İsoflavon DHT nin etkisini önler

İsoflavon soya fasulyesinden elde edilen fito östrojendir,vücutta östrojenin etkisini taklit eder ama östrojenin yan etkilerine sebep olmaz.Bu etkisi sayesinde erkek tipi saç dökülmelerinde anti androjenik etkisiyle ve DHT nin saç köküne zararlarını engellemesiyle saç dökülmesini, durdurur.Saç dökülmesi tedavisi yanı sıra aynı nedenlerle ortaya çıkan prostat hastalıklarının tedavisinde de kullanılmaktadır.

Bilimsel yayın 1

Isoflavone supplements stimulated the production of serum equol and decreased the serum dihydrotestosterone levels in healthy male volunteers

M Tanaka, K Fujimoto, Y Chihara, K Torimoto, T Yoneda, N Tanaka, A Hirayama, N Miyanaga, H Akaza and Y Hirao

Department of Urology, Nara Medical University, Kashihara, Japan

Department of Urology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan

Correspondence: Dr M Tanaka, Department of Urology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan. E-mail: masa-t@naramed-u.ac.jp

Received 28 October 2008; Revised 13 March 2009; Accepted 13 March 2009; Published online 14 July 2009.

The aim of this study was to evaluate the effect of supplementing healthy men with soy isoflavones on the serum levels of sex hormones implicated in prostate cancer development. A total of 28 Japanese healthy volunteers (18 equol producers and 10 equol non-producers) between 30 and 59 years of age were given soy isoflavones (60 mg daily) supplements for 3 months, and the changes in their sex hormone levels were investigated at the baseline and after administration. The serum and urine concentrations of daidzein, genistein, and the levels of equol in the fasting blood samples and 24-h stored urine samples were also measured. All 28 volunteers completed the 3-month supplementation with isoflavone. No changes in the serum levels of estradiol and total testosterone were detected after 3-month supplementation. The serum levels of sex hormone-binding globulin significantly increased, and the serum levels of free testosterone and dihydrotestosterone (DHT) decreased significantly after 3-month supplementation. Among the 10 equol non-producers, equol became detectable in the serum of two healthy volunteers after 3-month supplementation. This study revealed that short-term administration of soy isoflavones stimulated the production of serum equol and decreased the serum DHT level in Japanese healthy volunteers. These results suggest the possibility of converting equol non-producers to producers by prolonged and consistent soy isoflavones consumption.

Bilimsel yayın 2

Isoflavone-Rich Soy Protein Isolate Suppresses Androgen Receptor Expression without Altering Estrogen Receptor-b Expression or Serum Hormonal Profiles in Men at High Risk of Prostate Cancer

Jill M. Hamilton-Reeves,

Salome A. Rebello,

William Thomas,

Joel W. Slaton,

and Mindy S. Kurzer

Department of Food Science and Nutrition;

Division of Biostatistics in the School of Public Health; and

Department of Urologic

Surgery, University of Minnesota, Minneapolis, MN 55455 and

Department of Urology, Veterans Administration Medical Center,

Minneapolis, MN 55417

Abstract

The purpose of this study was to determine the effects of soy protein isolate consumption on circulating hormone profiles

and hormone receptor expression patterns in men at high risk for developing advanced prostate cancer. Fifty-eight men

were randomly assigned to consume 1 of 3 protein isolates containing 40 g/d protein: 1) soy protein isolate (SPI1) (107

mg/d isoflavones); 2) alcohol-washed soy protein isolate (SPI2) (,6 mg/d isoflavones); or 3) milk protein isolate (0 mg/d

isoflavones). For 6 mo, the men consumed the protein isolates in divided doses twice daily as a partial meal replacement.

