USDA/ARS Children’sNutrition Research Center, Department of Pediatrics, BaylorCollege of Medicine, TX.

Cessation of estrogen production with menopause results inhypercholesterolemia, hypertension and elevated bone loss inpostmenopausal women. Estrogen replacement therapy lowersblood lipids, increases arterial compliance and minimizes boneloss in postmenopausal women but increases the risk of cancer.Isoflavones, found in abundance in soybeans, are weak estro-gens and were shown to inhibit mammary cancer cell forma-

tion and growth. To determine the effects of soy isoflavones onblood lipids, blood pressure and biochemical markers of bonemetabolism in postmenopausal women, the fasting blood lipidconcentrations, blood pressure and biochemical markers ofbone metabolism of six postmenopausal women [ages 55.46 3.5 y, (mean 6 SD); body weight, 75.0 6 10.7 kg; height,164.7 6 5.2 cm] ingesting 160 mg/d of soy isoflavones weremeasured before and after 6 wk of treatment. After isoflavonetreatment, no significant changes were observed in bodyweight (20.8 6 2.1%, P 5 0.40) or in blood concentrationsof total cholesterol (0.5 6 7.5%, P 5 0.88) or LDL cholesterol(20.5 6 9.3%, P 5 0.90). However, an increase of 10.26 10.1% in apolipoprotein B100, the major lipoprotein inLDL cholesterol, was detected (P 5 0.06). Blood concentra-tions of HDL cholesterol and its major lipoprotein, apoli-poprotein A1, were elevated by 5 6 14% and 3 6 11%,respectively, although these increases were not significant (P0.45). No significant change in systolic blood pressure wasobserved; however, there was a significant reduction in dia-stolic blood pressure of 12 6 9% (P ,0.03). Isoflavone treat-ment affected biochemical markers of bone resorption; urinaryconcentrations of deoxypyridinoline and calcium, serum con-centration of parathyroid hormone and urinary calcium excre-tion were modified by 27 6 41%, 212 6 46%, 133 6 60%and 25 6 29%, respectively. Biochemical markers of boneformation also were altered by isoflavone treatment; serumconcentrations of osteocalcin, bone alkaline phosphatase andinsulin-like growth factor I were reduced by 9 6 15, 16 6 23and 8 6 27%, respectively. However, the changes in biochem-ical markers of bone metabolism were not significant. Al-though many of the changes in blood lipids and biochemicalmarkers of bone metabolism induced by the soy isoflavonetreatment were not significant because of the small sample size,the size of many of these changes was similar to that reportedin postmenopausal women receiving estrogen replacementtherapy. Therefore, further studies with a larger number ofsubjects, a longer treatment period, measurement of lumbarspine bone mineral content and bone density and measure-ment of calcium kinetics using the dual-tracer technique arewarranted. [Funded by the USDA/ARS; the soy isoflavonetablets were provided by Schouten USA Inc., and thePyrilinks-D kits, for the measurement of urinary Dpd, werekindly provided by Metra Biosystems, Inc.]

Cancer

Molecular Effects of Soy-Isoflavone Genistein in ProstateCancer Cells. Joanne N. Davis,* Omer Kucuk*† and FazlulH. Sarkar.** Departments of *Cancer Biology, †InternalMedicine, and **Pathology, Karmanos Cancer Institute,Wayne State University School of Medicine, Detroit, MI.

Prostate cancer is the second leading cause of cancer-relateddeaths in men in the United States, accounting for 36% of allmale cancers and 13% of cancer-related deaths in men. Epi-demiologic data provide convincing evidence that dietaryfactors play an important role in the etiology of cancer. Wepreviously demonstrated that the dietary isoflavone genisteininhibits proliferation, induces apoptosis and modulates impor-tant cell cycle regulatory molecules, particularly p21WAF1and cyclin B, in prostate cancer cells, and therefore may be apotential chemopreventive or therapeutic agent. To furtherelucidate the molecular mechanism by which genistein elicitsits effects, we first investigated the role of a transcription factorNF-kB; second, we measured prostate-specific antigen (PSA)

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levels in prostate cancer cells. NF-kB was shown to protectcells against apoptosis by initiating prosurvival mechanisms.We investigated whether genistein modulates NF-kB, partic-ularly the inactivation, which may lead to the apoptosis ob-served in genistein-treated cells. Here we show that genisteindecreases NF-kB activity in prosate cancer cells in a dose-dependent manner. Prostate cancer cells treated withgenistein at 30 and 50 mmol/L for 24 h resulted in reducedNF-kB DNA binding. Using confocal microscopy, we showedthat genistein blocks the translocation of NF-kB p50 and p65subunits from the cytoplasm to the nucleus, preventing NF-kBactivation and prohibiting DNA binding. Additionally, wedemonstrated that genistein abrogates NF-kB activation bytwo known inducers, H2O2 and tumor necrosis factor-a (TNF-a). Prostate cancer cells pretreated with 50 mmol genistein/Lfor 48 h inhibited NF-kB DNA binding and blocked translo-cation of NF-kB subunits to the nucleus when stimulated witheither H2O2 or TNF-a. These results suggest that the inacti-vation of NF-kB by genistein may lead to the cell growthinhibition and apoptosis observed in genistein-treated cells.The most valuable tumor marker used for the detection andmonitoring of prostate cancer is PSA. PSA, a member of thekallidrein family, is a serine protease secreted by prostateepithelial cells. PSA, which is able to cleave the predominantseminal vesicle protein, has been proposed as a candidategrowth factor, cytokine or growth factor regulator and hasbeen linked to tumor progression. Therefore, we investigatedwhether genistein has any effect on PSA expression and se-cretion in the androgen-sensitive prostate cancer cell line,LNCaP. LNCaP cells were treated with genistein at 0, 30 and50 mmol/L for 3 d. The medium was collected and assayed forthe presence of PSA. We observed that treatment withgenistein at 30 mmol/L reduced PSA secretion by 50% and 50mmol/L reduced PSA by 80% compared with control. Usingimmunohistochemistry and Western blot analysis, we deter-mined that genistein inhibits PSA protein expression levelsbut did not affect the protein expression levels of anothertumor-associated antigen, prostate-specific membrane antigen.These results indicate that genistein lowers the PSA levels inprostate cancer cells in vitro. In conclusion, the inactivationof NF-kB and downregulation of PSA by genistein provideencouraging evidence to support genistein’s role as a chemo-preventive and therapeutic agent for prostate cancer. Theseresults also indicate that NF-kB may play a pivotal role ingenistein-induced apoptosis, providing a mechanism by whichgenistein promotes cell death.

The Specific Role of Genistein in Estrogen Metabolism.N. B. Kumar, K. Allen, A. Cantor, G. Shaw and C. E. Cox.H. Lee Moffitt Cancer Center and Research Institute at theUniversity of South Florida, Tampa, FL.

Our goal was to evaluate the individual effectiveness of sup-plementing a group of premenopausal, breast cancer–freewomen with a dietary supplement of the isoflavone genistein(40 mg/d) in producing a change in sex hormones that areimplicated in the initiation and promotion of breast cancer.Consecutively recruited premenopausal omnivorous women (n5 68), of all races and ethnicities, aged 25–55 y, were admit-ted to the study and randomly assigned to an experimentalgroup supplemented with soy (40 mg genistein/d) or to acontrol group consuming a placebo for a 12-wk period.Changes in their anthropometric, nutritional and hormonalbiomarkers from the early follicular phase were analyzed atbaseline and after intervention. Preliminary analysis indicated

that hormonal concentrations of free estradiol decreased by78% in the group consuming genistein compared with 44% inthe placebo group. Serum estrone and sex hormone–bindingglobulin levels were elevated in 47 and 46% of the subjects,respectively, in the experimental group compared with 26 and40%, respectively, in the placebo group. In the experimentalgroup, the menstrual cycle of 58% of the subjects increased bymore than 2 d compared with 36% in the placebo group.These data suggest that increased genistein intake affects es-trogen metabolism by altering the sex hormone concentrationsthat are implicated in breast cancer promotion or inhibition.[Supported by an NIH NCI grant RO3 CA72588-01A1.]

Effect of Soybean Saponins on the Growth and AntioxidantDefense of Human Hepatocarcinoma Cells. M.-K. Sung andM.-Y. Park. Department of Food and Nutrition, SookmyungWomen’s University, Seoul, Korea.

Carcinogenesis is a multistep process including initiation, pro-motion and progression. Recent studies indicated that oxygenfree radicals, by-products of normal cellular respiratory pro-cesses as well as lipid peroxidation processes, induce cellularDNA damage, which is the most plausible mechanism for theinitiation of carcinogenesis. Lipid peroxides also were shownto promote tumor cell growth. Saponins are amphiphilic com-pounds present in a variety of edible and nonedible plants.Recent studies indicated that saponins extracted from soy-beans inhibit the formation of lipid peroxides in corn oilsamples; this may be due to their ability to scavenge radicals.In this study, effects of soybean saponins on the growth,cellular lipid peroxidation and antioxidative enzyme activitiesof HepG2 cells were investigated. Effects of saponins werecompared with a-tocopherol and ascorbic acid. Cells (1–23 107) were incubated for 24 h and then treated with tert-butylhydroperoxide (0.5 nmol/L for 45 min) to initiate lipidperoxidation followed by saponin treatment (300 mg/plate for48 h). Cellular superoxide dismutase (SOD), glutathione per-oxidase (GPX) and glutathione S-transferase (GST) activitieswere measured. Results showed that tert-butylhydroperoxidetreatment significantly increased cellular malondialdehydecontent. Cell growth was significantly decreased with saponin,a-tocopherol and ascorbic acid treatment. Malondialdehydecontent was significantly reduced by saponin (72%) and a-to-copherol (40%). Soybean saponins significantly increased cel-lular SOD, GPX and GST activities. Ascorbic acid signifi-cantly decreased GPX activity. However, the activity of GSTwas not affected by either a-tocopherol or ascorbic acid. Theseresults indicate that soybean saponins possess considerableantioxidative capacity, exerting antiproliferative effects ontumor cells.

Soybean Saponins Inhibit the Formation of DNA Adducts inHuman Colon and Liver Cells. H.-S. Jeon and M.-K. Sung.Department of Food and Nutrition, Sookmyung Women’sUniversity, Seoul, Korea.

Numerous chemical carcinogens, activated to form electro-philic agents, react with DNA, which explains the inductionof a heritable change in a cell leading to malignant transfor-mation. This may be a main event in the initiation of carci-nogenesis. Soybeans contain up to 2% saponins. Soybeansaponins were shown to inhibit the growth of human coloncarcinoma cells with low toxicity. Also, they were shown todecrease the ornithine decarboxylase activity that is directlyrelated to cancer cell proliferation. These results indicate that

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soybean saponins are important modulators in the promotionstage of carcinogenesis. This study was performed to examinethe effects of soybean saponins on DNA adduct formation,which is the most important reaction of carcinogens withcellular macromolecules initiating carcinogenesis. CCD-18Coand HepG2 cells were used as models for colon and liver cells,respectively. Cells (4–5 3 105) were seeded and allowed toattach. After 18 d (CCD-18Co) and 2 d (HepG2) in culture,soybean saponins at a concentration of 0–50 mg/mL wereadded and incubated for 1 h. Preincubated tritiated aflatoxinB1 (12 nmol/L; specific activity 25 Ci/mmol) was added toeach plate and incubated for a further 48 h. DNA was purifiedand aliquots of DNA samples were used to measure radioac-tivity by liquid scintillation counting. Results showed thatsoybean saponins significantly inhibited the formation ofDNA-aflatoxin B1 adducts in CCD-18Co cells by 23.7, 50.7and 49.4% when 10, 30 and 50 mg saponins/mL, respectively,were used. Amounts of DNA adducts in HepG2 cells were alsosignificantly decreased by 37.3, 50.1 and 49.8%, respectively.These results indicate that soybean saponins may effectivelyreduce cellular DNA damage by carcinogens and can be re-garded as potential chemopreventive agents.

Effects of Genistein on the Activities of Antioxidant En-zymes in Strenuously Exercised Rats. C. Chen and R. M.Bakhit. Department of Human Nutrition, Foods and Exercise,Virginia Polytechnic Institute and State University, Blacks-burg, VA.

Male Sprague-Dawley rats (n 5 48), aged 1 y, were assigned tofour groups as follows: normal diet/sedentary, normal diet/exercise, genistein diet/sedentary and genistein diet/exercise.The AIN complete basal diet was supplemented with 500 mggenistein/kg. After 4 wk of consuming the experimental diets,the rats in the exercise groups underwent an acute exerciseprotocol of 22 m/min at 12° inclination for 1 h. Immediatelyafter the exercise, blood was drawn from each rat and tested forantioxidant enzymes [superoxide dismutase (SOD), catalase(CAT) and glutathione peroxidase (GPX)]. Genistein con-centration was measured in the plasma, liver and muscle. Theaverage feed intakes were not significantly different among thefour groups (P 0.05). Plasma, liver and muscle genistein con-centrations were significantly higher in the rats undergoingacute exercise than in the nonexercised rats (P , 0.05). Inaddition, in the exercised rat groups, the genistein-fed rats hadsignificant increases in the activities of two antioxidant en-zymes, GPX and CAT, compared with exercised rats fed thenormal diet (P , 0.05). GPX and CAT levels in the exercisedgenistein-fed rats were comparable to those of the sedentaryrats. No significant changes were observed for SOD. Theresults suggest that acute strenuous exercise might lead to therelease of relatively more genistein from storage tissue intocirculation than would occur with no exercise. There is also anindication that 500 ppm genistein from dietary supplementa-tion might diminish the disadvantages of increased productionof free radicals resulting from acute exercise by maintainingthe antioxidant defense systems in the acutely exercised rats.[Funded by the Virginia Soybean Association.]

Familial Breast Cancer Dietary Prevention DemonstrationTrial. Cristina Bellati and Franco Berrino. Istituto NazionaleTumori, Milan, Italy.

High serum levels of sex hormones and insulin-like growthfactor I (IGF-I) usually precede breast cancer. We recently

conducted a randomized dietary trial (the DIANA study) inwhich a comprehensive modification of Western dietary habitssignificantly reduced the bioavailability of such hormones (tes-tosterone and estradiol decreased by 18%; sex hormone–bind-ing globulin and IGF binding protein increased). The dietaryintervention aimed at reducing insulin resistance and increas-ing the intake of phytoestrogens, both soy isoflavonoids andlignans from various sources. Insulin inhibits the liver synthe-sis of sex hormone–binding globulin and IGF-BP and stimu-lates the ovarian production of sex hormones, whereas phy-toestrogens have the opposite effect. The penetrance of genesresponsible for hereditary breast cancer is likely to be affectedby several environmental factors, including ionizing radiation,tobacco smoking, oral contraceptive use and nutritional hab-its. The latter refer mainly to fruit and vegetable consumptionand the dietary regulation of the availability of sex steroidhormones and IGF and related peptides. Evidence is increasingthat these nutritional factors are more strongly related togenetics than to sporadic breast cancer. We are presentlycarrying out a case-only study (the COS Study) to test thisinteraction in several thousand European women who werediagnosed with breast cancer before age 40. Seven countriesare involved in the study. In Italy, patients who most likelycarry high penetrance mutations together with their healthysisters are being invited to participate in a dietary preventiontrial. The dietary modification strategy for the interventiongroup includes kitchen courses and behavioral and psycholog-ical support. Families randomly assigned to the control groupreceive the usual cancer prevention dietary recommendations,but active intervention will be postponed for several years.The endpoint will be breast cancer in healthy women andcontralateral breast cancer in patients. A study including 400mutation carriers per group has 80% statistical power to detecta 50% reduction in breast cancer incidence.

Urinary Phytoestrogens and Breast Cancer Risk in a Pro-spective Study in Postmenopausal Dutch Women. P.H.M.Peeters,* I. den Tonkelaar,*† P. Van’t Veer,** L. Keinan-Boker,* C.M.J. Arts,‡ H. Adlercreutz†† and J.H.H. Thijs-sen.‡‡ *Julius Center for Patient Oriented Research, Univer-sity Medical Center, Utrecht, The Netherlands;†International Health Foundation, Utrecht, The Netherlands;**Division of Human Nutrition and Epidemiology, Wagenin-gen University, Wageningen, The Netherlands; ‡TNO Nutri-tion and Food Research Center Institute, Zeist, The Nether-lands; ††Department of Clinical Chemistry, University ofHelsinki, Helsinki, Finland; and ‡‡Department of Endocrinol-ogy, UMCU, Utrecht, The Netherlands.

Phytoestrogens, naturally occurring compounds in many foodsand especially in soy products, are defined as plant substancesthat are structurally or functionally similar to estradiol. Twoformer retrospective studies assessed the relationship betweenurinary phytoestrogen excretion and breast cancer risk; theirresults indicated a protective role for phytoestrogens. Urinesamples in those studies were collected after breast cancerdiagnosis, and urinary phytoestrogen levels (a short-term in-dicator) might have been affected by the disease state. Wechose, therefore, to study the associations of certain phy-toestrogens with breast cancer risk by using urinary specimenscollected several years before breast cancer was diagnosed.Subjects were 88 postmenopausal women with breast cancer(cases) and 286 postmenopausal women without breast cancer(controls) selected from a cohort of women (n 5 14,697) whoparticipated in a breast cancer screening program, the DOM

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Project, in Utrecht, The Netherlands. Levels of genistein andenterolactone were determined by time-resolved fluorescentimmunoassay and expressed in micromoles per mole creati-nine. For each subject, the mean value for genistein andenterolactone was computed from two urinary samples col-lected 1 y apart. Odds ratios of the highest to the lowesttertiles of urinary phytoestrogen per creatinine concentrationswere computed. Higher urinary genistein excretion was weaklyassociated with a reduced breast cancer risk, i.e., the odds ratiofor the highest compared with the lowest tertile was 0.83; the95% confidence interval (CI) was 0.46–1.51. In contrast,higher urinary enterolactone excretion was weakly and non-significantly associated with an increased breast cancer risk,i.e., the odds ratio for highest compared with lowest tertile was1.43, 95% CI, 0.79–2.59. Tests for trends for both phytoestro-gens were nonsignificant. We were not able to detect thepreviously reported firm protective effect of phytoestrogens.Such an effect may be smaller than expected or limited topremenopausal women.

Phytoestrogen and Androgen Levels in Australian Post-menopausal Women Diagnosed with Breast Cancer. A. L.Murkies, F. S. Dalais,* E. M. Briganti,† D. L. Heal, H. G.Burge, M. L. Wahlqvist* and S. R. Davis. The Jean HailesFoundation, Clayton, VIC, Australia; *International Healthand Development Unit, Monash University, Clayton, VIC,Australia; †Department of Epidemiology and Preventive Med-icine, Monash University, The Alfred Hospital, Prahran, VIC,Australia.

Isoflavones are phytoestrogens, plant-derived compounds, thathave been implicated as potential anticarcinogenic com-pounds by in vitro, animal, epidemiologic and human case-control studies. The role of endogenous androgens is still beinginvestigated. The aim of this study was to assess the associationbetween urinary excretion of isoflavones (daidzein andgenistein), dietary composition and circulating androgens andtheir 24-h urinary metabolites in postmenopausal women andthe risk of breast cancer. Cases (n 5 18) and controls (n 5 20)completed a detailed dietary questionnaire; a blood sample anda 24-h urine sample were collected. Analysis and detection ofphytoestrogens were carried out by HPLC and UV. Nonpara-metric analysis was undertaken (Mann-Whitney U test) tocompare groups. There were no significant differences in life-style and reproductive variables between groups. Urinary daid-zein excretion was significantly lower in breast cancer patients[0 nmol/24 h; 0, 1375.5 (median and interquartile ranges)]than in control subjects (1558.2 nmol/24 h; 498, 2773.2; P5 0.02), and there was a trend for decreased genistein (P5 0.08) for patients compared with control subjects. Serumtestosterone was elevated in the patients (1.2 nmol/L; 1, 1.6)more than in the control subjects (1 nmol/L; 0.7, 1.25; P5 0.05), whereas no significant differences were observedbetween the two groups for other hormonal measures or totalfat, fiber and vegetable consumption. Women with breastcancer were found to have lower urinary isoflavone levels. Thelower urinary phytoestrogens could be attributed to a reduceddietary intake due to future surgery for breast cancer. Therewas no difference in the usual diet as reported; however, thefood intake questionnaire may not have been sufficiently spe-cific for phytoestrogens and may have had limitations in eval-uating actual phytoestrogens intake from known sources, suchas processed foods. Larger studies are required to examine theassociation between phytoestrogens and breast cancer morethoroughly while addressing the issues of bias.

Serum Insulin-Like Growth Factor I Is Unaffected byGenistein in Wild-Type or Estrogen Receptor-a KnockoutMice. Ruth S. MacDonald, William H. Thornton, Jr., J.Kevin Day and Dennis B. Lubahn. Nutritional SciencesProgram, University of Missouri, Columbia, MO.

