Article

Effects of the phytoestrogen genistein on bone metabolism in osteopenic postmenopausal women: a randomized trial. Ann Intern Med

Azienda Ospedaliera Universitaria Policlinico G. Martino, University of Messina, Messina, Italy.
Annals of internal medicine (Impact Factor: 17.81). 07/2007; 146(12):839-47.
Source: PubMed

ABSTRACT

Observational studies and small trials of short duration suggest that the isoflavone phytoestrogen genistein reduces bone loss, but the evidence is not definitive.
To assess the effects of genistein on bone metabolism in osteopenic postmenopausal women.
Randomized, double-blind, placebo-controlled trial.
3 university medical centers in Italy.
389 postmenopausal women with a bone mineral density (BMD) less than 0.795 g/cm2 at the femoral neck and no significant comorbid conditions.
After a 4-week stabilization period during which participants received a low-soy, reduced-fat diet, participants were randomly assigned to receive placebo (n = 191) or 54 mg of genistein (n = 198) daily for 24 months. Both the genistein and placebo tablets contained calcium and vitamin D.
The primary outcome was BMD at the anteroposterior lumbar spine and femoral neck at 24 months. Secondary outcomes were serum levels of bone-specific alkaline phosphatase and insulin-like growth factor I, urinary excretion of pyridinoline and deoxypyridinoline, and endometrial thickness. Data on adverse events were also collected.
At 24 months, BMD had increased in genistein recipients and decreased in placebo recipients at the anteroposterior lumbar spine (change, 0.049 g/cm2 [95% CI, 0.035 to 0.059] vs. -0.053 g/cm2 [CI, -0.058 to -0.035]; difference, 0.10 g/cm2 [CI, 0.08 to 0.12]; P < 0.001) and the femoral neck (change, 0.035 g/cm2 [CI, 0.025 to 0.042] vs. -0.037 g/cm2 [CI, -0.044 to -0.027]; difference, 0.062 g/cm2 [CI, 0.049 to 0.073]; P < 0.001). Genistein statistically significantly decreased urinary excretion of pyridinoline and deoxypyridinoline, increased levels of bone-specific alkaline phosphatase and insulin-like growth factor I, and did not change endometrial thickness compared with placebo. More genistein recipients than placebo recipients experienced gastrointestinal side effects (19% vs. 8%; P = 0.002) and discontinued the study. Limitations: The study did not measure fractures and had limited power to evaluate adverse effects.
Twenty-four months of treatment with genistein has positive effects on BMD in osteopenic postmenopausal women. ClinicalTrials.gov registration number: NCT00355953.

Full-text

Available from: Marco Atteritano, May 17, 2014
Effects of the Phytoestrogen Genistein on Bone Metabolism in
Osteopenic Postmenopausal Women
ARandomizedTrial
Herbert Marini, MD; Letteria Minutoli, MD; Francesca Polito, PhD; Alessandra Bitto, MD; Domenica Altavilla, PhD; Marco Atteritano, MD;
Agostino Gaudio, MD; Susanna Mazzaferro, MD; Alessia Frisina, MD; Nicola Frisina, MD; Carla Lubrano, MD; Michele Bonaiuto, MD;
Rosario D’Anna, MD; Maria Letizia Cannata, MD; Francesco Corrado, MD; Elena Bianca Adamo, MD; Steven Wilson, PhD; and
Francesco Squadrito, MD
Background: Observational studies and small trials of short dura-
tion suggest that the isoflavone phytoestrogen genistein reduces
bone loss, but the evidence is not definitive.
Objective: To assess the effects of genistein on bone metabolism in
osteopenic postmenopausal women.
Design: Randomized, double-blind, placebo-controlled trial.
Setting: 3universitymedicalcentersinItaly.
Patients: 389 postmenopausal women with a bone mineral density
(BMD) less than 0.795 g/cm
2
at the femoral neck and no signifi-
cant comorbid conditions.