Serum samples collected at 0, 3, and 6 mo were analyzed for circulating estradiol, estrone, sex hormone-binding globulin,

androstenedione, androstanediol glucuronide, dehydroepiandrosterone sulfate, dihydrotestosterone, testosterone, and

free testosterone concentrations by RIA. Prostate biopsy samples obtained pre- and postintervention were analyzed

for androgen receptor (AR) and estrogen receptor-b expression by immunohistochemistry. At 6 mo, consumption of

SPI1 significantly suppressed AR expression but did not alter estrogen receptor-b expression or circulating hormones.

Consumption of SPI2 significantly increased estradiol and androstenedione concentrations, and tended to suppress AR

expression (P ¼ 0.09). Although the effects of SPI2 consumption on estradiol and androstenedione are difficult to

interpret and the clinical relevance is uncertain, these data show that AR expression in the prostate is suppressed by soy

protein isolate consumption, which may be beneficial in preventing prostate cancer. J. Nutr. 137: 1769–1775, 2007.

Introduction

Steroid hormones modulate growth of the prostate gland, and

elevated levels of androgens have been associated with prostate

cancer risk (1,2). Consumption of soy foods is thought to contribute to prostate cancer prevention as a result of the hormonal

properties of soy isoflavones, either through altered endogenous

circulating hormones or hormone-receptor signaling. Cell culture studies have suggested that the isoflavonoids, genistein and

equol, exert the most noteworthy hormonal effects. Genistein

inhibits the activity of 5a-reductase and 17b-hydroxysteroid dehydrogenase, enzymes required for androgen synthesis (3,4). The

isoflavonoid equol, a bacterially derived metabolite of the iso-

flavone daidzein, sequesters dihydrotestosterone (DHT)from

the androgen receptor (AR) in rat prostate tissue (5). Both isoflavonoids accumulate in the prostate gland (6–9) and may mimic or

modulate endogenous hormones relevant to prostate carcinogenesis.

Despite evidence from in vitro studies, human intervention

studies report inconsistent effects of soy or isoflavone consumption on circulating hormone profiles in men. Although reports

show statistically significant suppression of total testosterone

(10,11), sex hormone binding globulin (SHBG) (12), DHT (13),

dehydroepiandrosterone (14), estrone (15), and free androgen

index (13), and increased concentrations of SHBG (16) and DHT

(17), the majority of the 22 intervention studies to date have not

found significant changes in circulating sex steroid hormones (10–

31). Generally, the studies that report significant changes were

The Soy and Prostate Cancer Prevention (SoyCaP) trial was supported by grant

DAMD 17-02-1-0101 (M.S.K.) and W81XWH-06-1-0075 (J.H.R.) from the United

States Army Department of Defense Prostate Cancer Research Program. The

protein isolates were donated by The Solae Company, St. Louis, MO. Neither

sponsor was involved in writing this report.

Author disclosures: J. M. Hamilton-Reeves, S. A. Rebello, W. Thomas, J. W.

Slaton, and M. S. Kurzer, no conflicts of interest.

Supplemental Tables 1 and 2 are available with the online posting of this paper

at jn.nutrition.org.

* To whom correspondence should be addressed. E-mail: mkurzer@umn.edu.

Abbreviations used: 3a-AG, androstanediol glucuronide; AR, androgen receptor; DHEAS, dehydroepiandrosterone sulfate; DHT, dihydrotestosterone; ERb,

estrogen receptor-b; MPI, milk protein isolate; SHBG, sex hormone-binding

globulin; SPI2, alcohol-extracted soy protein isolate; SPI1, isoflavone rich soy

protein isolate.

0022-3166/07 $8.00 ª 2007 American Society for Nutrition. 1769

Manuscript received 15 January 2007. Initial review completed 7 March 2007. Revision accepted 11 April 2007.

by guest on November 14, 2012 jn.nutrition.org Downloaded from

C1.html

http://jn.nutrition.org/content/suppl/2007/06/21/137.7.1769.D

Supplemental Material can be found at:carried out in older men for a relatively long duration. None of the

published studies reported equol-excretor status effects on circulating hormone response to soy isoflavone interventions in men.