Consumption of soy foods is ecologically associated with areduced risk of breast cancer. On the basis of animal experi-ments, the phytoestrogen genistein is suggested to be onecomponent of soy that provides protection. Recent evidencesuggests that the estrogen receptor interacts with growth factorreceptors in several cell types to mediate cellular function. Incultured cells, genistein inhibits tyrosine kinase activity,which is an essential component of the insulin-like growthfactor-I (IGF-I) signaling pathway. Hence, we examinedchanges in serum IGF-I in wild-type (WT) and estrogen re-ceptor-a knock-out (ERKO) mice fed genistein. WT andERKO mice were fed a semipurified diet containing 0 (control)or 1 g genistein/kg diet beginning at age 3 wk. The objectiveof the study was to examine the protective effect of genisteinon breast cancer development; hence two medroxyprogester-one pellets (40 mg each) were implanted subcutaneously intoeach mouse at 6 wk, and DMBA (1 mg/dose) was given orallyat 9, 10, 12 and 13 wk. The mice continued to receive theirrespective dietary treatments for 14 mo. Body weight gain wasslightly less in mice fed genistein than in controls throughoutthe study because of reduced food intake. Tumors developed inall of the WT mice between 29 and 45 wk, but none of theERKO mice developed tumors. In the WT mice, no protectiveeffect of genistein on breast tumor formation was found. SerumIGF-I concentrations were 199 6 69, 512 6 98, 262 6 60 and252 6 85 ng/mL in the WT control, ERKO control, WTgenistein and ERKO genistein groups, respectively. Whenanalyzed as a 2 3 2 factorial by ANOVA, there were nosignificant differences by diet or genotype; however, there wasa diet-genotype interaction. The range of serum IGF-I con-centration in the WT mice was 13–281 ng/mL and in theERKO mice was 127–935 ng/mL, including two mice withvery high levels. The trend for higher IGF-I in the ERKO micefed the control diet, which was suppressed by feedinggenistein, suggests that genistein influenced serum IGF-I con-centrations in these mice. We are continuing to examine therelationship between genistein consumption and the IGF-Iaxis in both WT and ERKO mice. This animal model providesa useful tool for examining the relationships among estrogenreceptors, the IGF-I axis and dietary phytoestrogens. [Fundedby Missouri Agricultural Experiment Station InterdisciplinaryRegional Research Grant.]

Inositol Hexaphosphate Has an Antioxidant Function ThatReduces GST-P1 Foci on Hepatocarcinogenesis in Rats.Hae-Jeoung Lee, Hyeon-duck Kim, Sang-A Lee and HaymieChoi. Seoul National University, Department of Food andNutrition, Seoul, Korea.

Inositol and inositol hexaphosphate (phytate), which arefound in plant foodstuffs such as seeds, grains, fruits andvegetables, were demonstrated to have anticancer, anti-cell-proliferation, and antioxidant functions. This study was de-signed to determine the effects of phytate on rat hepatocarci-nogenesis and whether supplementing with inositol wouldenhance anticarcinogenic and antioxidant effects. Rats re-ceived a single intraperitoneal injection of diethylnitrosamine,were subjected to two-thirds hepatectomy 3 wk later and werekilled 8 wk after the injection. One week before the partial

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hepatectomy, the rats were divided into three groups, andinositol or phytate was added to the drinking water (adjustedpH. 7.4); one group received 2% phytate, one received 2%inositol and one received 1% phytate 1 1% inositol. In allthree groups, the numbers and the areas of glutathione S-transferase–placental positive (GST-P1) foci were signifi-cantly decreased. Thiobarbituric acid–reactive substances(TBARS) content and catalase and GST activities were sig-nificantly reduced in rats fed inositol phosphates. TBARScontent and catalase activities were the lowest in the phytategroup because phytate potently inhibited the formation of theiron-derived hydroxyl radical and suppressed lipid peroxida-tion. Catalase and GST activities were not induced likelybecause of the antioxidant function of inositol phosphates.TBARS content was positively correlated with GST-P1 foci.It seemed that inositol phosphates helped the endogenousdefense system during carcinogenesis by decreasing TBARSand H2O2. Therefore, the preventive effect against hepatocar-cinogenesis can be explained in part by the antioxidant func-tion of inositol phosphates, and there were no differences inGST-P1 foci, activities of catalase and GST, and TBARSwith or without the inositol supplement.

Soy Intake and Colorectal Cancer in Chinese in NorthAmerica and China. Anna H. Wu, Alice S. Whittemore,Marion Lee, Richard P. Gallagher and Zheng Shu. Universityof Southern California, Department of Preventive Medicine,Los Angeles CA.

Epidemiologic studies suggest that a high intake of soy foodsmay reduce the risk of certain types of cancer, including thoseof the breast, prostate and endometrium. However, the role ofsoy foods in colorectal cancer is less clear. Our purpose was toexamine the relationship of soy foods to risk of colorectalcancer, with a focus on consistency in findings in NorthAmerica and China, in men and women, or colon and rectalcancer. Cases included persons with histologically confirmedcolorectal cancer who were identified through the population-based tumor registries of Los Angeles, San Francisco, Vancou-ver and Ontario (n 5 432 in North America) and in Hang-zhou, China (n 5 473). Control subjects matched to cases byage, sex and study area were interviewed (n 5 1296 in NorthAmerica, n 51192 in China). Dietary and nondietary (includ-ing physical activity and body size characteristics) informationwas collected during home interviews. Odds ratios were esti-mated by using conditional logistic regression stratified jointlyby age, sex and study area. The intake pattern for soy will bepresented by continent, sex, and disease status. Risk of colo-rectal cancer was 40% lower in association with a high soyintake (at least two times per week compared with fewer thantwo times per month) for men in North America, but a similarreduction in risk was not observed for women in North Amer-ica. In China, the data showed no clear patterns of altered riskfor colorectal cancer with intake of individual soy foods or allsoy foods combined. Further adjustment for other dietary andnondietary factors did not change these findings. Thus, noconsistent or strong associations between colorectal cancerrisk and consumption of soy foods were found in this study.

Radical-Scavenging Activity in Brown-Colored SoybeanSeed Coat. Yashurio Takahata, Shu Furuta, Masakazu Ta-kahashi and Ikuo Suda. Kyushu National Agricultural Exper-iment Station, Ministry of Agriculture, Forestry and Fisheries,Nishigoshi, Kumamoto, Japan.

Soybeans having a brown- or black-colored seed coat havebeen used in the daily diet traditionally and occasionally inJapan. However, when the effect of soybeans on human healthis discussed, most of the background data are derived from anormal soybean that has a yellowish-white–colored seed coat.Here we investigated the 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical-scavenging activities of a brown-colored soy-bean seed coat by using indigenous varieties and breedingmaterials. Among four indigenous varieties, Akita-Zairaishowed the highest radical-scavenging activity. We crossedAkita-Zairai and a normal soybean cultivar to obtain progenyseeds as breeding materials. In the F3 seed generation, seedswere classified roughly into the following four phenotypes onthe basis of their seed coat phenotype: yellowish-white, lus-terless pale brown, lustrous pale brown and dark brown. DPPHradical-scavenging activity in the lustrous pale brown F3 seedcoat was higher than that in dark-brown F3 and the originalparental Akita-Zairai. The yellowish-white seed coat showednegligible activity as did the lusterless pale brown. The differ-ence in luster seemed to be a key trait in the intensity ofradical-scavenging activity. DPPH radical-scavenging activi-ties paralleled the content of polyphenolics. Using an LH-20column, we fractionated compounds demonstrating radical-scavenging activities and showed that DPPH radical-scaveng-ing activities and proanthocyanidin content were concomitantin each fraction. We could demonstrate that proanthocyani-dins are possible candidates for radical-scavenging activities inthe brown soybean seed coat. Soymilk made from cotyledonsof normal cultivar and seed coats of Akita-Zairai had 2.5-foldhigher radical-scavenging activity than did soymilk made fromnormal soybean alone. This result indicates that the radical-scavenging activity in brown seed coats is retained after pro-cessing.

Effect of Estrogen and Estrogen Mimics for the Growth ofMCF-7 Cells in Nude Mice. Keiji Sugi, Takahiko Gotoh,Yin Hong and Akihiro Ito. Department of Cancer Research,Research Institute for Radiation Biology and Medicine, Hiro-shima University, Hiroshima, Japan.

The estrogen-responsive human breast cancer cell MCF-7 wasexamined for its growth in nude mice under various doses of17-b-estradiol (E2), bisphenol A and a miso diet. Seven-week-old nude mice were inoculated subcutaneously in six sites eachwith 5 3106 MCF-7 cells/site. Mice were treated with intra-muscular injection of E2 at 0.001, 0.01 and 0.1 mg/mouse; oraladministration of bisphenol A at 10, 100 or 1000 ppm; or adiet containing 10% miso. Twelve weeks later, inoculatedtumor sites and weight were scored. Tumor take (incidence)was highest in control mice (47%, 223 mg). For E2 adminis-tration, they were 22%, 194 mg with 0.001 mg; 25%, 393 mgwith 0.01 mg; and 39%, 257 mg with 0.1 mg. Similarly,bisphenol A showed a dose-dependent increase by the inci-dence of 29%, 565 mg with 10 ppm; 40%, 616 mg with 100ppm; and 42%, 869 mg with 1000 ppm. For the miso group,the values were 17% and 185 mg. Immunohistochemical stain-ing for antibodies to pS2 estrogen-regulated protein and BrdUshowed high frequencies in tumor tissues.

Meta-Analysis of Soy Intake and Breast Cancer Risk. BruceTrock, Leslie White Butler, Robert Clarke* and Leena Hi-lakivi-Clarke.† Departments of Human Oncology, *Physiolo-gy and Biophysics, and †Psychiatry, Lombardi Cancer Center,Georgetown University Medical Center, Washington, DC.

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High soy intake in Asian countries was proposed as a factorcontributing to the low breast cancer risk for Asian women.However, soy is being marketed and recommended to thepublic as if a clear protective effect was established when, infact, the epidemiologic data are rather limited. Because in vivoand in vitro data show estrogenic effects for genistein, themajor component of soy, it is important that associationsbetween soy and breast cancer risk be evaluated before recom-mendations can be made with safety, especially for womenwho already have breast cancer. Therefore, we performed ameta-analysis of epidemiologic studies examining soy andbreast cancer risk. A literature search was based on key wordsassociated with soy or specific isoflavones and phytoestrogens,and breast cancer. An Internet search was also conducted toidentify unpublished data. We analyzed data by using themeasures of soy intake provided in the studies and also nor-malized intake to daily grams of soy protein. Odds ratios (OR)were pooled by using Mantel-Haenszel methods, and randomeffects models were used in the presence of significant hetero-geneity of OR across studies. A total of nine studies (eightcase-control, one cohort) were included in the analysis. Fivestudies were done in Asian women living in Asian countries;one was conducted in women with Asian ancestry living in theWest; and three were conducted in non-Asian populations. Agreat deal of variability was observed in the size of the OR andin the level of soy intake that was defined as high intake. Amodest significant reduction in risk was associated with highsoy intake over all studies [OR 5 0.87; 95% confidence inter-val (CI): 0.80, 0.96]. However, this effect was confined topremenopausal women (OR 5 0.80; 95% CI: 0.71, 0.90).There was no protective effect at all in postmenopausalwomen (OR 5 1.01; 95% CI: 0.86, 1.19). There was also nosignificant effect of soy in women in Asia (OR 5 0.95; 95%CI: 0.83, 1.09). Although there is some evidence of a smallreduction in premenopausal breast cancer risk associated withsoy intake, the number of studies is small, measurement of soyintake is crude and control of confounding factors is inconsis-tent. Interpretation of these results is further complicated bythe similar reductions in risk associated with widely varyingsoy intakes and the low percentage of subjects consuming soyin studies of non-Asians. There was also a lack of consistencyin the effects on hormonal measures observed in soy feedingstudies. Coupled with the fact that some studies have sug-gested potentially adverse effects of soy, these data suggest thatrecommendations for women to increase their soy intake toprevent breast cancer or prevent its recurrence are prematureand that larger, more rigorously controlled studies are required.

Premenopausal Equol Excretors Show Plasma Hormone Pro-files Associated with Lowered Risk of Breast Cancer. A. M.Duncan, B. E. Merz-Demlow, X. Xu, W. R. Phipps* and M. S.Kurzer. Department of Food Science and Nutrition, Universityof Minnesota, St. Paul, MN and *Department of Obstetrics-Gynecology, University of Rochester, Rochester, NY.

Increased urinary excretion of equol, a metabolite of theisoflavone daidzein, has been associated with lowered risk ofbreast cancer (Ingram et al. 1997). This risk reduction hasgenerally been presumed to be a consequence of increasedisoflavone consumption. However, only 30–40% of the pop-ulation excretes more than trace amounts of equol regardless ofisoflavone intake. Accordingly, we hypothesized that the ob-served protective effect of equol may be due to hormonaldifferences between equol excretors and nonexcretors. Toevaluate the effects of equol status per se, we compared plasma

hormone and binding globulin concentrations between pre-menopausal equol excretors (n 5 5) and nonexcretors (n 5 9)consuming identical isoflavone doses (10, 64 and 128 mg/d) ascomponents of soy protein isolates for 3.5 menstrual cycleseach. P , 0.05 was considered significant. Urinary equol forexcretors far exceeded that of nonexcretors, even at the lowestdose. At all doses, equol excretors generally had lower con-centrations of estrone, estrone sulfate, testosterone, andro-stenedione, dehydroepiandrosterone, dehydroepiandrosteronesulfate and cortisol and higher concentrations of sex hor-mone–binding globulin and midluteal progesterone, a hor-monal pattern generally consistent with lowered breast cancerrisk. Thus, the association of lowered breast cancer risk withequol excretion may largely reflect the tendency of equolexcretors to have more favorable hormonal profiles rather thanmerely reflecting increased isoflavone intake. Equol may be amarker for the presence of colonic bacterial enzyme activitythat increases fecal steroid excretion. Alternatively, equolitself, even with very modest isoflavone intake, may exertbeneficial effects on the regulation of endogenous hormones.[Supported by NIH grants CA-66016 and MO1-RR00400, anda gift from Protein Tech. International.]Ingram, D., Sanders, K., Kolybaba, M. & Lopez, D. (1997) Case-control study of

phyto-estrogens and breast cancer. Lancet 350: 990–994.

Phytoestrogen Effect on Human Breast Epithelium. N. J.Bundred, D. F. Hargreaves,* W. R. Miller,† M. Morton,‡ I.McFadyen, A. Howell,** S. A. Roberts* and C. S. Potten.*Academic Department of Surgery, South Manchester Univer-sity Hospital, Manchester, UK; *Paterson Institute of CancerResearch, Manchester, UK; †Department of Surgery, Edin-burgh Royal Infirmary, Edinburgh, Scotland; **Academic De-partment of Medical Oncology, Christie Hospital, Manches-ter, UK; and ‡Tenovus Institute, Cardiff, Wales.

Epidemiologic studies suggest that phytoestrogen consumptionprevents breast cancer, but the mechanism is unclear. Todetermine the effect of dietary soy protein supplementation onestrogenic- and androgenic-induced proteins in breast secre-tions, we conducted two randomized controlled trials of 14 dand 3 mo of treatment with either placebo or soy protein inpremenopausal women. Levels of phytoestrogens rose in bothserum (P # 0.001 for genistein and daidzein) and nippleaspirate (genistein: 387 6 433 ng/mL before treatment; 4306 430 ng/mL after 14 d of treatment) after supplementation.Nipple aspirate pS2 (an estrogen-induced protein) rose andapolipoprotein D (apo D) fell after 14 d and 3 mo of supple-mentation (Table 1). Phytoestrogens exert an estrogenic stim-

ulus on human breast as determined by increased secretion ofestrogenic proteins.Antimitotic and Cancer Preventive Properties of a SoybeanPeptide. Ben O. de Lumen and Alfredo F. Galvez. Nutri-

TABLE 1

Change after treatment

Baseline Placebo 14 d soy 3 mo soy

PS2 (ng/mL) 2092 6 2054 800 15725* 11725Apo D (ng/mL) 42.8 6 30 15.8 212.2** 22.1pS2-Apo D ratio 46.1 113 1209.5** 159**

* P # 0.001, ** P # 0.01.

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tional Sciences and Toxicology, University of California atBerkeley, Berkeley, CA.

Epidemiologic evidence suggesting a correlation between dietshigh in soybean and overall low cancer mortality rates, espe-cially those of colon, breast and prostate, has given impetus toidentifying components in soybeans responsible for their an-ticancer properties. We isolated a soybean cDNA encodingthe small subunit peptide of a cotyledon-specific 2S albumin(Gm2S-1). The peptide (named lunasin) has a unique, highlyacidic carboxyl end. A chimeric gene encoding the lunasinpeptide tagged with green fluorescent protein arrested celldivision and caused abnormal spindle fiber elongation, chro-mosomal fragmentation and cell lysis when transiently trans-fected into murine embryo fibroblast, murine hepatoma andhuman breast cancer cells. Deletion of the acidic carboxyl endabolished the antimitotic effect. Immunolocalization of luna-sin and an immunobinding assay using synthetic peptidesrevealed the preferential adherence of lunasin to chromatin.Lunasin is the first antimitotic peptide whose cDNA wascloned and the first from a common food source. We alsoshowed that the exogenous application of lunasin peptide to aslow as 125 nmol/L inhibits the in vitro transformation ofmouse embryo fibroblast cells (C3H 10T 1/2) into tumorousfoci by the carcinogens 3-methyl cholanthrene and 7,12-dimethylbez[a]anthracene. The lunasin gene has potential ap-plication as an antimitotic cancer therapeutic agent, and thelunasin peptide may be an important cancer-preventive com-pound in soybeans. Furthermore, its antimitotic property sug-gests that lunasin could play a native role in arresting mitosisthat initiates the cell expansion phase of seed development inwhich DNA endoreduplication and synthesis of storage pro-teins, lipids and carbohydrates occur.

Potential Effects of Combined Genistein Isoflavone and theGlucocorticoid Dexamethasone on Testicular Cells. JamesKumi-Diaka, Andre Butler and Vu Nguyen. Florida AtlanticUniversity, Davie, FL.

A series of experiments was performed to assess the response oftesticular cells TM3, TM4 and GC-1 spg to the exposure ofgenistein, dexamethasone and a combination of genistein anddexamethasone. The trypan blue exclusion assay was used todetermine the percentage of viability, and the lactate dehy-drogenase cytotoxicity test was used to assess the degree oftreatment-induced cytotoxicity on each cell type. A secondseries of experiments was performed to study and determinethe percentage of treatment-induced apoptotic cell death oneach cell line with the DNA-Tunnel assay ApopTagR kit(TdT-enzyme and digoxigenin-dUTP reaction). Genistein in-duced a concentration-dependent dual effect of growth pro-motion (mg/mL) and inhibition (10 mg/mL) on testis cells,with a significant degree of sensitivity between the cells.Genistein, dexamethasone and the genistein-dexamethasonecombination induced significant apoptosis on testicular cells;there was synergism in induction of apoptosis in testis cellswith the genistein-dexamethasone combination treatment.The synergistic actions of genistein and dexamethasone in theinduction of apoptosis may be of clinical and pathophysiologicresearch interest because of the chemopreventive-therapeuticpotential of genistein and the clinical and pharmacologicapplication of dexamethasone.

Effects of Chronic vs. Acute Phytoestrogen Supplementa-tion on Antioxidant Status, DNA Damage and Insulin-Like

Growth Factor. Jayne V. Woodside, Erica E. Denholm,Maeli J. Campbell, Ian S. Young* and Anthony J. C.Leathem. Department of Surgery, University College London,London, UK and *Department of Clinical Biochemistry, TheQueen’s University of Belfast, Institute of Clinical Science,Belfast, Northern Ireland.

Phytoestrogens, a group of plant compounds, may play a pre-ventative role in endocrine-responsive cancers and coronaryheart disease. However, the range of physiologic effects thatthese compounds may possess remains to be fully elucidated.Phytoestrogens are proposed to act as antioxidants and protectagainst DNA damage, but this work has been carried out onlyin vitro. In addition, like tamoxifen, phytoestrogens may beable to lower insulin-like growth factor (IGF) levels. Weconducted a feeding study examining the effects of acute (asingle 80-mg load) vs. chronic (80 mg/d for 7 d) administra-tion of phytoestrogens in healthy female volunteers (n 5 10)on various indices of antioxidant and lipid status, backgroundDNA damage assessed by using the COMET assay, and IGF-1and binding protein-1 levels. There were no significantchanges in antioxidant status, as measured by FOX1 assay, andlag time to oxidation or total cholesterol, HDL cholesterol ortriglyceride concentrations in either phase of the study. Levelsof background DNA damage in buccal cells were also un-changed after supplementation. However, the mean DNAdamage in lymphocytes was nonsignificantly reduced after 1wk of supplementation (mean 6 SD baseline and postweek,respectively: 4.07 6 2.81 and 2.10 6 0.86, P 5 0.066). Levelsof IGF-1 were unchanged after the single 80-mg load. How-ever, after 1 wk of supplementation, IGF-1 concentrationswere raised (mean 6 SD baseline and postweek, respectively:190.9 6 85.0 and 280.1 6 95.4 ng/mL, P ,0.01). Levels ofIGF binding protein-1 were not altered by phytoestrogens ineither the acute phase or the chronic phase of the study. Thisstudy provides a rationale for carrying out further placebo-controlled studies looking at phytoestrogen consumption andlevels of IGF and DNA damage.

Soy Protein Has an Anticarcinogenic Activity by ReducingGST-P1 Foci but Not by Apoptosis in Rat Hepatocarcino-genesis. Haymie Choi, Hyeon-duck Kim, Jo-hye Hwang andHae-jeoung Lee. Seoul National University, Department ofFood and Nutrition, Seoul, Korea.