Intervention: After a 4-week stabilization period during which par-
ticipants received a low-soy, reduced-fat diet, participants were
randomly assigned to receive placebo (n ! 191) or 54 mg of
genistein (n ! 198) daily for 24 months. Both the genistein and
placebo tablets contained calcium and vitamin D.
Measurements: The primary outcome was BMD at the antero-
posterior lumbar spine and femoral neck at 24 months. Secondary
outcomes were serum levels of bone-specific alkaline phosphatase
and insulin-like growth factor I, urinary excretion of pyridinoline
and deoxypyridinoline, and endometrial thickness. Data on adverse
events were also collected.
Results: At 24 months, BMD had increased in genistein recipients
and decreased in placebo recipients at the anteroposterior lumbar
spine (change, 0.049 g/cm
2
[95% CI, 0.035 to 0.059] vs. "0.053
g/cm
2
[CI, "0.058 to "0.035]; difference, 0.10 g/cm
2
[CI, 0.08 to
0.12]; P # 0.001) and the femoral neck (change, 0.035 g/cm
2
[CI,
0.025 to 0.042] vs. "0.037 g/cm
2
[CI, "0.044 to "0.027]; dif-
ference, 0.062 g/cm
2
[CI, 0.049 to 0.073]; P # 0.001). Genistein
statistically significantly decreased urinary excretion of pyridinoline
and deoxypyridinoline, increased levels of bone-specific alkaline
phosphatase and insulin-like growth factor I, and did not change
endometrial thickness compared with placebo. More genistein re-
cipients than placebo recipients experienced gastrointestinal side
effects (19% vs. 8%; P ! 0.002) and discontinued the study.
Limitations: The study did not measure fractures and had limited
power to evaluate adverse effects.
Conclusion: Twenty-four months of treatment with genistein has
positive effects on BMD in osteopenic postmenopausal women.
Ann Intern Med. 2007;146:839-847. www.annals.org
For author affiliations, see end of text.
ClinicalTrials.gov registration number: NCT00355953.
P
ostmenopausal osteoporosis is caused by a sharp de-
crease in estrogen levels that leads to an increased rate
of bone remodeling (1–3). Currently available treatments
for postmenopausal osteoporosis include hormone replace-
ment therapy; calcitonin; bisphosphonates; and selective
estrogen receptor modulators, such as raloxifene (4, 5).
Although hormone replacement therapy is effective in
reducing postmenopausal bone loss (6 8), it is associated
with a higher risk for breast, endometrial, and ovarian can-
cer; cardiovascular disease; venous thromboembolism; and
stroke (8 –10). Epidemiologic data indicate that women
who ingest high amounts of phytoestrogens, particularly
isoflavones in soy products, have less risk for osteoporosis
than do those who consume a typical Western diet (11–
13). Consequently, many women use phytoestrogens to
maintain bone density.
Genistein, an isoflavone phytoestrogen that is abun-
dant in soybean products, structurally resembles 17
!
-estra-
diol (14). As a natural selective estrogen receptor modula-
tor, genistein may positively regulate bone cell metabolism
without harmful estrogenic activity in the breast and
uterus. This safe profile results from the greater affinity of
genistein for estrogen receptor-
!
, which is more abundant
in bone, than for estrogen receptor-
"
, which is abundant
in reproductive tissue. Observational studies suggest that
postmenopausal Asian women who consume diets high in
isoflavones have a lower rate of fracture than that in other
groups (15, 16). However, the mechanism of action of
genistein on bone is not yet fully understood.