Circulating hormone profiles may fail to accurately reflect

prostate tissue exposure, and evaluating hormone receptor expression patterns in the prostate may provide additional evidence concerning the role of soy as a cancer preventive dietary

agent. The AR mediates the action of androgens, and AR expression is a potential marker for prostate cancer prognosis (32).

Dietary genistein has been shown to downregulate AR mRNA

expression in rodents (33,34), and genistein has been shown to

suppress AR activity through an estrogen receptor-b (ERb)-

dependent mechanism in LNCaP cells (35). Despite these data,

to our knowledge, there are no studies published to date that

evaluate the effects of soy protein isolate consumption on AR

and ERb expression in men, although one study reported that an

isoflavone extract derived from red clover failed to alter AR

expression compared with historically matched controls (26).

The objective of this project was to evaluate the effects of

isoflavone-rich soy protein isolate consumption on circulating

concentrations of reproductive hormones and prostate tissue

markers of estrogen and androgen receptor expression in men at

high risk of prostate cancer. The effects of an isoflavone-rich soy

protein isolate were compared with those of an isoflavone-poor

soy protein isolate to determine whether the isoflavones are the

responsible bioactive constituents. The underlying hypothesis

was that isoflavone-rich soy protein isolate consumption would

reduce circulating hormones, downregulate AR expression, and

upregulate ERb expression.

Material and Methods

Subjects. Fifty-eight men, aged 50–85 y, were recruited at the

Minneapolis Veteran’s Administration Medical Center Urology Clinic

from a group of patients that had already undergone a transrectal

ultrasound and biopsy. Patients in this study were either at high risk for

developing prostate cancer (n ¼ 53), or had low-grade prostate cancer

that was being followed by active surveillance (n ¼ 5). Subjects were

considered high risk if they had high-grade prostatic intraepithelial neoplasia (PIN) (n ¼ 50) and/or atypical small acinar proliferation (ASAP)

(n ¼ 14). The subjects with prostate cancer had Gleason scores of ,6 and

were not receiving any other prostate cancer therapy. Subjects were

recruited by urologic physicians, and the research nurse reviewed the

patients’ medical records to determine that eligibility criteria were met.

Exclusionary criteria included BMI .40 kg/m

, prostate cancer that required medical treatment, prostatitis, alcohol consumption .14 drinks/

wk, soy or milk allergy, regular antibiotic use, or renal insufficiency.

Eighty-seven subjects were screened for the study; 21 chose not to

participate after attending the orientation session, and 66 subjects began

the study. Eight subjects withdrew from the study before their 3-mo

appointment [disliked the study treatment powder (n ¼ 3), inconvenienced by study demands (n ¼ 2), gastrointestinal discomfort (n ¼ 1),

chose conventional prostate cancer treatment (n ¼ 1), weight gain (n ¼

1)]. Three subjects completed 3 mo of the study with good compliance

but chose not to finish due to inconvenience of the study demands, and

55 subjects completed the full 6-mo study.

Data from 58 subjects were included in the serum hormone analysis,

and 42 subjects were included in the hormone receptor expression

analysis. Fewer participants were eligible for the hormone expression

analysis because 3 subjects did not undergo the final prostate biopsy

[liver cancer diagnosis (n ¼ 1), heart condition (n ¼ 1), not clinically

indicated (n ¼ 1)], and 13 subjects had insufficient biopsy tissue at either

baseline or postintervention for the analyses. All 58 subjects who completed the study were Caucasian.

Study design. The University of Minnesota Institutional Review Board

Human Subjects Committee, the Minneapolis Veterans Affairs Institutional Review Board, and the U.S. Army Medical Research and Materiel