This study was designed to examine the effect of dietaryproteins in rat hepatocarcinogenesis. Six-week-old Sprague-Dawley rats were fed diets containing soy protein or casein at10 and 20%. Two weeks after the diet feeding, hepatocarci-nogenesis was initiated by diethylnitrosamine and promotedby 2-acetylaminofluorene and two-thirds partial hepatectomy.The rats were killed at 4 d or 8 wk after the diethylnitrosamineinjection. The areas and numbers of glutathione S-transferase–placental positive (GST-P1) foci and apoptotic hepatocytes,the contents of thiobarbituric acid–reactive substances(TBARS) and total glutathione (GSH), and the activities ofGST and glucose 6-phosphatase (G6Pase) were measured toinvestigate the anticarcinogenic effect of soy protein. Theareas and numbers of GST-P1 foci were significantly lower inthe soy protein groups than in the casein groups. The areas andnumbers of apoptotic hepatocytes were significantly increasedin the casein groups and decreased in the soy protein groups.From these results, apoptosis cannot be the reason for thedecrease in GST-P1 foci in soy protein groups. TBARS andGSH contents were not significantly different between groups.

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G6Pase activity, a biomarker of membrane stability, was in-creased at 4 d and 8 wk in the soy protein groups. Theseincreased G6Pase activities were inversely related to the de-crease of GST-P1 foci. Soy proteins seem to be more anticar-cinogenic than casein by decreasing the preneoplastic lesions,which may be explained in part by an increase in membranestability rather than by apoptosis during the initiation andpromotion periods.

Postprandial Changes in Male Sex Hormones after MealsContaining Soy (as Tofu), Lean Meat or Fatty Meat. R.Habito and M. J. Ball. School of Biological Sciences, DeakinUniversity, Burwood, VIC, Australia.

Prostate disease is considered to be sex hormone dependent,and dietary and lifestyle factors could affect sex hormoneconcentrations in a manner that may alter the risk for prostatedisease. Because individuals spend a considerable portion oftheir time in a postprandial state, we investigated the effect ofdifferent meals on sex hormone concentrations, including theeffect of one meal with soy protein containing isoflavones. Theeffects of three isocaloric meals were evaluated sequentially in15 healthy men at 7-d intervals. The meals were a lean meatmeal, a tofu meal (both containing 28% energy from proteinand 20% energy from fat) and a fatty meat meal (54% energyfrom fat). Blood samples were obtained at baseline (fasting,premeal) and at 2, 3 and 6 h after each meal for analysis ofserum testosterone, sex hormone–binding globulin (SHBG)and free androgen index. There was a significant fall in tes-tosterone within 2 h after both the tofu and lean meat meals.The changes in SHBG concentrations were significantly morepositive after the tofu meal than the lean meat meal at 2 and3 h (P 5 0.04 and 0.01, respectively). The 2-h serum testos-terone and the decremental area under the curve were signif-icantly less negative after the fatty meat meal in which therewas little change from baseline. Lower biologically active sexhormone concentrations after the lean meat and particularlyafter soy protein, as tofu, compared with fatty meats mayprovide long-term benefits in reducing the risk of a diseasesuch as prostate cancer that appears to be sex hormone de-pendent.

Regulation of Male Sex Hormone Levels by Isoflavone In-take. Jong-Sang Kim, Hyun-Mi Son and Chong-suk Kwon.*School of Food Science, Inje University, Kimhae, Korea and*Department of Food Science and Nutrition, Andong Na-tional University, Andong, Korea.

Soy isoflavones were reported to inhibit 5-a-reductase, whichis involved in the conversion of testosterone into dihydrotes-tosterone, an active form of male sex hormone. We hypothe-sized that feeding soy or isoflavones should cause a change inthe plasma dihydrotestosterone level in male rats. Rats fed adiet containing either soy flour or semipurified isoflavones for1 wk showed a significantly lower level of plasma dihydrotes-tosterone, but not testosterone, than did the rats fed thecontrol diet. This hormonal change caused by soy flour orisoflavones may be partially responsible for the prevention ofprostate cancer resulting from the consumption of soy.

Effects of Genistein and Radiation on HeLa Cells. Shung-jun Yang, Mingliang Jiang and Sameer Rafla. Department ofRadiation Oncology, New York Methodist Hospital, WeillMedical College, Cornell University, Brooklyn, NY.

Much effort has been made toward enhancing cure rates ofadvanced tumors by combined use of radiotherapy with anti-neoplastic drugs, including natural compounds of plant origin.Genistein, one of the major flavonoids isolated from soybeans,has been linked to the low incidence of breast, prostate andcolon cancers observed in some Asian countries. Moreover,increased public awareness of the importance of a healthfuldiet has led to the widespread use of soybean products andother natural nutritional supplements. The ability of genisteinto inhibit the activities of protein-tyrosine kinase and DNAtopoisomerase II, enzymes involved in DNA synthesis and cellproliferation, is well documented. In addition, topoisomeraseII plays an important role in repair of DNA damage. Genisteinis thus a promising candidate for clinical application as ananticancer agent. Thus, understanding possible interactions ofgenistein with conventional medical treatment such as radia-tion would be beneficial in treating cancer or other disorderssuch as arteriovenous malformation or arterial restenosis. Inthis work, we treated human HeLa cells, which are cervicalcancer derived, with genistein and X-rays, individually or incombination. Modifications in cell cycle distribution, certaincell cycle checkpoint genes and single-cell clonogenic survivalwere studied. Genistein cytotoxicity was found to be depen-dent on drug concentration in the culture medium and theduration of cell contact. For a 24-h treatment, concentrationsof genistein mmol/L did not consistently reduce cell survival,whereas those 100 mmol/L killed most cells. Cells preincu-bated with genistein at 20 mmol/L for the same period andthen irradiated with graded doses of X-rays showed lowersurvival levels than did counterpart cells preincubated in nor-mal medium containing no drug and similarly irradiated. Forexample, a 3- and 5-Gy dose of radiation reduced survival to30 and 14%, respectively, in control cells and 18 and 7%,respectively, in cells preincubated with genistein. Genisteinappeared to enhance radiation lethality, suggesting beneficialapplication of combined use to achieve increased cell kill.Studies on other cellular changes are ongoing.

A Double-Blind Randomized Study on the Effects of theIsoflavone P-07 on the Endometrium. Georgina E. Hale,Sanjay Agarwal, Claude Hughes, Stanley J. Robboy andMarcia Bievre. Cedars-Sinai Medical Center, Los Angeles,CA.

Endometrial cancer is the most common malignancy of thefemale genital tract and the fourth most common malignancyafter breast, lung and colon cancer. It is well established thatAsian women have a much lower incidence of endometrialcancer than do Caucasian women and that Asian womenconsume much higher amounts of pulses (tofu and other soyproducts) in their diet. Many of the known risk factors asso-ciated with endometrial cancer are related to prolonged expo-sure to estrogen such as estrogen hormone therapy, early age atmenarche, late age at menopause and obesity. How dietaryfactors can influence this estrogen exposure has been of inter-est to many investigators, and in one case-control study inHawaii, a high consumption of tofu and other soybean prod-ucts was found to be associated with a decreased risk ofendometrial cancer independently of other known risk factors(Goodman et al.1997). Pulses are rich in isoflavones, in par-ticular, genistein. As early as 1966, it was shown that subcu-taneously administered genistein partially reversed the utero-tropic effect of estrogen in rats (Folson et al. 1989), and morerecently, dietary soy protein isolate was shown to partiallyreverse the uterotropic effects of estrogen in monkeys (Foth

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and Cline 1998). This reversal was reflected in a decrease inthe Ki-67 antigen (a nuclear antigen marker of proliferation)but not a decrease in endometrial thickness. The Ki-67 anti-gen is an excellent marker of endometrial proliferation. Inves-tigators have shown a decrease in Ki-67 antigen staining withprogesterone and increased staining in association with endo-metrial hyperplasia and histological atypia compared withnormal endometrium. To investigate the direct effect of isofla-vones on human endometrium, we designed a randomizedplacebo-controlled study in women of late reproductive age tolook at the Ki-67 antigen staining of endometrial samplesbefore and after isoflavone administration. We are includingwomen between the ages of 45 and 50 y. This age group isassociated with having decreasing levels of progesterone rela-tive to estrogen, making them theoretically at increased risk ofthe proliferative influence of endogenous estrogen. Exclusioncriteria include the use of the oral contraceptive pill or hor-mone replacement therapy within the past 12 mo, a current(and within the past 3 mo) intrauterine device, a regularisoflavone-rich diet over the past 6 wk, cigarette smokingduring the past 12 mo and chronic disease. The isoflavonesupplement we chose is a red clover extract (P-07; NovogenLimited); each tablet contains a predominance of the isofla-vones biochanin A (49-methoxy-5,7-dihydroxyisoflavone) andgenistein (49,5,7-trihydroxyisoflavone) and smaller amounts ofdaidzein (49,7-dihydroxyisoflavone) and formononetin (49-methoxy-7-hydroxyisoflavone), totaling 43 mg unglycosylatedisoflavones. The following tests will be performed at baselineand 3 mo: serum estrogen, progesterone, hormone-bindingglobulin and inhibin B; fasting lipids, glucose and insulin; fullblood count, urea and electrolytes; fasting urinary bone mark-er; and transvaginal ultrasound estimation of endometrialthickness, uterine artery doppler and endometrial biopsy. Par-ticipants will be required to take a tablet of placebo or the redclover extract daily and adhere to a specified low isoflavonediet. Isoflavone excretion will be measured in two 24-h urinecollections at baseline and 3 mo.Folsom, A. R., Kaye, S. A., Potter, J. D. & Prineas, R. J. (1989) Association of

incident carcinoma of the endometrium with body weight and fat distributionin older women: early findings of the Iowa Women’s Health Study. CancerRes. 49: 6828–6831.

Foth, D. & Cline, M. J. (1998) Effects of mammalian and plant estrogens onmammary glands and uteri of macaques. Am. J. Clin. Nutr. 68 (suppl.):1413S–1417S.

Goodman, M. T., Wilkins, L. R., Hankin, J. H., Lyu, L. & Wu, A. H. (1997)Association of soy and fiber consumption with the risk of endometrial cancer.Am. J. Epidemiol. 146: 294–306.

Urinary Isoflavonoids Are Reliable Biomarkers for Soy In-take and Indicate Reduced Breast Cancer Risk. Adrian A.Franke, M.C. Yu,* W. Zheng,† L. Le Marchand and L. J.Custer. Cancer Research Center of Hawaii, University ofHawaii, Honolulu, HI; *University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA; and†School of Public Health and Cancer Center, University ofSouth Carolina, Columbia, SC.

Increasing evidence suggests that certain dietary phytoestro-gens, particularly isoflavonoids and lignans, may protectagainst chronic disorders including heart disease, bone loss.and breast, prostate and colon cancer. Epidemiologic studiesconcerned with the assessment of the role of phytoestrogens inhealth and disease require fast, reliable and affordable tech-niques to measure these phytochemicals in humans favorablythrough noninvasive protocols. We developed fast and selec-tive HPLC methods to analyze isoflavonoids, including theirconjugates and metabolites, from various human fluids and

from nonhuman primate tissues suitable for metabolic, clini-cal, epidemiologic and pharmacologic studies. Analytes wereidentified and quantitated by photodiode array, fluorometricand electrochemical detection and, most recently, by massspectrometric analysis after negative electrospray ionization.Applications of this technique showed that isoflavone levels inhuman milk, plasma and urine increase linearly with increas-ing soy doses and disappear (depending on the dose) 1–2 dafter soy exposure. Urine was preferred over other body fluidsas a biological matrix of isoflavone analysis for epidemiologicstudies because of the noninvasive nature of its collection, itslow biologic hazard, good subject compliance, the fact thaturinary isoflavones reflect a greater time period of soy exposureand its high concentration of isoflavones. Total urinary isofla-vone excretion was found to correlate very well with intake ofsoy products as assessed by a 1-y food-frequency questionnairein 50 multiethnic Hawaiians (daidzein: r 5 0.47, genistein: r5 0.34, glycitein: r 5 0.37, equol: r 5 0.50). A very similarresult was observed by a food-frequency questionnaire in pop-ulations known to consume larger amounts of soy products,including 60 Chinese women from Shanghai (total isofla-vones: P trend 5 0.018) and 147 Chinese from Singapore(total isoflavones: P trend 5 0.04). In a breast cancer casecontrol study with 120 Chinese women from Shanghai, weobserved a protective effect of isoflavones and, to a lesserextent, of lignans. Women in the tertile of highest urinaryisoflavone excretion experienced a 46, 30, 59 and 50% de-creased risk to develop breast cancer vs. those in the tertilewith lowest daidzein, genistein, glycitein and total isoflavoneexcretion, respectively. These findings support a potentialbreast cancer preventive effect achieved by soy consumptionin populations that eat soy foods habitually.

Heart disease and lipids

Dietary Soy Isoflavones Reduce Plasma LDL Cholesteroland Atherosclerosis in a Human Apolipoprotein B Trans-genic Mouse Model. F. M. Steinberg and M. M. Braun.Department of Nutrition, University of California, Davis, CA.

This study used a novel mouse model of atherosclerosis to testthe hypothesis that a soy diet containing isoflavones would becardioprotective, resulting in lower plasma LDL cholesterol(LDL-C) and smaller atherosclerotic lesion area comparedwith a soy diet lacking isoflavones. The 1IF-Soy diet con-tained 0.93 mg total isoflavones/g diet, whereas the compari-son diet (2IF-Soy) used ethanol-washed soy protein contain-ing 0.04 mg total isoflavones/g diet. The humanapolipoprotein B transgenic (h ApoB tg) mouse overexpressesthe h ApoB gene, resulting in a very human-like lipoproteinprofile with high levels of circulating LDL-C. Female weanlingh ApoB tg mice (1IF-Soy; n 5 6, 2IF-Soy; n 5 6) werehoused individually and fed for 20 wk. Blood samples weretaken at baseline and at 20 wk for lipid and lipoproteinseparation by HPLC and colorimetric analysis of cholesterol.The aortic sinus region (150 mm) was evaluated for fattystreak lesions after serial sections were stained with oil red Oand counterstained with Harris’ hematoxylin. Baseline valuesfor total and LDL-C were not different between groups (102.1vs. 83.6 mg/dL, and 56.2 vs. 47.2 mg/dL for 2IF-Soy and1IF-Soy diets, respectively). Final values for total-C were notsignificantly different between groups (291.3 vs. 249.1 mg/dLfor 2IF-Soy and 1IF-Soy diets, respectively ). However, finalLDL-C values were significantly higher in the 2IF-Soy group(204.4 vs. 135.2 mg/dL, P , 0.05). Atherosclerotic lesion

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analysis of the aortic sinus area showed greater mean lesionarea per section in the 2IF-Soy group than in the 1IF-Soy(83,454 vs. 34,766 mm2). These results demonstrate that iso-lated soy protein that contains isoflavones is more beneficialthan isolated soy protein without isoflavones (and other pos-sible components) with regard to indices of cardiovasculardisease in this mouse model of human atherosclerosis.

Lipoprotein Effects of Dietary Soy Protein Isolate before andafter Alcohol Extraction. Hans Meinertz and Karin Nil-ausen.* Department of Medicine B, National University Hos-pital, Copenhagen, Denmark and *Department of MedicalAnatomy, University of Copenhagen, Copenhagen, Denmark.

Animal studies show beneficial effects of alcohol-extractablecomponents of soy protein isolate on both atherogenic andantiatherogenic plasma lipoproteins. We tested the hypothesisthat those components produce beneficial lipoprotein effectssimilarly in humans, in which case soy protein might beprepared to contain optimal amounts of alcohol-extractablecomponents for use in preventive diets. Twelve healthy,normolipidemic subjects, six premenopausal women and sixmen, consumed three liquid-formula diets of identical compo-sition except that they contained either unextracted or etha-nol-extracted soy protein or casein. After 28–32 d, the unex-tracted soy protein diet did not lower total, LDL and VLDLcholesterol or apo B nor did it increase HDL cholesterolsignificantly compared with the extracted soy protein diet,although it did raise plasma apo AI 10% (P , 0.05). Further-more, in women, VLDL cholesterol, total triglycerides and apoAII increased 40, 36 and 6% (P , 0.05), respectively. In men,however, the unextracted soy protein diet decreased the ratioof LDL to HDL cholesterol by 22% (P , 0.05). Most remark-ably, unextracted soy protein increased the atherogenic li-poprotein(a) ;335% (P , 0.001) in both women and men.We conclude that alcohol-extractable components of soy pro-tein increase several lipid risk factors and that in youngwomen, in particular, the total lipid risk factor level appears tobe increased. These data suggest that extracted rather thanunextracted soy protein should be used in antiatherogenicdiets.

Effect of Genistein Injections on Borderline Hypertension inConscious Rats. Nikolai P. Breitkopf, Kathleen M. Eyster,John L. Williams and Douglas S. Martin. South Dakota SoyCardiovascular Research Consortium, Division of Physiologyand Pharmacology, School of Medicine, University of SouthDakota, Vermillion, SD.

Evidence indicates that soy isoflavones decrease plasma lipids,reduce atherosclerosis and alter coronary vascular reactivity.Although the effect of soy isoflavones on blood pressure hasreceived comparatively little attention, direct injection ofgenistein attenuated hypertension development in the spon-taneously hypertensive rat. The goal of this study was todetermine whether genistein has an antihypertensive effect inthe borderline hypertensive rat and to determine the optimaldose for this effect. Female borderline hypertensive rats wereobtained at age 4 wk and treated with intraperitoneal injec-tions of genistein in three doses (1, 10 and 25 mg/kg) orvehicle (dimethyl sulfoxide 10%). Injections were given dailyfor 4 mo. The rats then had arterial and venous cathetersimplanted chronically for the recording of mean arterial pres-sure (MAP) and intravenous drug administration, respectively.MAP reactivity was assessed by graded intravenous infusion of

norepinephrine [0.05–1 mg/(kg z min)]. In the borderline hy-pertensive rats treated with vehicle, MAP averaged 140 6 4mm Hg. There was a slight decrease in MAP to 135 6 9 mmHg in the rats treated with injections of genistein at 1 mg/kg.MAP was reduced further in the group treated with 10 mg/kg(123 6 4 mm Hg). No further decline in MAP was observedin the rats treated with 25 mg/kg (122 6 3 mm Hg). Thus,there is an optimal dose of genistein beyond which no furtherblood pressure–lowering effect is obtained. In addition, weinvestigated the hypothesis that the blood pressure–loweringeffect of genistein injections was due to a reduction in thevascular sensitivity to norepinephrine. The dose-responsecurves for norepinephrine were displaced only slightly bygenistein treatment. The maximal effect was obtained withthe genistein dose of 25 mg/kg, which increased the medianeffective dose for norepinephrine from 0.2 to 0.3 mg/(kgzmin).Nevertheless, the genistein-induced shifts in the norepineph-rine dose-response curves were not large enough or consistentenough to account for the blood pressure–lowering effect ofgenistein. In summary, direct injection of genistein attenuatedhypertension development in the borderline hypertensive rat.The antihypertensive effect of genistein reached a maximumat a dose of 10 mg/(kgzd) and was not mediated via a decreasein the pressor reactivity to norepinephrine. [Supported bygrants from the South Dakota Soybean Research and Promo-tion Council and the Faculty Development Funds of theUniversity of South Dakota School of Medicine.]

Cardiovascular Benefits of Foods Containing Soy Isoflavonesand a-Linolenic Acid. Lee Astheimer, Leisa Ridges, BarbaraMeyer, Rachel Sunderland, Katherine Moerman and PeterHowe. Smart Foods Centre (ARC Key Centre for Teachingand Research), University of Wollongong, NSW, Australia.

We examined the effects of 8 wk of consumption of a specific setof food products containing soy, linseed and canola on plasmalipids, fatty acid profiles of plasma and red blood cells, and plateletthromboxane production in 20 postmenopausal women (bodymass index 5 29.6 6 4.3 kg/m2). The daily supplement provideda total of 32 mg daidzein, 13 mg genistein and an estimated 6 ga-linolenic acid (LNA). Compliance was evaluated through un-scheduled diet recalls, food acceptability questionnaires and uri-nary isoflavone levels at 3 and 8 wk. Combined urinary isofla-vones (daidzein and genistein) increased dramatically from 0.236 0.094 to 6.36 6 1.15 mg/24 at 3 wk but fell to 2.58 6 0.507mg/24 h at 8 wk, coincident with reporting of reduced accept-ability of some high daidzein foods. Significant decreases in total(9.94 6 1.69%), LDL (12.54 6 2.31%), and non-HDL (9.816 2.24%) cholesterol were noted after 3 wk (paired t tests; P, 0.01), but these were no longer significant after 8 wk, perhapsas a result of reduced compliance. Slight but significant increasesin plasma LNA as well as the longer-chain n-3 fatty acids,docosahexaenoic and eicosapentaenoic acids, were noted inplasma after 8 wk. However, a concomitant increase in theincorporation of these long-chain n-3 fatty acids into red bloodcells was not evident. Similarly, platelet thromboxane produc-tion, known to decrease with long-chain n-3 supplementation,was unaffected. Because the predominant effect of n-3 supple-mentation on plasma lipids is reduction of triglycerides with apossible transient increase in LDL, the beneficial changes ofplasma lipids observed in this study are more likely to be attrib-utable to the increased consumption of phytoestrogens, that is,soy isoflavones and linseed lignans. Lignan levels and their po-tential effects on plasma lipids are now being assessed.