In postmenopausal women, treatment with genistein
(54 mg/d) increased bone mineral density (BMD) at the
lumbar spine and femoral neck with no clinically signifi-
cant adverse effects on the breast and uterus (17). In the
same cohort, genistein decreased the ratio of soluble recep-
See also:
Print
Editors’ Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840
Summary for Patients.......................I-34
Web-Only
Conversion of figures and tables into slides
Annals of Internal Medicine Article
© 2007 American College of Physicians 839
Page 1
tor activator of nuclear factor-
#
B ligand to osteoprotegerin,
which may partly account for its positive effects on BMD
(18). These investigations were the first to evaluate the
effects of purified, standardized genistein on bone health,
but they were short in duration and included only 90 pa-
tients. One published trial assessed the effect of isoflavones
on BMD in postmenopausal women, but it compared
isoflavone-rich soy milk (containing only a small amount
of genistein) with natural transdermal progesterone (19).
Other studies of pure genistein from our research group
have focused on cardiovascular outcomes or menopausal
symptoms rather than on bone health (20 –22).
We performed a randomized, placebo-controlled trial
of the effects of pure genistein on bone density and bone
metabolism over 24 months in a larger cohort of os-
teopenic postmenopausal women.
METHODS
Design and Setting
The study protocol is consistent with the principles of
the Declaration of Helsinki, and participants gave written
informed consent. Participants were recruited from women
reporting to the Center for Osteoporosis in the Depart-
ment of Internal Medicine and the Center for Menopause
in the Department of Obstetrical and Gynecological Sci-
ences, University of Messina (Messina, Italy), and to the
Department of Medical Physiopathology, University “La
Sapienza” (Rome, Italy). Three hundred eighty-nine
women met the inclusion criteria and agreed to participate
(Figure 1).
Participants
Participants were women 49 to 67 years of age who
had been postmenopausal for at least 12 months at base-
line, were in good general health, had not had a menstrual
period in the preceding year, had not undergone surgically
induced menopause, and had a follicle-stimulating hor-
mone level greater than 50 IU/L and a serum 17
!
-estradiol
level of 100 pmol/L or less ($27 pg/mL). At the start of
the study, a complete family history was obtained, physical
examination and laboratory evaluation (chemical analytes
and hematologic measurements) were performed, and
BMD was measured at the lumbar spine and femoral neck.
Exclusion criteria were clinical or laboratory evidence of
confounding systemic diseases, such as cardiovascular, he-
patic, or renal disorders; coagulopathy; use of oral or trans-
dermal estrogen, progestin, androgens, selective estrogen
receptor modulators, or other steroids; use of biphospho-
nates, cholesterol-lowering therapy, or cardiovascular med-
ications (including antihypertensive drugs) in the preced-
ing 6 months; smoking habit of more than 2 cigarettes
daily; treatment in the preceding year with any drug that
could have affected the skeleton; family history of estrogen-
dependent cancer; and BMD at the femoral neck greater
than 0.795 g/cm
2
(which corresponds to a T-score of "1.0
SD).
Randomization and Intervention
We assigned patients to groups by using a computer-
generated randomization sequence with a permuted block
size of 4, stratified by center. After a 4-week stabilization
period during which participants received a standard low-
soy, reduced-fat diet, participants were assigned to receive
genistein (n ! 198), 54 mg/d in 2 tablets (Laboratori
Plants, Messina, Italy), or placebo (n ! 191) (Figure 1).
The biological effects of phytoestrogen intake are described
elsewhere (23). No patient withdrew from the study during
the stabilization period. The purity of genistein was 98%.
Placebo and genistein tablets were identical in appearance
and taste. Both genistein and placebo tablets contained
calcium carbonate (500 mg) and vitamin D (400 IU).
All participants were counseled on an isocaloric, re-
duced-fat diet composed of 25% to 30% energy from fat,
less than 10% energy from saturated fatty acids, 55% to
60% energy from carbohydrates, and 15% energy from
protein, with a cholesterol intake less than 300 mg/d and
fiber intake of 35 g/d or greater. Recommended daily ca-
loric intake was based on body size and was calculated by
using the Harris–Benedict equation. We used this diet dur-
ing the stabilization period to ensure that all participants
had the same energy intake and to avoid interference with
the lipid profile. The intake of soy products, legumes, or
other nutrient supplements was prohibited. The isoflavone
intake before randomization, as assessed by using a food-
frequency questionnaire, was 1 to 2 mg/d. This intake has
been shown to be typical in Western populations (24).