Command’s Human Subjects Research Review Board approved the

study protocol, and all subjects provided informed consent, attended an

orientation session, and were provided with a study handbook. During

the study orientation, subjects were interviewed and prompted about

incidental exposure to dietary isoflavones (e.g., snack bars, shakes, soy

nuts, canned tuna, legumes, breads) to determine whether they were soy

consumers. Only one participant reported regular soy consumption, but

he did not consume soy-containing products for 1 mo prior to beginning

the study. The 6-mo intervention study used a randomized, singleblinded, placebo-controlled, parallel design. Free-living subjects supplemented their diets with 1 of 3 randomly assigned protein isolates: 1) soy

protein isolate high in isoflavones (SPI1); 2) soy protein isolate that had

most of the isoflavones removed by alcohol extraction (SPI2); or 3) milk

protein isolate (MPI) (The Solae Company). The protein isolates were

consumed in divided doses twice daily and contributed 40 g/d protein

and 200–400 kcal/d (1 kcal ¼ 4.184 kJ). The isoflavone content of the

protein isolates expressed as aglycone equivalents was 107 6 5.0 mg/d

for the SPI1; ,6 6 0.7 mg/d for the SPI2; and 0 mg/d for the MPI

(mean 6 SD). The mean distribution of isoflavones was 53% genistein,

35% daidzein, and 11% glycitein in SPI1, and 57% genistein, 20%

daidzein, and 23% glycitein in SPI2 as analyzed by Dr. Pat Murphy

(Department of Food Science and Human Nutrition, Iowa State University). The packets of protein isolate were numbered and patients were

unaware of the treatment protein isolate they had been assigned until all

subjects completed the intervention. Only the study coordinators who

administered the protein isolates knew the group to which each participant belonged. Compliance was assessed by counting the number of

times the patient consumed the protein isolate, as self-reported in recording calendars given to them, and mean compliance was 94%. Dietary and herbal supplements were allowed, and participants were asked

to avoid changing dosages or adding new supplements to their regimen

during the study. Subjects consumed their habitual diets, and received

detailed instructions to exclude soy products to minimize isoflavone

consumption from other sources.

Serum collection and analysis. Fasting blood was collected in the

morning at 0, 3, and 6 mo. Serum was separated and aliquots were frozen

at –70 C until analysis. All serum samples were analyzed for testosterone, free testosterone, DHT, androstanediol glucuronide (3a-AG),

androstenedione, dehydroepiandrosterone sulfate (DHEAS), SHBG, estradiol, and estrone. Steroid hormones were analyzed in duplicate by

RIA, and SHBG was analyzed by immunoradiometric assay (Diagnostics

Systems Laboratories). Hormone analyses were performed in 3 batches

and all assays required

I-labeled analyte. Intraassay variabilities were

3.7% for testosterone, 4.4% for free testosterone, 6.1% for DHT, 4.5%

for 3a-AG, 4.4% for androstenedione, 2.3% for DHEAS, 4.4% for

SHBG, 3.9% for estradiol, and 4.3% for estrone. An internal control

was utilized to determine variability among batches, and interassay variabilities were between 9 and 30% for all analytes. All 3 serum samples

for each participant were analyzed in the same batch.

Urine collection and analysis. To assess equol-producer status, 24-h

urine was collected in plastic containers containing 1 g/L of ascorbic acid

and separated into aliquots after the addition of sodium azide to a final

concentration of 0.1%. Aliquots were frozen at –20 C until analysis.

Equol was determined by HPLC and MS as previously described (36).

The intraassay CV for equol was 8.2%, and the interassay CV was

12.5%. Subjects were classified as equol excretors if 24-h urine equol

levels exceeded 1000 nmol/d.

Dietary intake and analysis. Food records were completed for 3 d

before each clinic visit. A registered dietitian taught study participants

how to keep accurate food records. Patients were encouraged to use

household scales and volumetric tools and to submit food labels from

unusual foods. Study coordinators reviewed each food record for

completeness and clarified ambiguities with the participant at each clinic

visit. Food records were analyzed with Nutritionist V, version 2.3 (37),

and, for each 3-d food record, mean intakes of energy, macronutrients,

saturated fat, cholesterol, fiber, vitamin D, vitamin E, calcium, selenium,

and zinc were calculated.