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Antihypertensive Effect of Dietary Soy: Functional Role ofNitric Oxide. Doug Martin, Kathleen M. Eyster, John L.Williams and Nikolai P. Breitkopf. South Dakota Soy Car-diovascular Research Consortium, Division of Physiology andPharmacology, School of Medicine, University of South Da-kota, Vermillion SD.

Phytoestrogens such as genistein may have beneficial effects incoronary artery disease, osteoporosis and breast cancer. Studiesin other laboratories showed that genistein reduced arterialresponses to angiotensin II, acute injection of genistein alteredcoronary artery reactivity in monkeys and chronic intraperi-toneal injection of genistein attenuated the development ofhypertension in the spontaneously hypertensive rat (SHR).We tested the hypothesis that dietary intake of genisteinthrough a soy-based diet would attenuate the development ofhypertension in SHR via a nitric oxide mechanism. FemaleSHR underwent ovariectomy at age 4 wk. The rats were fed acustom diet containing whole soy or casein (control) from age5 wk. Mean arterial blood pressure (MAP; mm Hg) and heartrate (HR; bpm) were recorded from conscious unrestrainedrats after 8–12 wk of consuming the diets. These variableswere measured before and after blockade of nitric oxide syn-thase with L-NAME (25 mmol/kg). ANOVA indicated asignificant effect of diet on MAP. Post-hoc analysis revealed asignificant reduction in MAP in the SHR fed soy. In thesoy-fed rats, MAP averaged 150 6 4 mm Hg (n 5 10), whereasin the casein-fed rats, MAP was 164 6 3 mm Hg (n 5 12).Injection of L-NAME into the soy-fed rats caused MAP toincrease by 34 6 3 mm Hg (n 5 8). In the casein-fed rats, theL-NAME–induced pressor response averaged 32 6 3 mm Hg(n 5 11). These values were not significantly different. Ac-cordingly, these data suggest that nitric oxide is not theprimary factor mediating the soy-induced decrease in bloodpressure. [Supported by a grant from the South Dakota Soy-bean Research and Promotion Council.]

Role of Amino Acid Composition of Dietary Proteins inRegulation of Hypercholesterolemia. Elzbieta M. Ku-rowska,*† Isabelle Giroux,† David J. Freeman† and KennethK. Carroll.*† Departments of *Biochemistry and †Pharmacol-ogy and Toxicology, University of Western Ontario, London,Ontario, Canada.

Substitution of dietary soy protein for animal proteins isknown to be associated with a reduction of plasma total andLDL cholesterol, especially in individuals with hypercholes-terolemia. This could be related to high content of arginine(Arg) and the low content of lysine (Lys) and methionine(Met) in soy protein compared with animal proteins. Ourprevious experiments showed that high dietary levels of Lys1 Met in rabbits induce hypercholesterolemia, and this iscounteracted in part by the addition of Arg. In this study, wepostulated that these effects could be mediated by the aminoacids themselves or by their metabolic products and that theregulation could occur directly in the liver. The results dem-onstrated that Met deamination products and a Met transam-ination intermediate, homocyst(e)ine, are unlikely to be im-portant in the hypercholesterolemia induced by Lys 1 Met.However, an involvement of other Met transamination prod-ucts was not excluded because feeding a diet rich in Lys 1 Metled to the accumulation of liver phosphatidylcholine, a com-pound that requires methyl groups from Met for its synthesis.Studies in the human liver cell line HepG2 showed increasesin net production of apolipoprotein B (apo B), the protein

component of LDL, after 24 h incubation of these cells withserum from rabbits fed the diet enriched with Lys 1 Met,whereas decreases were shown after a similar incubation withhigh levels of Arg. Medium apo B levels were not altered byexposure of cells to an excess of Lys 1 Met. The resultssuggested that an intact Arg and metabolites of Lys, Met orboth (rather than intact Lys, Met or both) could be involvedin regulation of LDL metabolism in the liver induced directlyby dietary amino acids. [Supported by the Heart and StrokeFoundation of Ontario and by the Ontario Soybean Growers’Marketing Board.]

A Prospective Study on Urinary Phytoestrogen Excretionand the Risk of Fatal Myocardial Infarction in Postmeno-pausal Women. Yvonne T. Van der Schouw, Jos H. H.Thijssen,* Herman Adlercreutz,† and Diederick E. Grobbee.Julius Center for Patient Oriented Research, University Med-ical Center, Utrecht, The Netherlands; *Endocrinology Lab-oratory, University Medical Center, Utrecht, The Nether-lands; and †Meilahti Hospital, Helsinki, Finland.

The purpose of this study is to investigate prospectivelywhether urinary excretion of genistein and enterolactone, as amarker for phytoestrogen intake, is associated with fatal myo-cardial infarction risk. Estrogen replacement therapy has beenassociated with a reduced cardiovascular disease risk. Phy-toestrogens are a family of plant compounds that have beenshown to have both estrogenic and antiestrogenic properties.Accumulating evidence suggests that phytoestrogens may con-fer health benefits related to cardiovascular diseases. We stud-ied the relation between phytoestrogen excretion and fatalmyocardial infarction in a prospective cohort study of 12,239women living in the city of Utrecht, who were initially agedbetween 52 and 67 y. The vital status of these women wasfollowed between 1976 and 1995 (168,513 y). Concentrationsof genistein and enterolactone in one overnight urine samplewere determined in urine samples of 250 women who died ofmyocardial infarction and in a reference group of 250 womenwho did not. Concentrations of genistein and enterolactonewere measured with time-resolved fluorescence immunoassaysand adjusted for creatinine excretion. Data were analyzed byusing a nested case-control analysis. With Poisson regression,we quantified the association between quartiles of genisteinand enterolactone excretion and death caused by myocardialinfarction. Higher urinary genistein excretion was not associ-ated with reduced myocardial infarction mortality risk; the riskratio for the highest compared with the lowest quartile was0.29, 95% confidence interval 0.76–1.04. Results for en-terolactone excretion were comparable. In conclusion, wewere not able to detect a protective effect of phytoestrogenexcretion on myocardial infarction mortality risk.

Oral Genistein Does Not Retard the Development of Hy-pertension in Growing Spontaneously Hypertensive Rats.Roger A. King and Richard J. Head. CSIRO Health Sciencesand Nutrition, Adelaide, SA, Australia.

Genistein has been shown to impair the constrictor responseof isolated blood vessels to the neurotransmitter norepineph-rine in vitro (Toma et al. 1995). Oral treatment with isofla-vones from soy (Nestel et al. 1997) and clover (Nestel et al.1999) for 5–10 wk has also been shown to improve vascularcompliance in women, but blood pressure (BP) was not re-duced in normotensive menopausal women (Nestel et al.1999) or in men with high normal BP (Hodgson et al. 1999).

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The spontaneously hypertensive rat (SHR) is a commonmodel of human essential hypertension in which BP risessharply after ;4–5 wk of age accompanied by hypernordren-ergic innervation of blood vessels and vascular hypertrophy,with BP ;50–70 mm Hg above their normotensive WistarKyoto (WKY) controls at 3 mo of age (King et al. 1992). TheSHR model allows the study of the entire period of hyperten-sion development, which is not possible in human studies. Inthis study, we sought to confirm that genistein impairs theconstrictor response of isolated rat aortic rings to norepineph-rine and to determine whether genistein, administered orallyduring the period of development of hypertension, reduced BPor aortic hypertrophy in SHR. In Experiment 1, the effect ofgenistein at 50 mmol/L on the constrictor response of aorticrings to graded doses of norepinephrine from 0.12 nmol/L to0.8 mmol/L was studied in WKY rats. Genistein decreased thetension developed by ;50% at all but the lowest concentra-tion of norepinephrine (P , 0.05). In Experiment 2, SHRwere fed an isoflavone-free purified diet at age 5 wk, when BPis similar to that of WKY controls. Genistein treatment wascommenced immediately at a rate of 3 or 6 mg/kg body weightas a once-daily oral gavage in 25 mmol Na2CO3/L. Controlrats received vehicle. Blood pressures were measured by atail-cuff method before the commencement of treatment andafter 4 and 8 wk of treatment, at which time the rats werekilled. Heart weight and abdominal aorta weight-length ratiowere measured as indices of hypertrophy. There was no signif-icant difference in any of the measured indices between thetwo treatment doses; thus the results were combined. Therewas no effect of genistein on BP, body weight, heart weight oraortic weight-length ratio (Table 1). We confirmed thatgenistein impaired the constrictor response of isolated rataortae to norepinephrine; however, oral genistein did notinfluence the development of hypertension or aortic hypertro-phy in SHR.Hodgson, J. M., Puddey, I. B., Beilin, L. J., et al. (1999) Effects of isoflavonoids

on blood pressure in subjects with high-normal ambulatory blood pressurelevels—a randomized controlled trial. Am. J. Hypertens. 12: 47–53.

King, R. A., Smith, R. M., Krishnan, R., et al. (1992) Effects of enalapril andhydralazine treatment and withdrawal upon cardiovascular hypertrophy instroke-prone spontaneously hypertensive rats. J. Hypertens. 10: 919–928.

Nestel, P. J., Pomeroy, S., Kay, S., et al. (1999) Isoflavones from red cloverimprove systemic arterial compliance but not plasma lipids in menopausalwomen. J. Clin. Endocrinol. Metab. 84: 895–898.

Nestel, P. J., Yamashita, T., Sasahara, T., et al. (1997) Soy isoflavones improvesystemic arterial compliance but not plasma lipids in menopausal and peri-

menopausal women. Aterioscler. Thromb. Vasc. Biol. 17: 3392–3398.Toma, C., Jensen, P. E., Prieto, et al. (1995) Effects of tyrosine kinase inhibitors

on the contractility of rat mesenteric resistance arteries. Br. J. Pharmacol.114: 1266–1272.

The Effect of Isolated Soy Protein on Total HomocysteineLevels in Adult Peritoneal Dialysis Patients. Carol J.

Boushey, Sara Long Anderson* and M. Rene Malinow.†

Department of Food and Nutrition, Purdue University, WestLafayette, IN; *Food and Nutrition Program, Southern IllinoisUniversity, Carbondale, IL; and †Oregon Regional PrimateResearch Center, Oregon Health Sciences University, Beaver-ton, OR.

The purpose of this investigation was to examine the effects ofisolated soy protein on total homocysteine (tHcy) concentra-tions in individuals treated with peritoneal dialysis. Twenty-six patients (58% males) were originally recruited. Agesranged from 36 to 73 y with an average of 55 y. Subjects wereprovided 35 g of isolated soy protein in the form of a beveragepowder and baked products to consume every day in additionto their usual diets. Nonfasting serum samples were collectedat baseline, 6 and 12 mo. Analyses of serum samples includedtHcy, folate, pyridoxal 59 phosphate (pyridoxine) and cobal-amin (vitamin B-12). Similar to results from other studies ofdialysis-dependent, end-stage renal disease (ESRD) patients,the average tHcy level in this group at baseline, 28.7 mmol/L,was greater than expected in the general population. Valuesfor individuals with a tHcy level in the upper tertile at baseline(i.e., 31.6 mmol/L) were compared with those for individualsbelow the upper tertile (i.e., #31.6 mmol/L). Individuals inthe upper tertile for tHcy had lower levels of folate, vitaminB-12 and pyridoxine; however, none reached statistical signif-icance. Only 14 individuals completed the protocol up to mo6 of measurement and only eight individuals completed theprotocol up to the last month (mo 12). Individuals with a tHcylevel in the upper tertile at baseline were compared withindividuals below the upper tertile to examine reduction intHcy. The average reductions in tHcy in the highest tertilegroup (16.6 mmol/L at 6 mo and 33.5 mmol/L at 12 mo) weresignificantly greater than the other tertiles: 3.7 mmol/L at 6 mo(P 5 0.085) and 2.1 mmol/L at 12 mo (P 5 0.025 ). This mayindicate that individuals with ESRD may not be able to lowertheir homocysteine levels to those of the general population byusing the current therapy. For these individuals, that interven-tion may be feasible only for those exceeding tHcy levels of;30 mmol/L. When nutrient metabolites were examined bytertiles of homocysteine, the nutrients that increased in con-centration were folate and vitamin B-12. This suggests thatthese two nutrients may have contributed to the reductions intHcy. This is of great interest because the isolated soy productfed to these subjects is considered to be only a fair source offolate. It is possible that other substances present in the soyproduct may protect individuals from the loss of folate andvitamin B-12 through the dialysate. In addition, exploration ofadditional therapies (e.g., very high supplementation of folicacid) may be appropriate.

Soy Intake Pattern and Relation with Serum Cholesterol inthe Hong Kong Chinese Population. Suzanne C. Ho, JeanWoo, Sophie Leung and Aprille Sham. Department of Com-munity and Family Medicine, Chinese University of HongKong, Shatin, Hong Kong.

The study aims to describe the pattern of soy intake in theHong Kong Chinese population and the influence of socio-economic status on soy intake. It is hypothesized that highersoy intake is associated with lower levels of cholesterol andLDL cholesterol. The importance of the hypothesis is thatalthough geographical variation of some common diseasessuch as coronary heart diseases have been attributed to thedifferences in soy intake, few data on the soy consumption

TABLE 1

Effect of genistein treatment

Treatment Body wt Heart wtAortic weight-length

ratioBlood

pressure

g g mg:mm mm Hg

Control 312 6 5 1.10 6 0.01 1.13 6 0.01 207 6 3Genistein 313 6 7 1.05 6 0.03 1.15 6 0.02 210 6 1

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pattern and its association with the risk factors for heartdisease in the Asian setting have been reported. The study willcontribute to the understanding of the soy-disease relation-ship. A dietary survey was conducted as part of a territory-widepopulation-based cardiovascular risk factor study. A total of500 men and 510 women completed the dietary intake study.The dietary assessment was based on a semiquantitative food-frequency questionnaire comprising 253 food items; 10 soyfoods commonly consumed in the local population wereamong the items included. Of the study population, 88% hadconsumed some form of soy in the past week; 68% of thesoy-derived isoflavones were obtained from tofu, 8% were fromfried tofu and 8% were from soymilk (vitasoy). The meanamount of isoflavone intake per week in the whole populationwas 135.2 mg (6 155.7); and 154.8 mg (6 157.3) in thosewho had consumed any of the soy items. Men had a higherintake of isoflavones than women, but no difference was ob-served after adjustment was made for the total energy intake(17.7 mg/MJ for men and 16.9 mg/MJ for women). Oldersubjects and subjects with higher levels of education tended tohave higher soy consumption. We observed a significant neg-ative linear relation between soy intake and LDL cholesterolin men. The overall intake of soy foods was modest only in theHong Kong Chinese population. There was apparent benefi-cial effect of soy intake in the improvement of lipid profile inmen.

Soy Protein Reduces Arterial LDL Concentration and LDLDelivery in Both Diabetic and Nondiabetic CynomolgusMonkeys. Janice D. Wagner, Li Zhang, Kathryn A. Greavesand Dawn C. Schwenke. Winston-Salem, NC.

We previously showed that soy protein consumption improveslipoprotein concentrations and reduces progression of athero-sclerosis in cynomolgus monkeys. The mechanism for thesebeneficial effects is unclear. The purpose of this study was todetermine potential mechanisms for the atheroprotective ef-fects and to determine whether these effects extend to diabeticmonkeys. As such, we designed an experiment with a 2 3 2factorial design in which adult male monkeys (n 5 23) werefed an atherogenic diet; the protein source was soy isolate (n5 11) or casein lactalbumin (n 5 12), and monkeys wereeither control (n 5 12) or streptozotocin-induced diabetic (n5 11). Diabetics had significantly increased fasting glucoseand glycated hemoglobin levels; this relationship was notaffected by protein consumption. Soy consumption signifi-cantly reduced total cholesterol and increased HDL choles-terol concentrations in both control and diabetic monkeys. Todetermine plasma and arterial LDL metabolism, radiolabeledLDL was injected before necropsy. 125I-LDL was injected 48 hand 131I-tyramine cellobiose-LDL was injected 1 h beforenecropsy to determine arterial LDL concentration and perme-ability, respectively. An increase in whole-body plasma LDLfractional catabolic rate was found with soy consumption. Soyreduced arterial LDL concentration across all arterial sites andLDL permeability in the carotid bifurcation and internal ca-rotid arteries. Arterial delivery of LDL was calculated as LDLpermeability 3 plasma LDL concentration and was also re-duced across all arterial sites. There was no additional effect ofdiabetes. In conclusion, these beneficial effects of soy on bothplasma and arterial LDL metabolism would be expected toreduce atherosclerosis progression and were found in bothcontrol and diabetic monkeys.

A Soy Diet Improves Vascular Reactivity in SpontaneouslyHypertensive Rats. Mark G. Lounsbery, Kathleen M. Ey-

ster, Douglas S. Martin and John L. Williams. South DakotaSoy Cardiovascular Research Consortium, University of SouthDakota School of Medicine, Vermillion, SD.

Recent studies demonstrated that a diet that contains soyfoods can lower blood cholesterol and may improve vascularreactivity during severe atherosclerosis. The goal of thesestudies was to determine whether a soy-based diet improvesvascular reactivity in spontaneously hypertensive rats (SHR).Four-week-old male, ovariectomized (ovx) female, or shamfemale SHR or normotensive ( Wistar Kyoto, WKY) rats werefed a diet containing either soymeal or casein for 10 wk.Posterior cerebral arteries (;250 mm) were removed fromanesthetized rats and placed in an arteriograph. The systemwas perfused with physiologic saline; pressure, flow, gases, pHand temperature were controlled. Cumulative doses (10210–1025 mol/L) of 2-methylthio-ATP (2MSATP), an agent thatcauses release of nitric oxide from the endothelium, wereadministered luminally for 10 min at each dose. Arterialdiameter was measured with a microscope and video system. Ingeneral, 2MSATP caused vasodilation at higher doses, but themagnitude of response varied considerably among groups. Di-lator responses were similar in male WKY rats consumingcasein vs. soy diets (14.4 6 3.3% vs. 10.9 6 1.8%, respec-tively; means 6 SEM). In contrast, in male SHR, vasodilationwas much greater (P , 0.05, repeated measures ANOVA) insoy-fed rats (17.8 6 4.0%) than in casein-fed rats (9.5 61.9%). Results were qualitatively similar in ovx rats, but themagnitude of response to 2MSATP was much smaller. Al-though increases in diameter were not significantly differentbetween dietary groups of ovx WKY rats, vasodilation wasgreater in soy-fed ovx SHR rats than in casein-fed ovx SHRrats (7.0 6 1.3% and 3.4 6 1.1%, respectively; P , 0.05). Inboth sham WKY and SHR rats, 2MSATP caused a smalldose-response effect that was similar in the soy and caseingroups. Our findings demonstrate that a diet containing soyfoods can improve or restore vasodilator function in hyperten-sive rats. Furthermore, our findings demonstrated sex differ-ences in dilator responsiveness to 2MSATP and in the abilityof soy foods to improve vascular reactivity. [Supported by agrant from the South Dakota Soybean Research and Promo-tion Council.]

Soy Isoflavone, Daidzein, Attenuates Postovariectomy BloodPressure Elevation by Accelerating Nitric Oxide Production.Y. Yamori,* T. Teramoto,*† T. Noguchi,* Y. Sekine,* Y.Nara** and K. Ikeda.* *Department of Environmental Pres-ervation and Development, Graduate School of Human andEnvironmental Studies, University of Kyoto, Kyoto, Japan;†Fujicco Co., Ltd., Kobe, Japan; and **Graduate School ofIntegrated Science, University of East Asia, Shimonoseki,Japan.

Recent studies suggested that estrogen-induced cardiovascularprotection might be mediated by an increased synthesis ofvascular nitric oxide (NO). In this study, we investigated theeffects of isoflavones with weak estrogenic activity on bloodpressure and vascular NO actions in ovariectomized stroke-prone spontaneously hypertensive rats. Female stroke-pronespontaneously hypertensive rats (n 5 32; 15 wk old) weredivided into four groups. Groups 1 and 2 were fed the standarddiet and groups 3 and 4 were fed daidzein (D)- and genistein(G)-mixed standard diets, respectively (0.16 mmol/100 g stan-dard diet). At age 19 wk, group 1 (Sham) was sham-operatedand groups 2 (ovx), 3 (ovx 1 D) and 4 (ovx 1 G) were

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ovariectomized. All rats were killed at age 22 wk and theirthoracic aortae were checked for vascular NO release indi-rectly by precontracting aortic rings with prostaglandin F2a

and by observing the effect on tone changes induced byN-monomethyl-L-arginine, an L-arginine analog that competesfor NO synthase and inhibits NO formation. Systolic bloodpressure and urinary NO metabolites were measured beforeand after surgery. Before surgery, systolic blood pressure andurinary NO metabolites did not differ among all groups. Aftersurgery, however, systolic blood pressure was significantlylower (P , 0.01) and urinary NO metabolites were signifi-cantly higher (P , 0.05) in ovx 1 D than in ovx. Basal releaseof NO in ovx 1 D was significantly higher than that in ovx (P, 0.005). These results indicate that daidzein attenuates thedevelopment of hypertension by accelerating vascular NOproduction. This new result supports the several epidemiologicand animal study findings suggestive of a possible cardiovas-cular protective effect of soy intakes.