Participants used this diet throughout the study, and ad-
Context
Women seeking alternative treatments to preserve bone
often use phytoestrogens, but evidence of their effective-
ness is lacking. Phytoestrogens are found in soy products.
Genistein is a phytoestrogen with a structure similar to
that of 17
!
-estradiol.
Contribution
This randomized trial compared genistein, 54 mg/d, with
placebo for 24 months in 389 osteopenic postmenopausal
women. Increases in bone mineral density were greater
with genistein than with placebo. Genistein also had fa-
vorable effects on markers of bone metabolism. Genistein
did not increase endometrial thickness, but it did cause
gastrointestinal side effects.
Implications
Genistein appears to have a favorable effect on markers
of bone health in osteopenic postmenopausal women.
Studies of its effect on fractures are needed.
—The Editors
Article Genistein and Bone Metabolism in Osteopenic Postmenopausal Women
840 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 www.annals.org
Page 2
herence was reinforced by a nutritionist. Diet and body
mass index were evaluated in all participants during follow-up.
Primary Outcome
The BMD at the anteroposterior lumbar spine and
femoral neck was measured by using dual-energy x-ray ab-
sorptiometry (Hologic QDR 4500 W, Technologic,
Turin, Italy) at baseline and after 12 and 24 months of
treatment. The instrument was calibrated daily according
to the manufacturer’s instructions. Reproducibility was cal-
culated as a coefficient of variation obtained by weekly
measurements of a standard phantom on the instrument
and by repeated measurements obtained in 3 patients of
different ages. The coefficient of variation of our instru-
ment is 0.5% with the standard phantom; in vivo, we
calculated a coefficient of variation of 1.1% for the lumbar
spine and 1.5% for the femoral neck.
Secondary Outcomes
Bone Resorption Markers
At baseline, 12 months, and 24 months, a 2-hour fast-
ing morning urine sample was collected at the same time of
day to assess urinary excretion of pyridinium crosslinks
(pyridinoline and deoxypyridinoline). Pyridinoline (nor-
Figure 1. Study flow diagram.
ArticleGenistein and Bone Metabolism in Osteopenic Postmenopausal Women
www.annals.org 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 841
Page 3
mal range, 26 to 91 pmol/
%
mol creatinine) and deoxypyr-
idinoline (normal range, 3 to 21 pmol/
%
mol creatinine)
were measured by using high-performance liquid chroma-
tography (Bio-Rad Laboratories, Hercules, California).
Bone Formation and Bone Growth Markers and Other Variables
After an overnight fast, venous blood samples were
collected between 8 a.m. and 9 a.m. through a polyethyl-
ene catheter inserted in a forearm vein. The serum was
separated from the blood corpuscles by centrifugation and
kept frozen at "70 °C until analysis for bone formation
and bone growth markers, calcium, intact parathyroid hor-
mone, 25-hydroxyvitamin D
3
, 17
!
-estradiol, follicle-stim
-
ulating hormone, total cholesterol, high-density lipopro-
tein cholesterol, low-density lipoprotein cholesterol, and
triglycerides.
Serum bone-specific alkaline phosphatase (normal
range, 8.5 to 17.9
%
g/L) and insulin-like growth factor I
(normal range, 9.6 to 21.2 nmol/L) were measured by us-
ing an immunoenzymatic assay (Pantec, Turin, Italy). Se-
rum calcium (normal range, 2.25 to 2.75 mmol/L [9 to 11
mg/dL]), serum phosphorus (normal range, 1.13 to 1.45
mmol/L [3.5 to 4.5 mg/dL]), and urinary creatinine (130
to 220
%
mol ! kg
"1
! d
"1
[14.71 to 24.89 mg/kg of body
weight per day]) were measured by using automated rou-
tine procedures. Parathyroid hormone (normal range, 12
to 100 pg/dL), 25-hydroxyvitamin D
3
(normal range, 1.25
to 7.5 nmol/L), and follicle-stimulating hormone (normal
range, 21 to 153 IU/L in the postmenopausal phase) were
measured by using high-performance liquid chromatogra-
phy (Bio-Rad Laboratories). 17
!