1770 Hamilton-Reeves et al.

by guest on November 14, 2012 jn.nutrition.org Downloaded from Tissue collection and analysis. Biopsies were performed before the

initial screening and at the 6-mo clinical visit. Biopsy cores were formalinpreserved for 24 h and paraffin embedded. The histological diagnoses

were determined during a routine pathological evaluation. Immunohistochemistry was performed to assess AR and ERb expression on primarily normal, hyperplastic, or preneoplastic glands collected from

eligible study participants. Antigen retrieval was achieved by pressure

cooking deparaffinized and rehydrated tissue sections at 103 kPa in

citrate buffer. Sections were treated in quenching solution (3% H2O2 in

100% methanol), and then incubated with a protein-blocking solution

(10% milk, 5% serum, and 1% BSA). Samples were incubated overnight

at 4 C with rabbit polyclonal anti-ERb antibody (ab3577; Abcam;

1:1000) for the ERb assay, or for 30 min at room temperature with the

mouse monoclonal anti-AR antibody (AM256–2M; BioGenex; RTU) for

the AR assays. Next, the avidin-biotin peroxidase method was carried

out (Vectastain Elite ABC kit, Vector Laboratories). Color reaction was

developed using diaminobenzidine as the chromagen. Appropriate positive and negative controls were included in all staining runs. Disrupted

glands and glands on the edge of tissue sections were excluded from

analysis to avoid false positives. A technician without prior knowledge of

histological grading scored both the intensity of immunostaining and

the percentage of immunopositive areas at 403 magnification using

the HSCORE system as previously described (38). The range of the

HSCORE is a minimum of 1 and a maximum of 4 (1 indicated absent

staining; 4 indicated intense staining). A mean of 6 intact glands (range:

2–15) per slide for ERb and a mean of 8 intact glands (range: 3–19) per

slide for AR were averaged to derive the HSCORE (Fig. 1).

Excluded from analysis. The following data were excluded from

statistical analysis: 6-mo dietary intake from one participant reporting

unusually low consumption (mean ,500 kcal) (1 kcal ¼ 4.184 kJ)

during the 3-d food diary as a result of illness; 3 mo DHEAS that was

above normal range (16 mmol/L) and inconsistent with the participant’s

baseline and 6-mo measurements; all DHEAS measurements from one

participant with abnormally high 3-mo and 6-mo DHEAS concentrations (9 and 10 mmol/L, respectively) compared with baseline; and all

SHBG measurements from one subject with undetectable SHBG in the

serum (,3 nmol/L). One subject did not consume the treatment powder

for 3 d prior to his 6-mo appointment as a result of illness, so he was

excluded from the 6-mo equol excretion analysis.

Statistical analysis. The data appeared normally distributed and had

similar variance among groups. Demographic comparisons between

groups were performed with 1-way ANOVA for continuous endpoints,

and chi-square for categories of prostate cancer markers. ANCOVA was

used to compare groups adjusted for the baseline value of the final

endpoint. For androstenedione, the model included a treatment by baseline interaction. Preplanned pairwise comparisons of all groups are

reported for each endpoint as dictated by the study hypotheses: each

group’s adjusted mean (least squares mean) was compared with the other

2 groups’ adjusted means. Paired t tests were used to test for significant

within-group changes over time. In addition, these covariates were

screened as adjusters: baseline body weight, equol excretor status, and

energy and nutrient intake. P , 0.05 was considered significant. All

analyses were performed using SAS, version 9.1 (39).

Results

Baseline. Baseline anthropometrics, cancer status, and dietary

intake did not differ among the groups (Table 1), except that the

MPI group had a higher body weight and the SPI2 group consumed significantly more protein, calcium, and zinc at baseline

(Table 2). Baseline prostate steroid receptor expression patterns

(Table 3) and serum hormone and SHBG concentrations (Table

4) did not differ among the groups.