Effect of Active Peptides from Soy Proteins on CholesterolHomeostasis in Cell Cultures. M. R. Lovati, C. Manzoni, E.Gianazza, E. M. Kurowska* and C. R. Sirtori. Institute ofPharmacological Sciences, Faculty of Pharmacy, Milano, Italyand *Centre for Human Nutrition, Department of Biochem-istry, University of Western Ontario, London, ON, Canada.

An activation of LDL receptors was recently described in ahuman hepatoma cell line (Hep G2) exposed to purified(a1a’) subunits from 7S soy globulin; b chains were ineffec-tive (Lovati et al. 1998). By using a mutant soy cultivar devoidof the a’ subunits (Keburi), further experiments in HepG2cells demonstrated a marked LDL-receptor activation by the a’vs. a subunit. A commercial isoflavone-free, heat-hydrolyzedsoy concentrate (Croksoy), found effective in type II hyper-cholesterolemic patients, showed an LDL-receptor activationsimilar to that of the 7S globulin when incubated under thesame conditions (Manzoni et al. 1998). To assess the finalidentity of the putative peptide (or peptides) responsible forthe biochemical effect, experiments were performed in HepG2 cells exposed to either synthetic peptides corresponding tospecific sequences differing among the a1a’ and b subunits orpeptides coming from the in vitro digestion (trypsin 1 pepsin)of Croksoy. Moreover, the ability of the whole 7S globulin, itsa1a’ and b subunits, and whole Croksoy to interfere in theapoB secretion in the medium as well as in sterol biosynthesiswas evaluated in the same model. Hep G2 cells were preincu-bated for 24 h at 37°C in minimal essential medium with 5%lipoprotein-deficient serum in the presence or absence of syn-thetic and in vitro–produced peptides at different concentra-tions (1024 or 1026 mol/L for synthetic peptides, 0.5 mg/mLfor digested Croksoy). After addition of 125I-LDL, cells wereincubated at 37°C for a further 4 h to determine the uptakeand degradation of LDL. Increased 125I-LDL uptake (141%)and degradation (110%) vs. controls were shown after HepG2 incubation with a synthetic peptide (1024 mol/L, MW2271) corresponding to the 127–150 positions of the consen-sus sequence present in the a’ and not in the b subunit of the7S globulin. Cells exposed to Croksoy enzyme digestion prod-ucts showed a marked up-regulation of LDL receptors vs. thatin controls as well as vs. HepG2 cells incubated with undi-gested Croksoy (125I-LDL uptake: 158% and 117%, respec-tively; 125I-LDL degradation: 1113% and 126%, respec-tively). Among soy-derived products, only the 7S soy globulinshowed an inhibitory activity on apolipoprotein B secretion(280% secretion) and 14C-acetate incorporation (270, 260

and 248% into free and esterified cholesterol and triacylglyc-erol, respectively) when tested in Hep G2 cells at a concen-tration of 1.0 mg/mL. These findings support the hypothesis(Sirtori et al. 1998) that one or more peptides escaping detec-tion in vivo may reach the liver after intestinal digestion, thuseliciting a cholesterol-lowering effect. Moreover, these dataindicate that the protein moiety, devoid of isoflavone compo-nents, is responsible for the biochemical effect.Lovati, M. R., Manzoni, C., Gianazza, E. & Sirtori, C. R. (1998) Soybean protein

products as regulators of liver low-density lipoprotein receptors. I. Identifica-tion of active b-conglycinin subunits. J. Agric. Food Chem. 46: 2474–2480.

Manzoni, C., Lovati, M. R., Gianazza, E., Morita, Y. & Sirtori,C. R. (1998) Soybean protein products as regulators of liver low-densitylipoprotein receptors. II. a-a’ rich commercial soy concentrate and a’ defi-cient mutant differently affect low-density lipoprotein receptor activation. J.Agric. Food Chem. 46: 2481–2484.

Sirtori, C. R., Lovati M. R., Manzoni C., Gianazza, E., Della Mura, N., Staels, G. &Auqerx, J. (1998) Reduction of serum cholesterol by soy proteins: clinicalexperience and potential molecular mechanism. Nutr. Metab. Card. Dis. 8:334–338.

Effect of Soy Protein on LDL Cholesterol in Hypercholes-terolemic Costa Rican Children. A. G. Arauz, G. Padilla, M.Rosello, S. Guzman, S. Rodriguez, L. Cunningham and J. M.Ordovas. Costa Rican Institute for Research and Education onNutrition and Health (Inciensa), Tres Rios, Costa Rica.

A diet low in cholesterol, total fat and saturated fat is the firststep in the treatment of hypercholesterolemia. However, manychildren cannot achieve plasma cholesterol goals by using thisdietary approach. It has been long recognized that there is ahypocholesterolemic effect of soy protein when it is partiallysubstituted for animal protein. The objective of our study wasto evaluate the effect of soy protein on LDL cholesterol re-duction when soy products are used as part of a regular diet athome. The product used were Archer Daniels Midland iso-lated protein and textured protein (soy meat and soymilk). Wecarried out a clinical random study including 26 hypercholes-terolemic children; 13 were in the control group and 13 werein the intervention group (mean age 10.3 6 2.04 y, range 7–15y). During the 8-wk experimental period, both groups con-sumed a Step 1 diet (,30% total fat, ,10% saturated fat,,250 mg cholesterol). The intervention group also consumedthe soy products as part of their regular meals. Food intake andadherence to the protocol were assessed during weekly homevisits. Daily soybean protein intake in the intervention groupwas 20.88 6 5.59 g/d, range 14.24–33 g/d. Preliminary datafrom 18 children (9 in each group) showed that the meanLDL-cholesterol reduction was significantly (P , 0.005)higher in the intervention group (15.5%) than in the controlgroup (5.5%). There were no significant differences in totalcholesterol reduction. The addition of soy protein to a Step 1diet in hypercholesterolemic children was associated with aspecific reduction in LDL cholesterol. We therefore proposethat soy protein may offer a nonpharmacologic alternative toreducing elevated LDL cholesterol in children. [Supported byInciensa-FODESAF, Costa Rica; American Soybean Associa-tion and United Soybean Board.]

Effects of an Isoflavone Supplement on Risk Factors forBreast Cancer and Coronary Heart Disease in Premeno-pausal Women. S. J. Smith, G.S.-M. Chan, S. Samman andP. M. Lyons-Wall. Human Nutrition Unit, Department ofBiochemistry, University of Sydney, Sydney, NSW, Australia.

Traditional soy-based Asian diets are associated with a lowerprevalences of breast cancer and coronary heart disease. One

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dietary component that could contribute to this effect is theisoflavones, found in especially high concentrations in soy.Isoflavones are diphenolic compounds with a structure similarto that of estradiol that may act as both estrogens and anties-trogens, depending on the target tissue and hormonal status ofthe individual. The aim of this study was to determine theeffects of an isoflavone supplement on plasma concentrationsof estradiol and androgen precursors, lipids and lipoproteins.We hypothesized that the isoflavones would produce favorablehormonal and lipid profiles. Participants were healthy women(n 5 14; mean age 27.5 6 8.2 y; body mass index: 21.3 6 0.5,measured as kg/m2) with regular menstrual cycles, stable bodyweight, low soy intake and not taking an oral contraceptivepill. The women consumed 86 mg/d of isoflavones in tabletform for two menstrual cycles followed by placebo for anequivalent period, or vice versa. Blood samples were collectedduring the second cycle of each treatment period for measure-ment of plasma steroid hormones (estradiol, progesterone,testosterone, androstenedione, dehydroepiandrosterone sulfateand sex hormone–binding globulin). Plasma lipids and li-poproteins were also measured. The isoflavone supplement hadno significant effects on concentrations of steroid hormones,sex hormone–binding globulin, total cholesterol or triacyl-glycerol, although there was a unexpected trend toward a 15%increase in mean estradiol concentration (P 5 0.06). Consis-tent with the rise in estradiol, there was a trend toward anincrease in HDL3 cholesterol; in the group of women whoreceived isoflavones followed by placebo, HDL3 cholesterolcontinued to rise further during the placebo period and at theend of the study, it was 11% higher than at baseline (P5 0.024). The results do not support the hypothesis thatisoflavones can reduce risk of breast cancer by lowering con-centrations of estradiol or precursor androgens per se. Howeverthe potential beneficial effect of isoflavones on HDL metabo-lism in relation to cardiovascular risk and, in particular, theinteraction with steroid hormones during the menstrual cyclerequire further investigation.

High Dietary Cholesterol Diets May Attenuate Hypocholes-terolemic Effects of Soy Consumption. Kathryn A. Greaves,Martha D. Wilson, Lawrence L. Rudel and Janice D. Wag-ner. Wake Forest University School of Medicine, Departmentof Pathology, Section on Comparative Medicine, Winston-Salem, NC.

We previously reported that one mechanism for the hypocho-lesterolemic effects of soy may be a reduction in cholesterolabsorption. In this abstract, we report that reductions in cho-lesterol absorption may be attenuated by high amounts ofcholesterol in the diet. Results from two studies examining 20wk of soy protein consumption in ovariectomized cynomolgusmonkeys are reported. All monkeys were fed either casein-lactalbumin (CAS) or isolated soy protein (SOY) as a proteinsource. In the first study, monkeys were fed diets containing0.28 mg cholesterol/kcal. In the second study, monkeys werefed diets containing 0.36 mg cholesterol/kcal. Cholesterolabsorption was determined by using the fecal isotope ratiomethod. Data are presented as means 6 SEM. A significantreduction in cholesterol absorption was found in monkeysconsuming isolated soy protein with 0.28 mg cholesterol/kcalcompared with monkeys consuming casein-lactalbumin pro-tein (SOY 66 6 3% vs. CAS 74 6 2%). However, there wasno significant difference between the SOY and CAS groupsconsuming 0.36 mg cholesterol/kcal (SOY 63 6 2% vs. CAS66 6 3%). Additionally, non-HDL cholesterol concentrations

were significantly lowered in the SOY group compared withCAS group consuming 0.28 mg cholesterol/kcal (SOY 1186 14% vs. CAS 326 6 37%). This was not the case in themonkeys consuming 0.36 mg cholesterol/kcal (SOY 3246 51% vs. CAS 403 6 51%). Although many studies inhumans suggest that soy protein is more beneficial in hyper-cholesterolemic individuals, most of these studies have in-cluded a low cholesterol diet in their design. The results fromthis study suggest that a threshold of dietary cholesterol con-sumption may exist above which soy protein may not beeffective in lowering plasma cholesterol.

Promoting Effect of Fermented Soy Flour on the AntioxidantActivity of Coenzyme Q-10. Joe Vinson and Jimmy Li.*Department of Chemistry, University of Scranton, Scranton,PA and *Department of Food Science, Rutgers University,Piscataway, NJ.

Soy food and fermented soy food have demonstrated manyhealth benefits, including hypocholesterolemic effects. Coen-zyme Q-10 (CoQ-10), the important antioxidant for energyproduction in human tissues, was also found to be beneficial tohuman health. Oral CoQ-10 supplementation was reported todecrease the oxidation of LDL. In our study, pure CoQ-10 wascombined into a fermentation medium at a concentration of7.5% (dry basis) in soy flour. Then the soy fermentationproduct and pure CoQ-10 were tested for their antioxidantactivity against the oxidation of human lipoprotein, mainlyLDL; the 50% inhibition concentration (IC50) was deter-mined to evaluate the antioxidant activity against lipoproteinoxidation. At the same CoQ-10 level, the fermentation prod-uct was found to be 9.6 times more active than pure CoQ-10.Accelerated storage stability in both solid and liquid form wasalso tested for those samples. CoQ-10 in the pure chemicalform decomposed much more quickly than it did in the soyfermentation product.

Effect of Soy Protein Foods on LDL Oxidation and Ex VivoSex Hormone Receptor Activity—a Controlled CrossoverTrial. David J. A. Jenkins,*† Cyril W. C. Kendall,** Mar-cella Garsetti,*†** Rachel S. Rosenberg-Zand,† Chung-JaJackson,‡ Sanjiv Agarwal,† A. Venket Rao,† Eleftherios P.Diamandis,†† Tina Parker,* Dorothea Faulkner,† VladimirVuksan*† and Edward Vidgen.*† *Clinical Nutrition andRisk Factor Modification Center, St. Michael’s Hospital, To-ronto, ON, Canada; †Department of Nutritional Sciences,Faculty of Medicine, University of Toronto, Toronto, ON,Canada; ** Department of Food Science Technology andMicrobiology, Division of Human Nutrition, University ofMilan, Milan, Italy; ‡Laboratory Services Division, Universityof Guelph, Guelph, ON, Canada; and ††Department of Pa-thology and Laboratory Medicine, Mount Sinai Hospital, To-ronto, ON, Canada.

Plant-derived estrogen analogs (phytoestrogens) may confersignificant health advantages, including cholesterol lowering,antioxidant activity and possibly reduced cancer risk. How-ever, concern has also been raised that phytoestrogens may beendocrine disrupters and major health hazards. We thereforeassessed the effects of soy foods as rich sources of isoflavonoidphytoestrogens on LDL oxidation and sex hormone receptoractivity. Hyperlipidemic subjects (n 5 31) consumed two lowfat metabolic diets for 1 mo in a randomized crossover study.Major differences between test and control diets were theincreases in soy protein foods (33 g/d of soy protein) providing

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86 mg/(2000 kcal z d) of isoflavones and the doubling ofsoluble fiber intake. Fasting blood samples were obtained atthe start, wk 2 and wk 4; 24-h urine samples were collected atthe end of each phase. Soy foods increased urinary isoflavoneexcretion in subjects consuming the test diet compared withthe control diet (3.8 6 0.7 vs. 0.0 6 0.0 mg/d; P , 0.001).The test diet lowered oxidized LDL concentrations measuredboth as conjugated dienes in the LDL fraction (56 6 3 vs. 636 3 mmol/L, P , 0.001) and as the ratio of conjugated dienesto LDL cholesterol (15.0 6 1.0 vs. 15.7 6 0.9, P 5 0.032),even in subjects already taking vitamin E (400–800 mg/d). Nosignificant difference was detected in ex vivo sex hormoneactivity between urine samples from the test and controlperiods. In conclusion, consumption of high isoflavone foodswas associated with reduced levels of circulating oxidized LDL,even in subjects taking vitamin E, with no evidence of in-creased urinary estrogenic activity. Soy consumption may re-duce cardiovascular risk without increasing risk for hormone-dependent cancers.

Combined Effect of Vegetable (Soy) Protein and SolubleFiber Added to a Standard Cholesterol-Lowering Diet. DavidJ. A. Jenkins,*† Cyril W. C. Kendall,** A. Venket Rao,†

Sanjiv Agarwal,† Peter J. H. Jones,** Mahmoud Raeini**Jon A. Story,‡ Edward Vidgen,*† Larry C Griffin,†† andStephen C. Cunnane.† *Clinical Nutrition and Risk FactorModification Center, St. Michael’s Hospital, Toronto, ON,Canada; †Department of Nutritional Sciences, Faculty of Med-icine, University of Toronto, Toronto, ON, Canada; **Schoolof Dietetics and Human Nutrition, McGill University, SteAnne de Bellevue, QC, Canada; ‡Department of Food andNutrition, Purdue University, West Lafayette, IN; ††LoblawBrands Ltd., Toronto, ON, Canada.

Our goal was to determine whether a combination of two plantcomponents, vegetable protein and soluble fiber, further re-duced serum lipids when incorporated into the currently ad-vocated low saturated fat diet. Hyperlipidemic men andwomen (n 5 31) consumed two low fat (7% of total energyfrom saturated fat), low cholesterol ,80 mg cholesterol/d)metabolic diets for 1 mo in a randomized crossover study.Major differences between test and control diets were theincreased amount of vegetable protein (93 vs. 23% of totalprotein), of which 23 g/d was soy, and the doubling of solublefiber. Fasting blood samples were obtained at the start and endof each phase. Fecal collections were obtained on the last 3 dof each phase. Compared with the low fat control diet, the testdiet lowered total cholesterol (6.2 6 1.2%, P , 0.001), LDLcholesterol (6.7 6 1.7%, P , 0.001), apolipoprotein B (8.26 1.2%, P , 0.001), and the ratios of LDL to HDL cholesterol(6.3 6 2.0%, P 5 0.004) and apolipoprotein B to apolipopro-tein A-I (5.4 6 1.5%, P 5 0.001). A combination of vegetableprotein and soluble fiber significantly improved the lipid-lowering effect of a diet low in saturated fat. The resultssupport expanding current dietary advice to include increasedvegetable protein and soluble fiber intakes so that the gap ineffectiveness between a good diet and drug therapy is reduced.

Effect on Serum Lipids and Oxidized LDL of SupplementingSelf-Selected Low Fat Diets with Soluble Fiber, Soy, andVegetable-Protein Foods. Cyril W. C. Kendall,* ** DavidJ. A. Jenkins* ** Edward Vidgen,* ** Marcella Garsetti,***‡ Larry C. Griffin,†† Sanjiv Agarwal,** A. Venket Rao,**Stephen C. Cunnane,** Philip W. Connelly,†‡‡# LawrenceA. Leiter,†** Vladimir Vuksan* ** and Robert Josse.†

*Clinical Nutrition and Risk Factor Modification Center and†Department of Medicine, Division of Endocrinology and Me-tabolism, St. Michael’s Hospital, Toronto, ON, Canada;**Department of Nutritional Sciences, Faculty of Medicine,University of Toronto, Toronto, ON, Canada; ‡Department ofFood Science Technology and Microbiology, Division of Hu-man Nutrition, University of Milan, Milan, Italy; ††LoblawBrands Limited, Toronto, ON, Canada; and Departments of‡‡Biochemistry and #Laboratory Medicine and Pathobiology,Faculty of Medicine, University of Toronto, Toronto, ON,Canada.

Increased intake of soluble fiber and soy protein may improvethe blood lipid profile and reduce oxidized LDL. The goal ofthis study was to assess any benefits of providing soy proteinand soluble fiber foods to hyperlipidemic subjects already con-suming low fat, low cholesterol therapeutic diets. Twentyhyperlipidemic men and postmenopausal women completed 8wk of tests and control dietary treatments in a randomizedcrossover design as part of an unlimited National CholesterolEducation Program Step-2 therapeutic diet (7% saturated fatand 200 mg/d of cholesterol). During the test phase, foods highin soy, other vegetable proteins and soluble fiber were pro-vided. During the control phase, low fat dairy and low soluble-fiber foods were provided. Fasting blood lipids and apolipopro-teins were measured at 4 and 8 wk of each phase. Subjectsreceiving the test diet consumed 12 6 2 g/d of soy protein fromthe foods they chose. Direct comparison of test and controltreatments indicated a rise in HDL cholesterol concentrationafter consuming the test diet (6.4 6 2.4%, P 5 0.013) and asignificantly reduced ratio of total to HDL cholesterol (25.96 2.3%, P 5 0.020). The proportion of conjugated dienes inthe LDL cholesterol fraction was significantly reduced (8.56 3.3%, P 5 0.020) as a marker of oxidized LDL. A combi-nation of acceptable amounts of soy, vegetable protein andsoluble fiber foods as part of a conventional low fat, lowcholesterol therapeutic diet is effective in further reducingserum lipid risk factors for cardiovascular disease.

Effects of Yeast Fermentation on the Antioxidant Activitiesof Soy Flour. Joe Vinson, Pratima Bose and Jimmy Li.*Department of Chemistry, University of Scranton, Scranton,PA and *Department of Food Science, Rutgers University,Piscataway, NJ.

Soy food and fermented soy food have shown many healthbenefits, including hypocholesterolemic effects. Much researchis being done on the bioactivities in soy food and fermentedsoy food. In our study, organic and nonorganic soy flours andtheir yeast fermentation products (Soynatto, Bio-Foods, WestPatterson, NJ) were tested for their total phenol levels andtheir antioxidant activities against the oxidation of humanlipoprotein (80% in LDL). The 50% inhibition concentration(IC50) was determined according to the inhibition of LDLoxidation vs. soy concentration curves. The fermentationproducts were found to be 5.5–10.0 times more active thannonfermented soy flours, and this depended on the differencebetween soy flours. On the other hand, the fermentation didnot change the total phenol content significantly.

Other topics

Daidzein Exerts a Marked Effect on Body Composition inMale Hamsters. Kathy B. Hanson, Tong T. Song,* CharlesT. Peterson,† Sun-ok Lee, Suzanne Hendrich, Patricia A.

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Murphy and D. Lee Alekel. Center for Designing Foods toImprove Nutrition; Department of Food Science and HumanNutrition; Iowa State University, Ames, IA; *Protein Tech-nologies International, St. Louis, MO; and †Department ofStatistics, Iowa State University, Ames, IA.