-Estradiol (normal range,
37 to 110 pmol/L in the postmenopausal phase) was eval-
uated by using a solid-phase immunoassay (Roche Diag-
nostics, Monza, Italy). Total cholesterol, high-density lipo-
protein cholesterol, and triglycerides were measured by
using a routine enzymatic method, and the low-density
lipoprotein cholesterol level was calculated by using the
Friedewald formula: [total cholesterol (mg/dL) " high-
density lipoprotein cholesterol (mg/dL) " triglycerides
(mg/dL)/5].
Genistein was measured in serum samples by using a
time-resolved fluorometric assay according to the manufac-
turer’s instructions (Labmaster TR-FIA test, Labmaster,
Turku, Finland) (25). The fluorescence was read by using a
Victor 1420 Multilabel Counter (PerkinElmer Life and
Analytical Sciences, Waltham, Massachusetts).
Adverse Events
Participants were asked about symptoms at clinic visits
every 3 months. Standard clinical evaluations and labora-
tory analyses, including hematologic, renal, and liver func-
tion tests, were done every 6 months. Endometrial thick-
ness was evaluated by using ultrasonography at baseline, 1
year, and 2 years. The endometrial thickness was measured
in the sagittal plane from 1 basal layer to the other. If the
endometrial thickness was 8 mm or greater or if uterine
bleeding occurred, hysteroscopy and endometrial biopsy
were performed. All unfavorable and unintended clinical
effects were considered adverse effects and were evaluated
for severity, duration, seriousness, and relation to the study
drug and outcome. We specifically checked all participants
for gastrointestinal symptoms, breast tenderness, vaso-
motor symptoms, depression, irritability, insomnia, and
vaginal bleeding. This evaluation was conducted by using a
checklist.
Statistical Analysis
We estimated that at least 97 participants in each
group would provide 80% power to detect a significant
expected absolute between-group difference in femoral
neck or lumbar BMD of 20%, assuming a 2-tailed
"
level
of 0.05. We estimated that genistein recipients would have
a statistically significant increase in BMD at both sites,
whereas BMD would continue to decrease in placebo re-
cipients, and that the change in BMD between the 2
groups would differ by at least 20% by the end of 2 years
of treatment.
The primary efficacy data on femoral neck and lumbar
spine BMD were analyzed according to the intention-to-
treat principle. These analyses included all 389 postmeno-
pausal women in whom BMD was measured at baseline.
Characteristics of women who withdrew from the study
were also investigated.
Descriptive data are given as the mean (SD), and 95%
CIs are provided where appropriate. The significance of
Table 1. Baseline Characteristics
Variable All Participants (
n
! 389) Genistein Group (
n
! 198) Placebo Group (
n
! 191)
Placebo
Group
(
n
! 191)
Genistein
Group
(
n
! 198)
Women Who
Completed
the Study
(
n
! 150)
Women Who
Withdrew
(
n
! 48)
Women Who
Completed
the Study
(
n
! 154)
Women Who
Withdrew
(
n
! 37)
Mean age (SD), y 54.2 (2.7) 54.7 (3.5) 54.9 (3.7) 54.3 (2.9) 54.2 (2.8) 53.9 (2.1)
Mean body mass index (SD), kg/m
2
25.1 (4.2) 25.0 (3.3) 24.8 (3.5) 25.9 (2.6) 25.5 (4.3) 23.4 (3.6)
Mean time since menopause (SD), mo 59.1 (38.4) 66.8 (45.8) 69.5 (47.4) 58.3 (39.6) 59.8 (38.8) 56.1 (37.4)
Mean bone mineral density (SD), g/cm
2
Anteroposterior lumbar spine 0.837 (0.099) 0.842 (0.08) 0.85 (0.08) 0.82 (0.06) 0.83 (0.10) 0.86 (0.07)
Femoral neck 0.674 (0.055) 0.667 (0.055) 0.67 (0.06) 0.66 (0.04) 0.67 (0.06) 0.68 (0.04)
Article Genistein and Bone Metabolism in Osteopenic Postmenopausal Women
842 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 www.annals.org
Page 4
between-group differences was assessed by using a mixed-
model, repeated-measures analysis that included fixed ef-
fects for treatment and visit and the interaction of these 2
terms, as well as random effects for intercept and slope.