Anthropometrics and dietary intake. Body weight did not

change from baseline to 3 or 6 mo in any group (Table 2), and

the significant differences in body weight among the groups at

baseline were maintained. Protein, calcium, and vitamin D intakes increased in all groups during the study as a result of their

concentrations in the protein isolates, and the differences in

protein, calcium, and zinc intake at baseline were not present at

3 and 6 mo. At 3 mo, total and saturated fat consumption were

reduced in the SPI2 group relative to baseline. During the study,

energy, carbohydrate, cholesterol, fiber, vitamin E, selenium, and

zinc intakes did not change for any group. Dietary and herbal

supplement usage did not differ among groups (data not shown).

Body weight and protein intake differences among groups were

unrelated to altered hormone concentrations or steroid receptor

expression patterns.

Steroid receptors. Baseline-adjusted AR expression was lower

in prostate biopsies after 6 mo in the SPI1 group compared with

the MPI group (P ¼ 0.04) and tended to be lower in the SPI2

group compared with the MPI group (P ¼ 0.09; Table 3). AR

expression significantly increased from baseline in the MPI group,

but not in the other 2 groups. There were no changes from

baseline in ERb expression among the groups (Table 3).

Serum estrogens. The serum estradiol concentration was

significantly increased in the SPI2 group at 3 and 6 mo relative

to baseline, and by 6 mo, baseline-adjusted estradiol concentrations were significantly higher in the SPI2 group compared with

the other 2 groups (Table 4). Serum estrone was also significantly

increased in the SPI2 group at 3 and 6 mo, and was significantly

higher than in the MPI group at 3 mo but not at 6 mo.

Serum androgens and SHBG. The serum androstenedione

concentration was significantly higher in the SPI1 group than in

the MPI group at 3 mo. At 6 mo it was significantly greater than

at baseline in the SPI2 group, resulting in a significantly higher

concentration than in the SPI1 group (Table 4). At both 3 and 6

mo, serum DHEAS was higher in the SPI group than in the other

2 groups, and at 3 mo, 3a-AG was higher in the SPI2 group than

the other 2 groups. At 3 mo, the DHT concentration decreased

from baseline in the SPI group. Serum SHBG concentrations

were decreased significantly from baseline at 3 and 6 mo in all

groups, with no difference among the groups.

Equol-excretor status and hormone profiles. Equol excretor

status was assessed only in the SPI1 group, because only they

consumed sufficient daidzein to excrete equol. At 3 mo, there

were 4 excretors and 15 nonexcretors, but of this group, only

1 excretor remained at 6 mo [dropped out after 3 mo (n ¼ 1),

TABLE 1 Baseline characteristics of subjects

Values are means 6 SD or n (%). Means in a row with superscripts without a

common letter differ, P , 0.05.

Subjects were categorized by most advanced prostate cancer marker.

Soy effects on hormones in men 1771

by guest on November 14, 2012 jn.nutrition.org Downloaded from apparently changed status (n ¼ 1), and excluded data (n ¼ 1)].

Baseline characteristics (Supplemental Table 1) and serum

hormone concentrations at 3 mo (Supplemental Table 2) did

not differ between excretors and nonexcretors.

Discussion

The present study evaluated men at high risk of prostate cancer

to determine the effects of soy protein consumption on serum

hormones and prostate tissue steroid receptor expression levels.

The major finding was lower AR expression levels and no differences in ERb expression or circulating hormones in men

consuming SPI1 compared with those consuming MPI.

AR increased significantly from baseline in the MPI group,

but did not change from baseline in the soy groups. Because AR

expression is expected to increase in this population (40), we

infer that SPI1 apparently prevented or suppressed a rise in AR

expression. Lower tissue AR expression in the SPI1 group is

consistent with research in which dietary phytoestrogens downregulated AR mRNA expression in adult male rats (33,34,41).

Our data differ, however, from those of Jarred et al. (26), who

reported no differences in AR expression patterns between radical prostatectomy patients treated with isoflavones and historically matched controls. The inconsistent results between the

2 studies can be explained by several methodological differences.