The purpose of this study was to determine whether dietaryisoflavones have an estrogen-like effect on bone, body com-position and food efficiency (weight gain/food intake) in grow-ing male and female hamsters. We hypothesized that dietaryisoflavones (either a mixture of genistein, daidzein, and gly-citein or daidzein alone) would increase food efficiency andexert a positive effect on bone mineral density and bodycomposition by decreasing the proportion of weight gain as fat,regardless of sex. Golden Syrian hamsters (n 5 80; aged 6–8wk) were randomly assigned to one of the following four dietregimens (10 males and 10 females per treatment): caseinwithout added isoflavones (CAS2), casein plus daizein (CAS1 DAID), soy protein isolate containing isoflavones (SPI1)and soy protein isolate deficient in isoflavones (SPI2). After10 wk of unlimited feeding, the hamsters were killed andcarcasses were analyzed by dual-energy X-ray absorptiometry.Statistical analysis using ANOVA indicated that sex (P,0.0001) exerted a predominant effect on all outcomes [bonemineral density, lean mass, percentage of body fat (%BF) andfood efficiency], with females having higher values for eachvariable except %BF. Contrary to our hypothesis, no treat-ment effects were found in females. However, in males, CAS1 DAID suppressed weight gain (P 5 0.03), food efficiency (P5 0.03) and %BF (P 5 0.003), whereas it increased lean mass(P 5 0.008) compared with SPI1. In contrast, %BF wasgreater in SPI1 than CAS2, illustrating that the mixture ofisoflavones had a different physiologic effect than did daidzeinalone. In estrogen-sufficient females, isoflavone intake had noeffect, whereas daidzein had a marked effect on body compo-sition in males.

Adipose Differentiation of 3T3-L1 Cells by Soybean Isofla-vones. Keizo Sekiya. Shikoku National Agricultural Experi-ment Station, Ministry of Agriculture, Forestry and Fisheries,Zentsuji, Kagawa, Japan.

Antidiabetic and hypoglycemic drugs were recently reportedto enhance adipose differentiation of 3T3-L1 preadipocytes. Inthis experiment, the effect of soybean constituents on adiposedifferentiation was investigated by using mouse 3T3-L1 pre-adipocytes. Active constituents were purified and identified.3T3-L1 cells were grown as monolayer cultures at 37°C inDME-medium supplemented by 10% fetal bovine serum underan atmosphere of 5% CO2/95% air. Confluent cell cultureswere then treated and converted to adipocytes by culture inthe presence of insulin and soybean extract samples. After;10 d, glycerol-3-phosphate dehydrogenase activity and tri-glyceride accumulation were measured as markers of adiposedifferentiation. Insulin-sensitive glucose uptake and lipopro-tein lipase activity were also assayed in the treated cells. Crudesoybean extract was found to enhance the differentiation.Active constituents were purified and identified as isoflavones(e.g., daidzein or genistein). Aglycones were more active thanglycosides. In the daidzein-treated cells, insulin-sensitive glu-cose uptake and lipoprotein lipase activity were increased. Inthis experiment, it is proposed that the activation of adiposefunction by newly differentiated adipocytes seems to be animportant factor in explaining the mechanism for the effect ofsoybean in the prevention and treatment of chronic diseases.

Vitamin E Content and Radical-Scavenging Activity ofSoymilk Made from Lipoxygenase-Lacking (Triple-Null)Soybean. Ikuo Suda, Yoichi Nishiba and Shu Furuta. KyushuNational Agricultural Experiment Station, Ministry of Agri-culture, Forestry and Fisheries, Nishigoshi, Kumamoto, Japan.

Soybeans constitute an important food ingredient because oftheir low cost and high nutritional quality with physiologicfunction. However, normal soybean seeds contain three li-poxygenase isozymes: L-1, L-2 and L-3. These enzymes areresponsible for the generation of undesirable grassy-beany fla-vors that limit the wide use of soybeans in food products.Because of their low level of beany flavor, lipoxygenase-lack-ing (triple-null) soybeans (e.g., Ichihime or L-star) can pro-duce excellent, tasty soymilk and soymilk-related products. Inthis study, we compared the vitamin E (tocopherol) contentand 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavengingactivity in soymilk made from normal soybeans and lipoxyge-nase-lacking (double-null and triple-null) soybeans. Vitamin Econtent and DPPH radical-scavenging activity were deter-mined by using HPLC and electron spin resonance spectrom-etry, respectively. Soymilk made from triple-null soybeansshowed a higher vitamin E content and radical-scavengingactivity than did normal and double-null soybeans. Vitamin Epotency of triple-null soybean soymilk was 1.4- to 2.8-foldhigher than that of parental normal soybean soymilk. Triple-null soybeans could improve the nutritional quality and func-tionality of soybean products in addition to improving theflavor and could offer advantages for the prevention of life-style-related disease (chronic disease) induced by recent foodhabits in Japan.

Radical-Scavenging Activities of Boiled Extracts of Soybean.Shu Furuta, Ikuo Suda, Yasuhiro Takahata and YoichiNishiba. Kyushu National Agricultural Experiment Station,Ministry of Agriculture, Forestry and Fisheries, Kumamoto,Japan.

Various soybean cultivars have different surface colors, such asyellow, green, red-brown and black. We investigated the rad-ical-scavenging activities of soybean cultivars in vitro. Theboiled soybean extracts obtained from cultivars with a blacksurface (containing anthocyanins) showed the highest scav-enging activities toward 1,1-diphenyl-2-picrylhydrazyl and su-peroxide radicals.

Beneficial Effects of Soy Protein on Renal Function in Type1 Diabetic Patients at Risk for Nephropathy. T. J. Hanna, P.Fanti and J. W. Anderson. Department of Internal Medicine,University of Kentucky, Lexington, KY.

Diabetic nephropathy is a primary contributor to illness anddeath in individuals with type 1 diabetes. An increase inurinary albumin excretion and elevated glomerular filtrationrate (GFR) are characteristics of early diabetic nephropathy.The objective of this research was to evaluate the potentialrole of soy protein as a therapeutic agent in the prevention ofrenal disease in type 1 diabetes. Seven young (29.6 6 1.9 y)volunteers with type 1 diabetes under good glycemic control(hemoglobin A1c 7.2 6 0.5%) participated in this 20-wk,crossover, dietary intervention pilot study. GFR was measuredby using technetium 99m pentetate (Tc99m-DTPA) clearanceand urinary albumin, and albumin-creatinine ratios were de-termined from 24-h urine collections. After a 4-wk run-inperiod to assess baseline characteristics, subjects consumed a

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diet containing 55 g/d of soy protein for 8 wk. Soy protein wasin the form of a soy patty, soy beverage and soy pasta. After 8wk of consuming the diet based on soy protein, subjects wereasked to resume their normal diet based on animal protein foran additional 8 wk. Subjects received extensive dietary coun-seling throughout the duration of the study. Specifically, theywere instructed to reduce animal protein intake during the soyperiod. However, dietary recalls indicated that soy proteinsupplemented rather than substituted animal protein duringthe soy period. Consequently, total protein intake was higherduring the soy period (1.4 g/kg body weight) compared withbaseline (1.1 g/kg body weight) and the control period (1.0g/kg body weight). Despite higher total protein intake duringthe soy period, the GFR was lower at the end of this period[147 612 mL/(min z 1.73 m2)] compared with baseline [1546 14 mL/(min z 1.73 m2)] and with the end of the controlperiod [162 6 16 mL/(min z 1.73 m2)]. When only the fivesubjects with GFR values consistent with hyperfiltration [GFR120 mL/(min z 1.73 m2)] were included in this computation,the decrease in GFR during the soy period was more pro-nounced. Reductions of 6.3 and 8.6% in GFR in these hyper-filtering subjects were noted when the soy period was com-pared with baseline and the soy-free control period,respectively. Only two subjects had abnormal urinary albuminexcretion at baseline, and these were the only individuals toexperience a reduction in the albumin-creatinine ratio duringthe soy period. Interestingly, both subjects had a reduction inGFR as well during this period. From our small sample popu-lation, it may be hypothesized that subjects with clinicallyrelevant microalbuminuria (30–300 mg/d) benefit the mostfrom a soy-based diet. Additionally, incorporating soy into thediet of these type 1 diabetes patients had a significant benefiton serum total and LDL cholesterol levels in all seven subjects.After 8 wk of consuming a diet based on soy protein, there wasan average 7% reduction in total cholesterol and 13% reduc-tion in LDL cholesterol. When subjects returned to theircontrol diet based on animal protein, both total and LDLcholesterol returned to values greater than those at baseline.In summary, our small sample population appears to demon-strate that subjects with clinically relevant microalbuminuria(30–300 mg/d) may benefit the most from a soy-based diet andthat soy protein exerts beneficial effects on lipid profiles intype 1 diabetes. In conclusion, this pilot study suggests thatincorporating soy protein into the diet of individuals with type1 diabetes may be of value in the prevention of nephropathy.

Development of a Well-Liked Muffin Containing High Lev-els of Soy Protein and Isoflavones. Sandra C. Shehadeh andRaga M. Bakhit. Virginia Polytechnic Institute and StateUniversity, Blacksburg, VA.

Soy protein isolates (SPI) and the isoflavones they containhave received much attention in recent literature for theireffectiveness in decreasing the risk of cardiovascular diseaseand cancer. These SPI are desirable because of their lightcolor, their bland flavor and the fact that they can be mixedinto bakery products accepted by the general public. A soyblueberry muffin developed at Virginia Tech underwent phys-ical evaluation along with sensory evaluation by a consumerhedonic survey. Each muffin contained 11.3 g protein (9.3 gSPI) and 15.82 mg total isoflavones (from HPLC analysis).Moisture content was 43.3% and water activity was 0.954 at22.0°C. Compression displacement was 13.0 mm with a max-imal load of 1.71 kg. Freezing and thawing did not affect anyphysical measurement. This amount of SPI per muffin is much

higher than the 5–6 g that is common in other clinical recipes,thus allowing consumers to eat small quantities of bakedproducts and still obtain desired levels of soy protein intake.Sensory evaluation by 170 subjects in southwest Virginia in-dicated that overall (appearance, flavor, consistency), the muf-fin was well-liked by 85% of the participants; 50% said thatthey would buy the soy muffins at least two times per week,and 95% said they would eat them again. The soy blueberrymuffin recipe that we used is reproducible for clinical studies inwhich SPI supplementation is desired. Developing soy prod-ucts that are well liked by the public is vital for long-termcompliance for maximal health benefit.

Positive Effects of Soy Supplementation on MacronutrientIntake in Postmenopausal Women. Janine E. Albers, RandallA. McCoy and Eric E. Thomas. South Dakota Soy Cardio-vascular Research Consortium, Department of Family Medi-cine, University of South Dakota School of Medicine, SiouxFalls, SD.

Protein needs increase and energy needs decrease as peopleage. Unfortunately, protein needs are often not met in theaging population. Soy supplementation may provide benefitsof increasing protein intake with minimal increases in energyintake in the elderly. The purpose of this study was to inves-tigate the effects of soy protein supplementation on dietarymacro- and micronutrient intake in an independently livingaging population. Thirteen postmenopausal women, $55 y ofage, were randomly assigned to receive 40 g soy protein withand without isoflavones along with their usual diet in a double-blind, randomized, crossover design. A baseline period wasconducted in which usual nutrient intake information wasobtained. The baseline period was followed by a 4-wk dietintervention period, an intervening 1- to 2-wk washout period,and a second 4-wk diet intervention. Soy was provided in theform of muffins. Measurements were taken by using 4-d foodrecords during each period of the study. Comparisons weremade between the two soy supplementation periods and be-tween the soy supplementation periods and usual diet intake.Supplementing the subjects’ diets with soy protein resulted ina significant increase (P , 0.001) in overall protein intakewhile producing a significant decrease (P 5 0.019) in theintake of fat. Energy intake did not change significantly be-tween study periods. Results from this study show that dietaryprotein can be increased without an increase in fat or energyintake in the diet of an aging population through the use of soyprotein.

Efficacy of a Food Mix Based on Soy Protein Isolate inImproving the Nutritional Status of Malnourished Children(Age 1–2 y). Usha Chandrasekhar, Rajammal P. Devadas,W. Hilda Sahayarani and M. Vimalarani. Department ofFood Science and Nutrition, Avinashilingam University, Co-imbatore, India.

The study was conducted to compare growth and developmentof malnourished children fed a porridge fortified with soyprotein isolate (SPI) with that of children fed the standardfood mix provided by the Nutrition Intervention Programmeof India’s Integrated Child Development Services (ICDS) tochildren 2 y of age. Malnourished children (n 5 1200) aged1–2 y, with grade II malnutrition, selected from Coimbatore,Tamil Nadu, India, participated in the study. The energy gapof the children’s existing diet was identified through a stan-dardized food intake and a 3-d weighed food record, which

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formed the basis for determining the level of SPI-fortifiedporridge for supplementation. Children were divided into fourgroups as follows: group I (SPI 62; n 5 200 ) fed porridgefortified with 62 g SPI to bridge the existing energy gap in thecommunity; group II (Control; n 5 400) unsupplementedcontrol; group III (SPI 49; n 5 400) fed porridge fortified with49 g of SPI to provide the same amount of energy as in ICDSfood mix; and group IV (ICDS; n 5 200) fed the standardICDS food mix. Monthly records of heights, weights, threemonthly assessment of head circumference, skinfold and chestmeasurements, initial and final hemoglobin levels, and numberof respiratory incidences (morbidity pattern) were evaluated.After 10 mo of the feeding program, the mean increments inheight and weight were as follows: group I, 3.76 cm, 3.48 kg;group II, 0.82 cm, 0.65 kg; group III, 2.15 cm, 1.76 kg; andgroup IV, 1.32 cm, 0.96 kg, respectively. These increments ingrowth parameters were significantly (P , 0.01) superior ingroup I compared with all other groups. Group III childrenexhibited the next highest increment, which was also signifi-cant (P , 0.01) compared with groups I and IV. Group IVchildren also had a significant (P , 0.01) increase in thesevariables compared with group II (Control) children. Thesetrends indicate the beneficial effect of intervention in generaland specifically bring out the possible role of SPI-fortifiedporridge in improving the nutritional status of young growingchildren.

Good Compliance and Few Side Effects with Consumptionof Natural Soymilk in a 2-y Study. Eva Lydeking-Olsen andJens-Erik Beck Jensen.* Institute for Optimum Nutrition,Copenhagen, Denmark and *Copenhagen University Hospi-tal, Hvidovre, Denmark.

Previous studies in animals and humans showed a beneficialeffect of soy products on bone metabolism and risk factors forheart disease. It is therefore important to evaluate the con-sumption of soy products. The aim of the study was to evaluatethe use of, compliance with and side effects of soymilk in a 2-ystudy from 6- and 12-mo data. Postmenopausal Caucasianwomen (n 5 106), mean age 58 y (range 41–75 y) wererecruited and randomly assigned to one of the following fourtreatment groups: placebo, soymilk with isoflavones (100 mg/d), natural transdermal progesterone or both soymilk withisoflavones and natural transdermal progesterone. All partici-pants received a broad-spectrum food supplement (Osforte)containing 680 mg calcium (citrate and carbonate), 300 mgmagnesium (amino chelate), 20 mg silicium (sodium metasili-cate), 15 mg zinc (amino chelate), 6 mg manganese (aminochelate), 3 mg boron (proteinate), 2 mg copper (amino che-late), 200 mg vitamin C, 40 mg pyridoxine, 200 IU vitaminD-3 and 1 mg vitamin K-1. The soymilk was enriched withcalcium to 120 mg/100 mL and the daily intake was 400 mL;total calcium intake from diet, soymilk and food supplementwas 1200–1500 mg/d. The study included courses on healthfulcooking, how to use soymilk, exercise, balance training andrelaxation techniques; one weekend course was followed bysingle-day courses every 3 mo for the first year and withindividual follow-up during the second year. Main endpointswere changes in bone measurements (dual energy X-ray ab-sorptiometry), lipids and biochemical markers of bone metab-olism. Six participants withdrew from the study within 3 mo;three withdrew for social reasons, one relapsed with nonestro-gen-receptor–positive cancer, one disliked the diet and onebecame severely nauseated from the soymilk. Mean compli-ance (controlled by pill-intake count, diaries recording the use

of soymilk and weighing of leftover progesterone cream) was97% (range 52–102%), 94% (range 50–117%) and 97%(range 39–100%) for food supplement, soymilk and progester-one cream, respectively. Participants were requested to call ifany undesirable events occurred. Intervention was usuallystopped until the undesirable events were over and then re-started to clarify whether the complaints were caused by theintervention. If this turned out to be the case, the reaction wasreproduced twice and a decision about cessation was made.Mild stomach upset was experienced by 11% of the partici-pants if the supplement was taken without food; 29% of theparticipants experienced mild side effects not leading to ces-sation of intake, predominantly digestive troubles, of whichhalf were temporary; included in this group, 9% had undesiredweight gain (1–7 kg), mainly from fluid retention. In addition,6% had side effects leading to cessation of the soymilk intake;these reactions occurred 2–11 mo into the study; two subjectsexperienced severe nausea, flatulence and diarrhea, and foursubjects had both digestive and generalized symptoms (tired-ness, fluid retention, feeling of malaise, sleep disturbances,shortness of breath and joint pains). Symptoms cleared withindays after cessation of soymilk. Two of these six subjects haddisturbed liver parameters that normalized within weeks aftercessation of soymilk. It is unclear whether the soymilk causedthese disturbances because one subject had inactive hepatitisC and the other developed a coagulation disturbance 2 wkafter she stopped the soymilk. Of the participants who re-ceived progesterone cream, 22% experienced mild side effectsof skin irritation and temporary breast tenderness. One subjecthad to stop using the cream because of severe local skinirritation. No changes were seen in hematological variables.Of 13 subjects with self-reported cow’s milk intolerance, 11tolerated soymilk. Beneficial effects occurred in 18, 14 and 8%for food supplement, soymilk and progesterone cream groups,respectively. The food supplement improved nail and hairquality; some subjects felt more energetic, and sleep qualityimproved. Soymilk relieved 12 subjects of chronic constipa-tion, irritable bowel symptoms or both. We conclude thatsoymilk is a suitable alternative or supplement to other cal-cium sources in the diet. It is well tolerated and side effects aremostly mild and temporary, with a prevalence similar to thosefor cow’s milk or calcium supplements. [Soymilk and placebowere provided by ALPRO, Belgium, Pro-derma and placebowere provided by Phillips Nutritionals, USA, and Osforte byGenese A/S, Denmark. The study is supported by grants fromthe Danish Ministry of Health, Fondation Idella, Glunz andJensen Foundation, Civilingenieur Frode V. Nyegaard andWife’s Foundation, and Director E. Danielsen and Wife Foun-dation.]

Isoflavone intake

Soy Consumption and Nutrient Intakes of American AdultsBased on the CSFII Data Base 1994–1996. V. Juturu, G.Hsheih and P. M. Kris-Etherton. Department of Nutrition,Pennsylvania State University, University Park, PA.

The purpose of this study was to evaluate the effect of con-suming soy products on the energy and nutrient intakes offree-living male and female adults and children relative to notconsuming soy products by using data from the ContinuingSurvey of Food Intake by Individuals (CSFII) conducted from1994 to 1996. More Caucasians (4.22%) reported soy con-sumption than did other groups (Asians, 1%; blacks, 0.3%;American Indians, 0.03%; and others, 0.25%). Of CSFII re-

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spondents, 5.8% consumed soy products. Compared with soynonusers, soy users had a higher intake of energy, protein,polyunsaturated fatty acids, dietary fiber and dietary carbohy-drate and a lower intake of saturated fatty acids (SFA) (P, 0.0001). Total fat, monounsaturated fatty acids, dietarycholesterol, calcium and vitamin B-12 intakes were similar forsoy users and nonusers. Energy intake was higher in adult soyusers than nonusers (P , 0.0001). Body mass index was 25.8and 24.9 kg/m2 for male and female soy users and 26.6 and 26.2kg/m2 for male and female nonusers, respectively (P, 0.0001). Mean adequacy ratio was higher in soy users (90)than nonusers (86; P , 0.001). The nutrient profile of the dietof soy users was superior to that of nonusers and met somedietary recommendations, mainly by reducing SFA and cho-lesterol, for the prevention of chronic diseases.

Intake of Dietary Phytoestrogens in Postmenopausal Women:The Framingham Heart Study. Miriam J. J. de Kleijn,Yvonne T. van der Schouw, Peter W. F. Wilson,* DiederickE. Grobbee and Paul F. Jacques.† Julius Center for PatientOrientedResearch,UniversityMedicalCenterUtrecht,Utrecht,The Netherlands; *Framingham Heart Study, Boston Univer-sity School of Medicine, Framingham, MA; and †Jean Mayer,U.S. Department of Agriculture Human Nutrition ResearchCenter on Aging at Tufts University, Boston, MA.