This analysis was repeated for secondary outcome mea-
sures.
Before analysis, all end points were tested for normal-
ity by using the Kolmogorov–Smirnov test, and normal
probability plots, box-and-whiskers plots, and plots of raw
scores against normal percentile plots were constructed.
These analyses revealed no pronounced deviations from
normality, and all analyses were therefore conducted on
raw scores.
A P value of 0.05 or less was considered statistically
significant, and 95% CIs were calculated wherever possi-
ble. Statistical analysis was performed by using SAS soft-
ware, version 9.1 (SAS Institute, Cary, North Carolina).
Role of the Funding Sources
The study was supported by a grant from the Italian
Ministry of Education, University, and Research and by
the University of Messina, Italy. The funding sources had
no role in the design, conduct, and analysis of the study or
in the decision to submit the manuscript for publication.
RESULTS
Patient Characteristics
Table 1 shows baseline characteristics of the partici-
pants. No differences were observed between groups. A
post hoc comparison was done of the baseline characteris-
tics of women who withdrew from the study before com-
pletion (n ! 85) and those of women who completed the
study and between the 2 treatment groups. Baseline BMD
at the lumbar spine was statistically significantly lower in
genistein recipients who withdrew, and body mass index
was statistically significantly lower in placebo recipients
who withdrew (Table 1). However, these findings were
judged to be unlikely to affect outcomes of interest. Twenty-
eight (58%) of 48 genistein recipients who withdrew and
18 (48%) of 37 placebo recipients who withdrew com-
pleted 1 year of follow-up. The BMD at 1 year did not
differ between these women and those who completed the
study.
Primary Outcome
Mean BMD at the femoral neck increased from 0.667
g/cm
2
at baseline to 0.683 g/cm
2
at 1 year and 0.702
g/cm
2
at 2 years in genistein recipients and decreased from
0.674 g/cm
2
at baseline to 0.659 g/cm
2
at 1 year and 0.638
g/cm
2
at 2 years in placebo recipients. Similarly, mean
BMD at the lumbar spine was 0.842 g/cm
2
, 0.866 g/cm
2
,
and 0.891 g/cm
2
at baseline, 1 year, and 2 years, respec
-
tively, in the genistein group and 0.837 g/cm
2
, 0.807
g/cm
2
, and 0.784 g/cm
2
, respectively, in the placebo
group.
To further investigate the changes in BMD at the fem-
oral neck and lumbar spine, we conducted a mixed-model
analysis in which the intercepts were allowed to vary. This
analysis treated missing data as missing at random and thus
used all available information to determine the underlying
covariance structure as well as mean and variance informa-
tion from nonmissing data. The expected mean difference
in change in femoral neck BMD (genistein values minus
placebo values) was 0.023 g/cm
2
(95% CI, 0.011 to 0.034
g/cm
2
)(P # 0.001) at 1 year and 0.062 g/cm
2
(CI, 0.049
to 0.073 g/cm
2
)(P # 0.001) at 2 years (Figure 2
and
Table 2). The expected mean difference in change in lum-
bar spine BMD (genistein values minus placebo values)
was 0.055 g/cm
2
(CI, 0.036 to 0.073 g/cm
2
)(P # 0.001)
at 1 year and 0.10 g/cm
2
(CI, 0.08 to 0.12 g/cm
2
)(P #
0.001) at 2 years (Figure 2 and Table 2).