In the study by Jarred et al. (26), the subjects, who consumed

160 mg/d of isoflavones in extracts derived from red clover, were

men with advanced prostatic neoplasms treated for short and

varied time periods (7–54 d). The tissue sections studied from

the radical prostatectomies taken from treated subjects represented cancerous glandular acinae and were compared with

sections of cancers from historically matched controls. Our

subjects consumed 107 mg/d of isoflavones in isoflavone-rich

SPI, were earlier in the carcinogenesis continuum, were treated

TABLE 2 Anthropometrics and dietary intake of men at high

risk of prostate cancer that consumed various

protein isolates for 6 mo

All values are means 6 SD. Means in a row with superscripts without a common

letter differ, P , 0.05. *Different from baseline, P , 0.05.

Sample sizes are for all time points except the following: 3 mo, MPI (n ¼ 17), and 6

mo, SPI1 (n ¼ 18), and SPI2 (n ¼ 18).

1 kcal ¼ 4.184 kJ.

FIGURE 1 Representative immunohistochemical staining of AR in

human prostate core biopsies for HSCORE. Arrow indicates stained

acinar cell in MPI control group (enlarged view inset in lower right).

1772 Hamilton-Reeves et al.

by guest on November 14, 2012 jn.nutrition.org Downloaded from for 6 mo each, and all biological samples were evaluated within

the same subject before and after the intervention. Furthermore,

the gland acinae studied presented either benign, hyperplastic, or

preneoplastic tissue.

Consumption of SPI1 did not affect ERb expression or

circulating hormones. The ERb expression results are inconsistent with studies in animals in which prolonged isoflavone exposure decreased ERb expression (33,42), and may be explained

by the variability in commercially available ERb antibodies (43).

Our hormone results, however, are consistent with most published reports from the clinical setting. The testosterone results

are consistent with numerous soy or isoflavone intervention

studies in which no change in total testosterone was observed

(12–31), but differ from 2 studies of short duration (10,11). Our

finding of no effect on directly measured free testosterone is

similar to published soy or isoflavone intervention studies to

date (11,14,15,20,22,24), and our finding of no effect on circulating DHT is consistent with most reports (10,14,16,19–

21,23,30), although it differs from results of 2 studies (13,17),

one of which used red clover extract (17). The lack of effect on

circulating estradiol or estrone is consistent with the literature

(10,11,15,16,19,22,29,30), although there is one report of decreased estrone in men consuming soymilk for 8 wk (15).

Serum SHBG decreased significantly from baseline in all

study groups. The finding that consumption of SPI1 decreased

SHBG is similar to a report by Mackey et al. (12); however, they

did not find a significant decrease in SHBG with an isoflavonepoor protein isolate as we did. In contrast to our findings,

Habito et al. (16) reported increased SHBG in men consuming

35 g of tofu daily for 2 wk, and others have reported no significant changes of SHBG with isoflavone-rich foods or extracts

(13,15,17,20–23,30). Decreased SHBG is a potentially harmful

effect because SHBG-bound hormones are less biologically available to stimulate hormone-sensitive cancers. Because high protein intake has been associated with decreased SHBG (44), it is

likely that the decrease in SHBG from baseline in all groups in

our study resulted from the subjects’ significantly increased

protein intake during the study (45).

The hormonal effects in the SPI2 group were unexpected.

Although AR expression was not significantly lower in the SPI2

group, AR expression appeared to be intermediate between that

of SPI1 and MPI groups. In addition, serum estradiol was increased in the SPI2 group. These results are similar to a study in

young men by Dillingham et al. (20) in which a low-isoflavone

protein isolate containing ,2 mg/d isoflavones significantly increased estradiol and estrone compared with a milk protein isolate after a 8-wk intervention. Our results differ, however, from a

study in older men by Goldin et al. (19) in which a low-isoflavone

soy protein isolate containing ,2 mg/d isoflavones did not change

estradiol or estrone concentrations after a 6-wk intervention.