The purpose of the study was to estimate the intake of dietaryisoflavones, coumestans and lignans in healthy Western post-menopausal women. Many plant foods contain isoflavones,coumestans and lignans. Only a few published studies exam-ined the dietary intake of phytoestrogens in the general West-ern population. The potentially positive health effects of phy-toestrogens are most relevant to postmenopausal women. Westudied 939 postmenopausal Caucasian women who enteredthe fifth examination of the Framingham Offspring Study(1991–1995) and completed the Willett Food FrequencyQuestionnaire (FFQ). By searching the medical and agricul-tural literature and contacting experts, we identified foodsources of phytoestrogens. We determined the concentrationsof the different isoflavones (daidzein, genistein, formononetinand biochanin A), coumestans and lignans (matairesinol andsecoisolariciresinol) for each food item on the FFQ. We scoredthe concentration of phytoestrogens in each food item intoseven categories and multiplied the score by the serving size ofthe food. The amounts in each food item were multiplied bythe frequency of the consumption of that food and thensummed across foods to obtain the total intake of each phy-toestrogen. Median total daily intake of the phytoestrogens ispresented with interquartile ranges in parentheses. The esti-mated daily median intake of the isoflavones was as follows:daidzein, 39 mg (24–57 mg); genistein, 70 mg (28–120 mg);formononetin, 31 mg (13–44 mg); and biochanin A, 6 mg(2–11 mg). The median total intake of isoflavones was 154 mg(98–235 mg), and the main source was beans and peas. Theestimated daily intake of coumestans was 1 mg (0–2 mg), andthe main source was broccoli. The estimated daily medianintake of matairesinol was 25 mg (16–36 mg ) and of secoiso-lariciresinol was 534 mg (383–763 mg). The median totalintake of lignans was 565 mg (407–788 mg), and the mainsource was fruits other than citrus fruits or berries. Dietaryintake of isoflavones, coumestans and lignans in healthy post-menopausal Caucasian women in the United States is low.Despite the low intakes, recommendations for changes in thediet of postmenopausal women to increase dietary phytoestro-

gens may be premature before the health benefits of phy-toestrogens are clearly demonstrated.

Soy Consumption in Taiwanese Children in Taipei. A.K.-F.Hsiao and P. M. Lyons-Wall. Human Nutrition Unit, De-partment of Biochemistry, University of Sydney, Sydney,NSW, Australia.

Soybeans have been a staple food in the traditional Asiancuisine for many centuries. Epidemiologic evidence showedthat lifetime soy consumption could contribute to the lowrates of heart disease and cancer observed in Asian commu-nities, yet controversy remains concerning whether eating soyis beneficial for children mainly because of the presence in soyof the weak estrogenic isoflavones. This study examined therange and frequency of intake of soy foods in a group of healthyTaiwanese children, aged 8–9 y living in Taipei (n 5 66;weight: 32.4 6 0.9 kg; height: 134 6 0.8 cm). The city ofTaipei was divided into three geographical areas, and withineach, one public school was randomly selected. Approximatelyequal numbers of boys and girls participated from each school,and parents were requested to fill in the questionnaire onbehalf of their children. The range and frequency of intake ofsoy foods were determined by using a specially designed fre-quency questionnaire that included soy products available inlocal shops and markets in Taipei. Seven frequency categorieswere offered ranging from ‘3 times/d‘ to ‘never,‘ and each itemwas allocated an appropriate serving size. The children con-sumed a diversity of soy products (23 different items), reflect-ing the use of soy as a traditional staple food in the Taiwanesecuisine. The most commonly consumed items were soy drink(mean intake 556 g/wk), soft tofu (303 g/wk), hard-pressedtofu (255 g/wk), soybean curd jelly (221 g/wk) and soy sauce(122 g/wk). Soy products were introduced at age 1.9 y (meanage) as soy drink (40%), tofu (40%), soybean curd jelly (9%)or other products (11%). Estimated total intake of isoflavonesin the group was 255 mg/wk. The average daily intake ofisoflavones in this study (36.6 mg/d) was comparable to intakesthat have resulted in physiologic effects in young women.However, no effects have been reported in children, eitheranecdotally or in the scientific literature. It is possible that thepotential estrogenic effects of isoflavones are lowered in chil-dren as a result of the first-pass effect in the liver, in whichisoflavones are absorbed and converted to inactive metabolitesin the liver before entering the peripheral circulation to act ontarget tissues. Alternatively, the tissues of children could beless sensitive than those of adults to the effects of isoflavones.

Urinary Isoflavone and Lignan Excretion by PremenopausalWomen in Seattle, Washington. H. Skor, S. Schwartz, E.Fitzgibbons, D. Scholes,† C. Chen, K. Wahala* and J.Lampe. Fred Hutchinson Cancer Research Center, PublicHealth Sciences, Seattle, WA; *University of Helsinki, De-partment of Chemistry, Helsinki, Finland; and †Group HealthCooperative of Puget Sound Center for Health Studies, Seat-tle, WA.

Soy foods are increasingly available to consumers, but few dataexist on the measurement of and levels of exposure to isofla-vones in the general U.S. population. Over a 24-mo period(4/97 to 3/99), we measured excretion of isoflavones andlignans by Seattle women participating in a case-control studyof uterine leiomyomata. Premenopausal women (n 5 217)provided two overnight urine collections 48 h apart. Isofla-vone and lignan excretion expressed as nanomoles per milli-

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gram of creatinine was highly correlated with excretion ex-pressed as nanomoles per hour (P 5 0.0001). Correlationsbetween the two measures of genistein, daidzein, O-desmeth-ylangolensin, total isoflavones (sum of isoflavonoids), and thelignans enterolactone and enterodiol (nmol/h) were 0.34,0.32, 0.76, 0.41, 0.65 and 0.41, respectively (P 5 0.0001).Geometric mean excretions (nmol/h) were as follows:genistein, 9.1; daidzein, 19.8; total isoflavones, 45.0; and totallignans (enterolactone plus enterodiol), 100.0. Isoflavone andlignan excretion was detectable in at least one collection inmost women as follows: genistein, 81%; daidzein, 93%;O-desmethylangolensin, 49%; enterolactone, 99%; and en-terodiol 96%. However, when excretion was examined sepa-rately for each collection, the frequency of detectable levels ofisoflavones in women decreased; for example, for genistein,63% of women excreted detectable levels on a single night butonly 46% excreted genistein on both nights. Asians (n 5 39)had higher levels of genistein (P 5 0.02), daidzein (P 5 0.04)and total isoflavones (P 5 0.03) than did non-Asians, but thenon-Asians excreted higher concentrations of lignans (P5 0.002). Despite increased soy food availability and con-sumer awareness of the possible benefits of soy, we did notobserve an increase in isoflavone excretion over the 24 mo inthe whole group or in non-Asians alone. In conclusion, in theSeattle area, a large proportion of women are exposed toisoflavones, even though at low levels.

Phytoestrogen Concentrations in Japanese Women and Men.M. S. Morton, O. Arisaka,* A. Miyake,* L. D. Morgan andB.A.J. Evans.† Bioclinical Services, International, Units 1–3Willowbrook Laboratories, Cardiff, UK; *Juntendo UniversitySchool of Medicine, Tokyo, Japan; and †Department of ChildHealth, UWCM, Cardiff, UK.

The soy-derived isoflavones daidzein and genistein have beenproposed to be cancer-protective agents in Japanese and otherAsian populations. This study reports the concentrations ofthe isoflavonoids daidzein, genistein and equol in the serum ofJapanese women (n 5 125) and men (n 5 102) as determinedby a robust gas chromatography–mass spectrometric method.The women were aged 40–89 y (mean 66.9 y), and the menwere aged 40–85 y (mean 64 y). For comparison, data are alsopresented for British women (n 5 60) and men (n 5 50). Themean serum levels of daidzein and genistein in Japanesewomen were 62.6 ng/mL (range 0–611 ng/mL) and 135.6ng/mL (range 0–1133 ng/mL), respectively. The mean equolconcentration was 13.8 ng/mL (range 0–241 ng/mL) with only32.5% of the women producing equol concentrations 5 ng/mL.In British women, the mean concentrations of daidzein andgenistein were 3.7 and 8.1 ng/mL, respectively. The meanconcentrations of daidzein and genistein in the sera of Japa-nese men were 71.1 ng/mL (range 0–577 ng/mL) and 133.2ng/mL (range 0–1160 ng/mL), respectively. The mean con-centration of the isoflavone equol was 23.8 ng/mL (range0–461 ng/mL), and 57 (56%) of these samples had equolconcentrations 5 ng/mL. For British men, the mean concen-trations of daidzein and genistein were 4.63 ng/mL (range0–16.4 ng/mL) and 9.2 ng/mL (range 0–31.8 ng/mL), respec-tively. Clearly, from this study, Japanese women and men havemuch higher levels of daidzein and genistein in their serumthan do British women and men. A life-long exposure to thesesoy-derived phytoestrogens may be responsible in part for thelower incidences of breast and prostate cancer observed inJapanese and other Asian populations.

Menopause symptom relief

A Comparison of the Effect of Treatment with Oral Micron-ized Estradiol or Phytoestrogen-Containing Soy Protein Iso-late on Tail Skin Temperatures of Ovariectomized Rats.Yuanlong Pan, Mary S. Anthony, Misti Kapps and ThomasB. Clarkson. Comparative Medicine Clinical Research Cen-ter, Wake Forest University School of Medicine, Winston-Salem, NC.

Whether phytoestrogen-containing soy supplements havebeneficial effects on hot flushes of postmenopausal women andhow those effects, if any, compare with estrogen replacementtherapy has been uncertain. Some researchers found a 22%reduction in severity of hot flushes in postmenopausal womentreated with soy supplements but no change in numbers of hotflushes. Small effects on hot flush frequency and severity werefound by one researcher. No significant differences betweensoy and control treatments were found in two other studies.These observations are in contrast to the 90% reduction in hotflushes with estrogen replacement. Uncertainty exists aboutwhether the dose of soy isoflavones (30–60 mg/d) was too low.We used ovariectomized retired breeder rats to approach theseuncertainties experimentally. The treatment groups were asfollows: the control group was fed a diet based on casein andlactalbumin; the Soy(2) group was fed alcohol-washed soyprotein isolate with the phytoestrogens extracted; the Soy(1)group was fed phytoestrogen-containing soy protein (equiva-lent to a woman’s dose of 144 mg/d of isoflavones), which istwo to three times higher than that in the studies with women;and the 17-b-estradiol (E2) group was fed oral micronizedestradiol at a dose equivalent to a woman’s dose of 1 mg/d. Atemperature transponder was taped to the surface of the tail tomeasure temperature. Tail skin temperature was significantlyincreased within 1 wk after ovariectomy. The animals werepair-fed during treatment (21 d). Soy(2) had no effect on skintemperature. E2 had a large effect on skin temperature(;1.4°C reduction from Control). Soy(1) was intermediatebetween the E2 and Control (;0.8°C reduction from Con-trol). In conclusion, in the rat model system, soy phytoestro-gens, even at high doses, have a modest effect on skin tem-peratures that is about half that of estradiol.

Effect of Isoflavone-Rich Soy Protein Isolate on MenopausalSymptoms in Perimenopausal Women. D. Lee Alekel, A. St.Germain and C. T. Peterson.* Department of Food Scienceand Human Nutrition, Human Metabolic Unit, Center forDesigning Foods to Improve Nutrition and *Department ofStatistics, Iowa State University, Ames, IA.

Soy isoflavones, estrogen-like substances structurally and func-tionally similar to 17-b-estradiol, may play a role in relievingmenopausal symptoms. In this double-blind, 24-wk study, weexamined the effects of isoflavone-rich (80.4 mg/d) soy proteinisolate in relieving menopausal symptoms and compared twomethods for collecting menopausal symptoms, i.e., a meno-pausal diary and a menopausal index. Subjects (n 5 69) wererandomly assigned to treatment as follows: isoflavone-rich soy(n 5 24), isoflavone-deficient soy (n 5 24) or whey control (n5 21) protein. At baseline, midtreatment and post-treatment,subjects used a diary to record hot flushes and night sweats.Every 6 wk, a menopausal index was used to assess symptoms.Urinary isoflavones indicated excellent compliance. Repeatedmeasures ANOVA indicated no treatment effect using themenopausal diary or menopausal index, respectively, on

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change in frequency of hot flashes (P 5 0.065 or P 5 0.18) andnight sweats (P 5 0.85 or P 5 0.92). Chi-square analysesindicated no treatment effect on severity of hot flushes ornight sweats at any time when either tool was used except fornight sweats at midtreatment (using the diary), which weresignificantly lower (P 5 0.036) in the control group. Chi-square analyses revealed no treatment effect on frequency orseverity of insomnia, limb numbness, headaches or fatigue atany time point. Similarly, there was no treatment effect onmood swings, libido, vaginal dryness or urinary frequency andurgency. However, all treatment groups in general reported adecline in menopausal symptoms, indicating a strong placeboeffect. On the basis of these results, isoflavones may notprovide the once-anticipated relief of vasomotor or othermenopausal symptoms.

A Randomized Trial of a Soy Beverage in the Treatment ofMenopausal Symptoms in Postmenopausal Breast CancerSurvivors. Cheri Kutynec, Ivo Olivotto, Jerilynn Prior,*Greg Hislop, Keith Chambers,† Karen Gelmon and EdithTempleton. BC Cancer Agency, Vancouver Cancer Centre,Vancouver, BC, Canada; *University of British Columbia,Canada; and †Vancouver Hospital and Health Sciences Cen-tre, Vancouver, BC, Canada.

Menopausal symptoms are often distressing and difficult totolerate in women who have undergone treatment for breastcancer. Treatments such as chemotherapy and tamoxifen mayinduce menopause and increase its symptoms, and conven-tional treatment using hormone replacement therapy is con-traindicated. In both postmenopausal and Asian women, theconsumption of soy-rich diets containing phytoestrogens hasbeen associated with a reduction in hot flushes. The purpose ofthis randomized, double-blind, placebo-controlled trial was toevaluate the acceptability and effectiveness of a soy beverage(containing 80 mg isoflavones) in the treatment of meno-pausal symptoms in women with a history of treated breastcancer. Eligible women were included in the study if they weresymptomatic for hot flushes, were 4 mo or more post-treatment(may be taking tamoxifen) and postmenopausal. Women wereexcluded if they had had a recurrence of cancer, had liver orkidney disease, were using hormone replacement therapy orantibiotics, or smoked. After enrollment, women completed avalidated instrument called the daily menopause diary for 4 wkat baseline and for 12 wk while consuming 500 mL/d of a soybeverage or placebo. At baseline and 4, 8 and 12 wk, womencompleted a questionnaire to determine their consumption ofsoy, alcohol and caffeine and use of alternative therapies. Themeasurement of serum follicle-stimulating hormone, alkalinephosphatase and gamma GT was obtained at baseline, andadditional samples of serum and urine were stored at baseline,6 and 12 wk for future analysis. At present, 135 out of a goalof 160 women are enrolled, and 60 women have completed thestudy. Results will be discussed. [Funded by The NationalCancer Institute of Canada and the Canadian Breast CancerResearch Initiative.]

Effect on Biochemical Parameters of an Oral Soy ExtractUsed in the Treatment of Vasomotor Symptoms in Meno-pausal Women. D. H. Upmalis, F. C. Cone and C. Lamia.Personal Products Company, North Brunswick, NJ.

The objective was to determine biochemical changes resultingfrom the use of an oral soy isoflavone extract for relief of hotflushes in menopausal women with the use of a double-blind,

randomized, balanced, parallel group, Phase II, outpatient,multicenter (16 sites) study. Menopausal women (n 5 177),mean age 55 y, with five or more hot flushes per day, wererandomly assigned to receive either two soy isoflavone extracttablets (containing a total of 25 mg genistin and daidzin pertablet) or two placebo tablets once daily for 90 d. Physical andgynecological exams and biochemical evaluations were per-formed at admission and completion. Soy isoflavone demon-strated a significant reduction in the severity of hot flushesover the 12 wk and there was a trend toward significance inreduction of frequency of hot flushes and night sweats. Therewas no effect on endometrial thickness. Mean biochemicalchanges are given in Table 1. Soy isoflavone extract waseffective in reducing frequency and severity of menopausal hotflushes but in this study showed little effect on biochemicalvariables that have been shown to be affected by dietary soy.The change in fasting glucose requires further investigation toconfirm and characterize this observation. The findings con-

firm the utility of soy isoflavone extract in the relief of meno-pausal hot flushes and underline the importance of character-ization of isoflavone extracts because their metabolic effect canbe markedly different from that of the parent dietary source.

Improvement of Menopausal Symptoms and Quality ofLife by Oral Soy Extract Tablets. David H. Upmalis,Brinda Wiita, Frederick L. Cone, Cathleen A. Lamia,Michelle Warren* and Lynn Bradley.† Advanced CareProducts, North Brunswick, NJ; *Columbia University,New York, NY; and †Johns Hopkins University, Baltimore,MD.

The objective was to characterize the effects of soy isofla-vone extract tablets on menopausal symptoms and qualityof life. Estrogen-deficient menopausal women (n 5 177)experiencing five or more hot flushes daily participated in adouble-blind, randomized, placebo-controlled, 12-wk mul-ticenter study of Optisoy soy extract tablets (a total 50 mg/dof genistin and daidzin). Symptom-based quality of life wasmeasured with the Women’s Health Questionnaire, a vali-dated menopausal quality-of-life scale. Hot flushes andnight sweats, recorded on diary cards, were the primaryefficacy criteria. Severity of hot flushes was reduced to asignificantly greater extent by soy compared with placebo.The number of hot flushes also decreased significantly in thefirst 6 wk. Patient responses to the questionnaire revealed asignificant (P 5 0.04) reduction from baseline in sleep

TABLE 1

Mean biochemical changes

VariableIsoflavone(n 5 56)

Placebo(n 5 62)

Follicle stimulating hormone, (mIU/mL) 7.3 5.9Total cholesterol, (mg/dL) 1.3 1.0LDL, (mg/dL) 21.1 21.9HDL, (mg/dL) 1.4 1.4Triglycerides, (mg/dL) 1.6 7.3Glucose, (mg/dL) 25.3 2.2*Sex hormone–binding globulin 5.7 6.1

* Significantly different from isoflavone, P 5 0.01.

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problems and a slight improvement in memory problems inthe soy group (P 5 0.06). Soy isoflavone extract tabletsrelieved menopausal symptoms effectively and improvedquality of life.

Safety issues

Long-Term Adverse Effects After Developmental Exposureto Genistein. Retha R. Newbold, Wendy N. Jefferson, Eliz-abeth Padilla Banks and Bill C. Bullock. DevelopmentalEndocrinology Section, Laboratory of Toxicology, NationalInstitute of Environmental Health Sciences, Research Trian-gle Park, NC.

Exposure to synthetic and naturally occurring estrogeniccompounds results in permanent alterations in the devel-oping organism if exposure occurs during the critical stagesof differentiation. Reports from our laboratory showed thatperinatal exposure of mice to diethylstilbestrol (DES) re-sults in impaired fertility, structural malformations and le-sions of the reproductive tract. In fact, long-term conse-quences of exposure to DES at 2 mg/(pup z d) on d 1–5include a high incidence (95%) of uterine carcinoma inanimals 18 mo of age. Doses mg/(pup z d) also result inlong-term adverse effects. Thus, the developing reproduc-tive tract appears to be extremely sensitive to perturbationby compounds with estrogenic activity. Because the nutri-tional and pharmaceutical use of phytoestrogens has in-creased over the past few years, mainly because of its re-ported beneficial effects, we investigated the potential risksposed by genistein if exposure occurs early in development.We tested the possibility that developmental exposure tothis compound would influence morphological, functionaland biochemical markers known to be estrogen sensitive.Uterine epithelial cell proliferation and induction of uter-ine lactoferrin and complement C3 were increased in re-sponse to genistein. Although lactoferrin was previouslyreported to be constitutively produced in the uteri of neo-natally DES-treated mice as early as 2 mo before the devel-opment of uterine carcinoma, the role of this protein in theinduction of neoplasia, either as a marker or contributingfactor, remains to be determined. However, lactoferrin andother marker proteins are induced by developmental expo-sure to genistein. The interaction of genistein with theestrogen receptors a and b was investigated. Further, micewere followed to evaluate a potential increased risk forhistological abnormalities, including uterine tumors later inlife. Many of the long-term effects observed after DEStreatment, including uterine adenocarcinoma, were ob-served after developmental exposure to genistein. Similar-ities between the effects of DES and genistein point to theneed for further mechanistic studies in phytoestrogens andthe role in long-term effects that follow developmentalexposure.

Estrogen Receptor a Estrogen Receptor b and LactoferrinExpression in Reproductive Tract Tissues after Treatmentwith Genistein or Diethylstilbestrol during Development.Wendy N. Jefferson, Sabine E. Kulling,† Elizabeth PadillaBanks, Jennifer Hagelbarger, John F. Couse* and Retha R.Newbold. Laboratory of Toxicology and *Laboratory of Re-productive Toxicology, NIEHS, Research Triangle Park, NCand †Institute of Food Chemistry, University of Karlsruhe,Karlsruhe, Germany.

Permanent alterations occur in both male and female miceafter exposure during development to synthetic and naturallyoccurring estrogenic compounds. Reports from our laboratoryshowed that perinatal exposure of mice to diethylstilbestrol(DES) results in impaired fertility and structural malforma-tions and lesions of the reproductive tract. Long-term conse-quences of exposure to DES on d 1–5 included a high inci-dence (95%) of uterine carcinoma. Similar long-term effectswere shown with genistein treatment. Thus, the developingreproductive tract appears to be uniquely sensitive to com-pounds with estrogenic activity. To study the developmentaleffects of genistein, which is found in many soy products andwas documented to have estrogenic activity, we tested thepossibility that neonatal exposure to this compound wouldalter estrogen-signaling pathways in estrogen target tissues andcompared the changes with tissues similarly exposed to DES.First, the ontogeny of estrogen receptor (ER) a, ER-b andlactoferrin (LF) from fetal d 14 through postnatal d 26 wasmapped in the developing reproductive tract of control femaleCD-1 mice by using a ribonuclease protection assay and im-munohistochemical analysis. ER-a was present in both theovary and reproductive tract; ER-b was present in the ovarybut not in the uterus. LF could not be detected in uterinetissue until about postnatal d 19. After neonatal treatment ond 1–5 with genistein (500–50,000 mg/kg) or DES (0.01–1000mg/kg), reproductive tract tissues were compared with those ofnonexposed control mice to determine whether the treatmentaltered the expression of these proteins. Mice exposed togenistein or DES showed an increase in LF expression in theuterus on d 5, the last day of treatment. Preliminary data fromneonatally exposed genistein or DES mice showed alterationsin both ER-a and ER-b expression in the ovary and reproduc-tive tract. The dose response and time course for ER-a andER-b expression during and after estrogen treatment are beingdetermined. The relationship of altered ER-a, ER-b and LFexpression and long-term adverse consequences in the femalereproductive tract is unknown; however it is currently beinginvestigated.