Figure 2. Change in bone mineral density (
BMD
) over time.
Estimates are expected means from mixed-effects models. *P # 0.001 vs.
placebo.
ArticleGenistein and Bone Metabolism in Osteopenic Postmenopausal Women
www.annals.org 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 843
Page 5
Secondary Outcomes
Mixed-model analyses were performed for secondary
measures (Table 2). At 1 year and 2 years, genistein levels
were increased from baseline values in the genistein group
but remained unchanged or decreased slightly in the pla-
cebo group. The between-group difference was statistically
significant at both time points.
Urinary excretion of pyridinoline and deoxypyridino-
line had statistically significantly decreased at 1 year and 2
years in the genistein group but did not significantly
change in the placebo group (Figure 3). Between-group
analyses showed that genistein use statistically significantly
decreased urinary excretion of pyridinoline and deoxy-
pyridinoline at both time points compared with placebo
(Table 2). Likewise, levels of bone-specific alkaline phos-
phatase and insulin-like growth factor I statistically signif-
icantly increased from baseline in the genistein group but
did not significantly change for the placebo group. Be-
tween-group differences were statistically significant for
each of these variables at 1 year and 2 years (Figure 3).
Levels of calcium and 25-hydroxyvitamin D
3
in
-
creased statistically significantly from baseline in both
groups, but the changes did not differ significantly between
the groups. The phosphorus level had increased statistically
significantly at 1 year and 2 years in the genistein group
but not in the placebo group; between-group comparison
of changes was not significant. Within-group and between-
group changes in parathyroid hormone level were not sta-
tistically significant (Table 2). Finally, body mass index did
not change significantly in either group after 1 year and 2
years of treatment.
Adverse Events, Uterine Safety, and Climacteric
Symptoms
Results on routine biochemical, liver function, and he-
matologic testing did not change over time in placebo or
genistein recipients. Eight placebo recipients and 16
genistein recipients withdrew because of adverse events in
the first 12 months of treatment (Figure 1). During the
second year, 7 placebo recipients and 21 genistein recipi-
ents had adverse events (Figure 1). In total, 37 (19%)
genistein recipients and 15 (8%) placebo recipients discon-
tinued therapy because of adverse events (P ! 0.002), all
of which were gastrointestinal (Table 3). No patient who
remained in the study experienced an adverse event.
Genistein at a dosage of 54 mg/d did not cause a
significant change in the endometrial thickness compared
with placebo (Table 3). At 2 years, genistein treatment
markedly reduced the mean number of hot flushes per day
compared with placebo (1.7 [SD, 0.21] vs. 3.9 [SD, 0.26];
P # 0.001).
DISCUSSION
We found that treatment with genistein, an abundant
soy isoflavone, prevents bone loss caused by estrogen defi-
ciency without affecting the uterus in osteopenic post-
menopausal women. Genistein decreased levels of bone re-
sorption markers and increased levels of markers of new
bone formation, producing a net gain in bone mass after 1
year and 2 years of therapy. Nevertheless, although BMD
and bone markers are considered good surrogates of bone
strength and bone quality, they may not correlate perfectly
with reduction in fracture risk.
Urinary excretion of pyridinoline and deoxypyridino-
line was decreased in genistein recipients compared with
placebo recipients. This finding confirms that the positive
effects of genistein on bone loss reduction are caused, at
least in part, by a constant decrease in bone resorption.