Interestingly, we found serum estradiol was significantly higher in

the SPI2 group than in the  SPI1 group, whereas in Dillingham

et al. (20) found that estradiol in the low-isoflavone group did not

differ from the high-isoflavone group (20).

Serum androstenedione and DHEAS concentrations were

increased in the SPI2 group compared with both SPI1 and MPI

groups. No other soy protein or isoflavone intervention study has

TABLE 4 Serum hormones and SHBG in men at high risk

of prostate cancer that consumed various protein

isolates for 6 mo

Baseline data are unadjusted means 6 SEM. All other data are least-squares means

adjusted for baseline measurement 6 SEM, except androstenedione, which is

additionally adjusted for interaction between treatment and baseline. Means in a row

with superscripts without a common letter differ, P , 0.05. *Different from baseline,

P , 0.05.

Sample sizes are for all time points except: 3 mo MPI (n ¼ 17), and 6 mo SPI1 (n ¼

18), and SPI2 (n ¼ 19).

Sample sizes differed from other hormones due to excluded data. At 3 mo, SPI1

(n ¼ 19) and SPI2 (n ¼ 19). At 6 mo, SPI1 (n ¼ 17) and SPI2 (n ¼ 19).

Sample sizes differed from other hormones due to excluded data. At 3 mo, SPI1

(n ¼ 19), and at 6 mo, SPI1 (n ¼ 18).

TABLE 3 Steroid receptor expression of men at high risk of

prostate cancer that consumed various protein

isolates for 6 mo

SPI1 SPI2 MPI

Androgen receptor HSCORE

Baseline data are unadjusted means 6 SEM. All other data are least-squares means

adjusted for baseline measurement 6 SEM. Means in a row with superscripts without

a common letter differ, P , 0.05. *Different from baseline, P , 0.05.

Soy effects on hormones in men 1773

by guest on November 14, 2012 jn.nutrition.org Downloaded from reported a change in circulating androstenedione (12,17,19,20,30),

but all other studies to date have intervened for a shorter duration. Higher DHEAS is consistent with other low-isoflavone

soy protein isolate interventions (19,20). Although DHEAS and

androstenedione can be converted by 17b-hydroxysteroid dehydrogenase to testosterone, no significant changes were observed

in circulating testosterone, free testosterone, or DHT. Instead,

our study population had low, but normal, testosterone concentrations throughout the study. Although DHEAS and androstenedione concentrations have been associated with aggressive

prostate cancer (46), our findings of unchanged testosterone and

a trend toward lower AR expression (P ¼ 0.09) suggest neutral effects of SPI2 consumption. In fact, because DHEAS and

androstenedione may be converted to estradiol and estrone in the

prostate gland (47), the increase in DHEAS and androstenedione

may have contributed to the observed increases in circulating

estradiol and estrone. The hormonal effects of SPI2 consumption are likely due to the effects of the alcohol extraction process

on SPI constituents.

In conclusion, we found that consumption of isoflavone-rich

soy protein for 6 mo lowered AR expression levels in the prostate,

but did not change ERb expression or circulating hormones in

men at high risk of prostate cancer. Although consumption of

the alcohol-extracted soy protein did not significantly lower AR

expression, its effect appeared to be intermediate to that of SPI1

and MPI consumption, suggesting that the isoflavones alone may

not be responsible for the AR expression decrease, or, alternatively, that the low level of isoflavones in SPI2 were sufficient to

alter the AR. Unexpectedly, consumption of SPI2, but not SPI1,

significantly increased estradiol and androstenedione concentrations. None of these results were influenced by equol excretion

status. These data suggest that consumption of isoflavone-rich

and isoflavone-poor soy protein isolate exert differing effects on

endogenous hormones and receptor expression, which may

mediate prostate cancer preventive effects.

Acknowledgments

Immunohistochemistry was performed by Kenji Takamura. The

authors thank Kayla Vettling and Ellie Wiener for tissue scoring,

and Lori Sorensen, Nicole Nelson, and Mary McMullen for

assistance with clinic visits and data entry.

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Soybean effect

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