Dietary Soy Phytoestrogens Decrease Brain Calcium-Bind-ing Proteins but Do Not Alter Hypothalamic Androgen-Metabolizing Enzymes in Adult Male Rats. Edwin D. Lep-hart, Kenneth D. Setchell,* Herman Adlercreutz† and K.Scott Weber. Neuroscience Center, Brigham Young Univer-sity, Provo, UT; *Clinical Mass Spectrometry Center, Chil-dren’s Hospital Center, Cincinnati, OH; and †Folkhalsan Re-search Center, Department of Clinical Chemistry, Universityof Helsinki, Finland.

Phytoestrogens, estrogen-like molecules found in many plants,recently have received a great deal of investigative attentionbecause of their potential protective effects against age-relateddiseases (e.g., cardiovascular disease and osteoporosis) andhormone-dependent cancers (i.e., breast and prostate cancer).Calbindin (CALB) and calretinin (CALRET) are calcium-binding proteins that are potentially important in the devel-opment and function of the central nervous system, acting aspotential neuroprotective factors against programmed celldeath (apoptosis) associated with neurogenerative diseases,such as Parkinson’s and Huntington’s diseases. On the otherhand, the aromatase and 5a-reductase enzymes represent themajor pathways of steroid hormonal action in the centralnervous system, especially in hypothalamic regions duringperinatal and postnatal development in which they play im-portant regulatory roles in neuroendocrine functions, repro-

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ductive endocrine physiology and sexual behaviors. To deter-mine whether a soy diet containing relatively high levels ofphytoestrogens may influence brain androgen-metabolizingenzymes and calcium-binding protein levels in adult male rats,we examined aromatase and 5a-reductase enzyme activities inthe medial basal hypothalamic and preoptic area (MBH-POA)and characterized MBH-POA and amygdala (AMY) CALBand CALRET levels from rats fed a phytoestrogen-free vs. aphytoestrogen-containing diet. Male Sprague-Dawley ratswere randomly assigned to two treatment groups as follows:phyto-free diet or phyto-600 diet. The phyto-600 diet con-tained phytoestrogens at 600 mg/g; the phytoestrogen contentin the phyto-free diet was below the limits of HPLC detection.After 4 wk of consuming the diets (ages 70–100 d), braintissue sites were collected, MBH-POA aromatase and 5a-reductase levels were determined, plasma and brain phy-toestrogen concentrations were measured, and MBH-POAand AMY CALB and CALRET levels were assayed by West-ern analysis. The total plasma phytoestrogen concentration inthe phyto-600 group was significantly higher (2460 ng/mL)than in the phyto-free group (32 ng/mL). The MBH-POAcontent of phytoestrogens was also significantly higher in thephyto-600 (69 ng/g) than in the phyto-free group (9 ng/g).Surprisingly, there were no significant differences in the MBH-POA androgen metabolizing enzymes (i.e., aromatase or 5a-reductase). However, there was a significant decrease in CALBlevels in the MBH-POA brain site (and CALRET in theMBH-POA and AMY brain sites) of the phyto-600 comparedwith the phyto-free–fed rats. These data suggest that dietaryphytoestrogens (or the lack thereof) for a relatively shortinterval (;4 wk) can significantly alter brain calcium-bindingproteins in adult male rats. This alteration may have impor-tant implications for the neuroprotective effects of theseCa21-regulating molecules at the time of neurogenesis, duringpostnatal development and especially in aged animals.

Normal Growth and Immune Function of Newborn TermInfants Fed Soy Formulas for One Full Year. Karin M.Ostrom and John B. Lasekan. Ross Products Division, AbbottLaboratories, Columbus, OH.

Healthy, newborn term infants fed soy-based infant formulas(SOY) in a randomized, masked parallel study were evaluated forgrowth, serum biochemistry values, antibody response to routinechildhood vaccinations and morbidity. For their first year of life,infants (n 5 186) were randomly fed one of two formulas (onecommercially available and one experimental) containing soyprotein isolate. A nonrandomized reference cohort of 81 infantswas fed mixed feedings (MF) consisting exclusively of humanmilk to at least age 2 mo followed by human milk, a commerciallyavailable cow’s milk–based formula or both to age 1 y. Groups didnot differ by sex, ethnicity or birth weight. Growth was normaland weight, length and head circumference were similar amongthe three groups. All serum biochemistry values for the threegroups were within the normal infant reference ranges. Theresponse to Hemophilus influenzae type b conjugate (Hib), diph-theria toxoid, oral polio and tetanus toxoid vaccines, differentialwhite blood cell count and lymphocyte subsets indicated normalmaturation of the immune system for all three groups of infants.For the infants fed SOY, the response to Hib vaccine was similarto or greater than that for infants fed MF, and the responses to theother vaccines were similar to those of infants fed MF. Of allinfectious morbidity and antibiotic usage data collected, onlyphysician- reported infectious diarrhea was less for infants fed MFthan for infants fed SOY. This 1-y study demonstrated that

growth and immune response of infants fed SOY was normal andwas similar to growth and immune response of infants fed HM.

Low Comparative Reactivity of Soy Proteins and Soy-BasedInfant Formulas in an Animal Model of Food Allergy. Chris-topher T. Cordle, Geralyn Duska-McEwen, Karen L. Cour-tad, Melissa R. Nameth and William T. Malone. Ross Prod-ucts Division, Abbott Laboratories, Columbus, OH.

The most common infant food allergen is cow’s milk, probablybecause cow’s milk–based infant formula is the most widelyused infant feeding substitute for human milk. Infants whodevelop allergies to cow’s milk react to its casein, whey proteinor both. The use of soy-based formula in infants allergic to cowmilk is widely practiced but not officially endorsed by pediatricsocieties. Clinical results for prophylactic use of soy in patientsat high risk to develop atopic disease conflict. When animalmodels of food allergy were used to compare cow’s milk–basedwith soy-based formulas and casein or whey with soy proteinisolate, soy formulas and soy proteins were consistently lessreactive. We used a hyperimmunization animal models to rankthe immunologic reactivity potential of several protein systemsthat might be used for infants allergic to cow’s milk or infantsat high risk to develop food allergy. Laboratory animals werehyperimmunized with casein, whey, rice protein, rice branprotein, pea protein, oat protein or soy protein. Immune serawere collected and antibodies to the immunizing proteins werequantitated by using sensitive, antigen-specific ELISA meth-ods. Preimmunization antibody titers were also determined ascontrols for the immunization process and ELISA specificity.Data were expressed as log immune response [IR; (log d 35titer) 2 (log d 0 titer)]. Geometric mean IR values for theimmunogens were compared by ANOVA; this analysis indi-cated that the proteins fall into three statistically distinctreactivity categories. Cow’s milk proteins are highly reactive,i.e., casein IR was 4.49 and whey IR was 4.46. Rice, rice branand pea proteins showed the same intermediate reactivities,i.e., the IR were 3.99, 3.96 and 3.63, respectively. Oat and soyproteins were least reactive and not different from each other;the IR were 2.71 and 2.22, respectively. The IR of a hypoal-lergenic casein hydrolysate was 1.28. It is clear that soy and oatproteins are allergenic for some patients. However, well-con-trolled and standardized animal model data support the currentpractice of using soy-based infant formula and oatmeal forinfants with cow’s milk intolerance. The reduced immunologicreactivity of soy and oat may represent intrinsic characteristicsof these proteins that might be further exploited in the man-agement of food allergies.

Immunological Study of a Soybean-Based Liquid Food. Leo-nardo M. Vanella, Oscar A. Brarda, Raul V. Boudet andMarcela Permigiani. Fundacion Medica Cientıfica Centro Re-publica, Cordoba, Argentina.

The purpose of this study was to evaluate the immunogenicityof a soybean-based liquid food (SBLF; AdeS Natural), and toestablish its antigenicity and allergenicity. Specific antiserawere obtained by hyperimmunization of 15 rabbits as follows:five rabbits were inoculated with SBLF, five were inoculatedwith an isolation of soybean, and five were inoculated with asoybean antigenic extract. The stimulation was done with anemulsion in equal parts of antigen and Freund’s incompleteadjuvant. The presence of the major immunoglobulins (Ig),anti-SBLF and antisoybean was investigated in sera of 213children by double diffusion gel and crossover immunoelectro-

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phoresis techniques. The levels of total serum IgE and thepresence of IgE antisoybean were determined by radioaller-gosorbent test. The allergenicity of SBLF and soybean wasevaluated by skin testing (skin prick) in 202 atopic children.The group of rabbits stimulated with SBLF demonstrated animmune response slightly lower than that of the other groups.The presence of IgG, IgM and IgD anti-SBLF or antisoybeanwas not observed in children’s sera. IgE antisoybean was de-tected in five sera. Eight atopic children showed a positivesoybean cutaneous test. The prick test with SBLF was negativein all children. The immunogenicity of SBLF was slightlylower than that of the soybean extract; SBLF shares twoantigenically related components with soybean; IgE anti-SBLFwas not observed in the children. It would be due to theindustrial elaboration of the SBLF that includes thermal de-naturalization of trypsin inhibitors that are strong allergenicfactors.

Biological Effects of Soybean Isoflavones on NutritionalStatus in the Rat Model. G. Sarwar, M. R. L’Abbe and S.Brooks. Nutrition Research Division, Health ProtectionBranch, Health Canada, Ottawa, ON, Canada.

The isoflavones, genistein and daidzein, and their glycosides,found in high concentrations in soybeans and soy-proteinfoods, may have beneficial effects in the prevention of manyhormone-dependent diseases. However, these bioactive phy-toestrogens possess a wide variety of hormonal and nonhor-monal activities. It has been suggested that potential adverseeffects may occur in infants fed soy formulas (Setchell 1997).The daily exposure of infants to isoflavones in soy-based for-mulas was calculated to be 6- to 11-fold higher on a weightbasis than the dose that has hormonal effects in adults con-suming soy foods. Circulating concentrations of isoflavones ininfants fed soy-based formulas have been reported to be13,000–22,000 times higher than plasma estradiol concentra-tions in early life that may exert biological effects (Setchell1997). However, The no observed adverse effect level(NOAEL) of soy isoflavones is not known. To address this, a16-wk feeding study was conducted to investigate the biolog-ical effects of the addition of graded levels (0, 50, 100, 200 and400 mg/kg diet) of soybean isoflavones to a casein control dieton growth, protein, mineral and hormone status and repro-ductive endpoints in male and female rats. An isoflavone richextract (Novasoy) and a soy-based infant formula were used asthe source of dietary isoflavones. Isoflavones in foods and inplasma samples were determined by the methods of Wang andMurphy (1994) and King et al. (1996), respectively, usingWaters HPLC linear gradient with UV detector monitored at254 nm. The commercial powder forms of soy-based infantformulas sold in Canada contained 178–292 mg/g of totalisoflavones (daidzin, genistin, daidzein and genistein), whereasNovasoy contained 300 mg/g of the total isoflavones. The ratfeeding study started on August 20, 1999, and will be com-pleted on December 10, 1999. Interim analyses (after 6 wk ofstudy) revealed an adverse effect on body weight of male ratsfed the highest two levels of isoflavones, 200 mg/kg fromformula and 400 mg/kg from Novasoy. There was a dose-related increase (up to fivefold in males and up to threefold infemales) in the levels of plasma isoflavones. The plasma datashowed that the absorption of isoflavones from formula waslower than that from Novasoy. There was also a dose-relatedincrease in the length of the estrous cycle in female rats. Thecycle in rats fed the infant formula diet (providing 200 mg/kgof isoflavones) was shorter, however, than that in those fed the

Novasoy diet providing the same amount of isoflavones. Thissuggests differences in the potency of endogenous and ex-tracted isoflavones.King, R. A., Broadbent, J. L. & Head, R. J. (1996) Absorption and excretion of

the soy isoflavone genistein in rats. J. Nutr. 126: 176–182.Setchell, K.D.R., Zimmer-Nechemias, L., Cai, J. & Heubi, J. E. (1997) Exposure

of infants to phyto-oestrogens from soy-based infant formula. Lancet 350:23–27.

Wang, H.-J. & Murphy, P. A. (1994) Isoflavone content in commercial soybeanfoods. J. Agric. Food Chem. 42: 1666–1673.

Transfer of Isoflavones from Mother to Suckling Pupsthrough Milk in Rats. Yoko Nakashima, Yoshiko Ishimi,*Keizo Umegaki* and Sachie Ikegami.* Seitoku University,Iwase, Matsudo-city, Chiba, Japan and *The National Insti-tute of Health and Nutrition, Toyama, Shinjuku-ku, Tokyo,Japan.

There is a general agreement that soy isoflavones can bebeneficial to health in adults. However, isoflavones have actedas endocrine-disrupting chemicals. Therefore, some consider-ation should be given to the possibility that soy foods mayadversely affect the reproductive system in infants. On d 5 ofpregnancy, we fed Sprague-Dawley rats one of three dietscontaining daidzein and genistein and having a total isofla-vone concentration of 0, 0.5 and 1.0 g/kg. Although the bodyweight and serum triiodothyronine concentration of dams inthe 1.0-g/kg group were lower than those in 0-g/kg group, nosignificant differences were observed among the suckling pupsin the three groups. We detected both daidzein and genisteinin the blood of dams and pups and the stomach contents(milk) of pups. However, small amounts of isoflavones trans-ferred from mother to suckling pups.

No Estrogenic Hormonal Effects in Children Fed Soy For-mula Long Term. L. Businco, G. Bruno, P. G. Giampietro,G. Furcolo and F. Lucaroni. Allergy and Clinical Immunol-ogy Division, Department of Pediatrics, University ‘La Sapi-enza,‘ Rome, Italy.

Phytoestrogens are present in high concentration in soybeans.Therefore, hormonal effects may occur in infants fed soyformula (SF). The aim of this study was to evaluate thehormonal effects of long-term SF feeding in a selected group ofchildren who were fed SF (Isomil) for more than 6 mo early inlife. We enrolled 50 children (30 boys and 20 girls), medianage 29 mo (range 7 mo–12 y). Every child had a physicalexamination with particular attention paid to signs and symp-toms of precocious puberty. In addition, the following wereevaluated: bone density (mineralometry with double photonabsorptiometry) in children aged 3 y; bone age (X-ray of carpusand metacarpus according to Tanner) in all of the children;bone metabolism urinary markers, i.e., urinary deoxypyridino-line (by chemiluminescence), calcium, creatine, phosphorus(by colorimetry), in all the children; and serum levels of bonealkaline phosphatase (by immunoenzymeassay), osteocalcin,17-b-estradiol (by RIA) and parathyroid hormone (by chemi-luminescence) in all the children. The same variables wereevaluated in 20 normal children of the same age and sex(controls) who were not fed any soy. No abnormalities wereseen in the evaluated variables. None of the enrolled childrenpresented signs or symptoms of precocious puberty; weight andheight were within the normal range; and bone age was in thenormal range. Serum levels of bone alkaline phosphatase,osteocalcin, 17-b-estradiol and parathyroid hormone and thelevels of the urinary markers of bone metabolism (deoxypyr-

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idinoline, calcium, creatine and phosphorus) were in the nor-mal range. Of the 20 children aged 3 y, 8 (40%) had miner-alometry values that were in the range for sex and age. Thepreliminary data of the present study suggest that prolonged SFfeeding does not induce estrogenic effects for the evaluatedvariables. To confirm these preliminary data, further studies,which are in progress in our department, on a larger cohort ofchildren fed SF for a prolonged period are required.

Effects of Dietary Genistein Exposure during Developmenton CD (Sprague-Dawley) Rats. K. Barry Delclos, Thomas J.Bucci,* Larry G. Lomax,* John R. Latendresse,* Alan War-britton,* Constance C. Weis and Retha R. Newbold.† Na-tional Center for Toxicological Research, Jefferson, AR; *Pa-thology Associates International, Jefferson, AR; and †NationalInstitute of Environmental Health Sciences, Research Trian-gle Park, NC.

Many of the reported beneficial health effects of soy productshave been suggested to be due, at least in part, to the soyisoflavones. Genistein is a major soy isoflavone that interactswith the estrogen receptor and multiple other targets; it hasbeen investigated for potential benefit in preventing cancers atmultiple sites and alleviating or preventing adverse effectsassociated with menopause. A study to find dose range wasconducted as a prelude to an on-going multigeneration bioas-say to assess toxicities potentially associated with genisteinconsumption. Genistein was administered in a soy- and alfalfa-free diet at 0, 5, 25, 100, 250, 625 and 1250 ppm to dams fromgestation d 7. Litters were standardized to 4 pups per sex onpostnatal d 2. Exposure of pups continued through lactationand after weaning until postnatal d 50. Body weight and foodconsumption of the dams showed a decreasing trend with asignificant reduction at the highest dose. Similarly, pups ofboth sexes in the high dose group showed significantly de-creased body weights. Absolute organ weights, relative organweights or both showed relatively minor dose-related changesin terms of main dose effects, trends or difference betweenspecific treatment groups and controls. In males, affected or-gans were liver, thymus, pituitary, preputial gland and ventralprostate. In females, affected organs were thymus, pituitary,uterus and vagina. Bone measures in both sexes showed treat-ment-related effects. Histopathologic examination of femalepups revealed ductal and alveolar hyperplasia and hypertrophyof the mammary glands at 625 and 1250 ppm; similar effectswere observed in males at $25 ppm. Estrous cycle morpho-logic abnormalities and abnormal ovarian antral follicles wereobserved at 1250 ppm. In males, degeneration of the seminif-erous tubules was observed at 1250 ppm. There was an appar-ent deficit of sperm in epididymal sections at 625 and 1250ppm, although testicular spermatid head counts and epididy-mal spermatozoa counts did not show significant differencesfrom controls at these doses. Dietary genistein thus producedpotentially adverse effects in multiple estrogen-sensitive tis-

sues in males and females that are generally consistent with itsestrogenic activity and occur within exposure ranges, as deter-mined by serum levels, achievable in humans.

Inactivation of Thyroid Peroxidase by Genistein and Daid-zein In Vitro and In Vivo: Mechanism for AntithyroidActivity of Soy. Daniel R. Doerge, Hebron C. Chang andMona I. Churchwell. National Center for Toxicological Re-search, Jefferson, AR.

The association between soybean consumption and goiter inanimals and humans has a long history. Current evidence forthe beneficial effects of soy requires a full understanding ofpotential adverse effects as well. We previously identified theisoflavones, genistein and daidzein, as the only antithyroidconstituents present in soy (Divi et al. 1997). Genistein anddaidzein caused time- and H2O2-dependent irreversible inac-tivation of bovine lactoperoxidase (LPO) and porcine thyroidperoxidase (TPO), the enzyme that synthesizes thyroid hor-mones. The inactivation kinetics were consistent with a sui-cide mechanism; the apparent dissociation constants and par-tition ratios for genistein and daidzein were 0.2 and 0.5mmol/L and 1 and 3, respectively. Radiolabeled genisteinbecame covalently bonded to LPO concomitant to loss ofenzyme activity (;3 mol genistein/mol enzyme inactivated).Minimal effects were seen on the prosthetic heme content.These data are consistent with potent mechanism-based inac-tivation of LPO and TPO in which radical products derivedfrom oxidation of genistein and daidzein inactivate the per-oxidases by covalent binding to critical amino acid residues.Antithyroid effects were also investigated in Sprague-Dawleyrats fed a diet supplemented with genistein aglycone (0–500mg/g dry food) through postnatal d 140 after exposure togenistein in utero and in maternal milk. Blood and thyroidglands from male and female rats were snap-frozen upon col-lection. Both aglycone and conjugated forms of genistein werequantified in blood and thyroid glands by using isotope dilu-tion liquid chromatography–electrospray mass spectrometry.A dose-dependent increase was observed in both compart-ments. In blood, the aglycone content was 1–2% of totalgenistein and in thyroid it was 18–28%. Furthermore, TPOactivity was depressed in a dose-dependent manner in thethyroids of both male and female rats. These data show thatgenistein is present in the thyroid at concentrations equivalentto those causing enzyme inactivation in vitro and that this issufficient to inactivate rat TPO in vivo. The consequences ofTPO inactivation await confirmation by histopathologic eval-uation, but the results are consistent with the reported goitro-genic effects of soy and underscore the potential for thyroidtoxicity from isoflavones at the levels actually observed inhuman blood.Divi, R.L., Chang, H. C. & Doerge, D. R. (1997) Anti-thyroid isoflavones from

soybean: isolation, characterization, and mechanisms of action. Biochem.Pharmacol. 54: 1087–1096.

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