Genistein-mediated effects on bone resorption markers are
explained by in vitro studies showing that genistein can
Table 2. Changes in Biochemical Variables*
Outcome Measure Genistein Group
Mean Value
at Baseline (SD)
Change in Value
at 1 Year (95% CI)
Change in Value
at 2 Years (95% CI)
Mean Value
at Baseline (SD)
Bone mineral density, g/cm
2
Femoral neck 0.667 (0.055) 0.016 (0.007 to 0.023) 0.035 (0.025 to 0.042) 0.674 (0.056)
Lumbar spine 0.842 (0.080) 0.024 (0.012 to 0.034) 0.049 (0.035 to 0.059) 0.837 (0.099)
Genistein level,
%
mol/L 0.149 (0.020) 0.580 (0.572 to 0.587) 0.603 (0.595 to 0.611) 0.146 (0.018)
Calcium level
mmol/L 2.39 (0.09) 0.063 (0.043 to 0.08) 0.06 (0.04 to 0.078) 2.40 (0.12)
mg/dL 9.54 (0.36) 0.25 (0.17 to 0.32) 0.24 (0.16 to 0.31) 9.58 (0.48)
Phosphorus level
mmol/L 1.17 (0.15) 0.03 (0.006 to 0.061) 0.04 (0.013 to 0.068) 1.16 (0.18)
mg/dL 3.63 (0.46) 0.10 (0.02 to 0.19) 0.13 (0.04 to 0.21) 3.60 (0.55)
Parathyroid hormone level, pg/mL 48.8 (16.0) 2.1 ("1.6 to 5.9) 0.75 ("3.2 to 4.7) 48.2 (16.7)
25-Hydroxyvitamin D
3
level, IU/dL
29.5 (10.2) 13.5 (11.4 to 15.6) 13.9 (11.7 to 16.1) 30.6 (10.9)
Bone-specific alkaline phosphatase level,
%
g/L 10.4 (2.1) 3.4 (3.2 to 3.6) 3.9 (3.7 to 4.2) 10.3 (1.9)
Insulin-like growth factor I level,
%
g/L 111 (27) 20.2 (14.3 to 26.1) 17.2 (10.9 to 23.4) 113 (31)
Pyridinoline level, pmol/
%
mol creatinine 92.0 (21.7) "13.3 ("17.2 to 9.3) "13.0 (17.2 to 8.7) 91.6 (31.9)
Deoxypyridinoline level, pmol/
%
mol creatinine 21.7 (5.5) "2.7 ("3.6 to 1.8) "2.5 ("3.5 to 1.5) 21.1 (5.8)
* Values were obtained from mixed-model analyses.
Article Genistein and Bone Metabolism in Osteopenic Postmenopausal Women
844 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 www.annals.org
Page 6
suppress osteoclast activity through several mechanisms
(26–31). Furthermore, in vivo studies indicate that
genistein prevents estrogen-deficiency bone loss in ovariec-
tomized animals (32, 33).
In a 6-month study, isolated soy isoflavones decreased
bone resorption in postmenopausal women (34). More-
over, our previous study (18) showed that 1 year of
genistein therapy (54 mg/d) had antiresorptive action in
postmenopausal women. This action was probably medi-
ated through direct effects on the soluble nuclear factor-
#
B
ligand–osteoprotegerin system (18).
We observed increases in the bone-specific alkaline
phosphatase level in the genistein group, which may be
caused by a direct genomic estrogen receptor–mediated
effect or a nongenomic action in target cells. Genistein
may act on de novo protein synthesis (35, 36) and on
amplification of the interaction between the estrogen re-
ceptor complex and nuclear DNA in osteoblasts. These
cells express both estrogen receptor-
"
and estrogen recep-
tor-
!
(37), and genistein may act on bone by a mechanism
involving estrogen receptor-
!
(14). Moreover, genistein
may stimulate osteoblast proliferation, although this phy-
toestrogen has been shown to inhibit both basal- and
growth factor–induced proliferation of several normal and
cancer cell lines (3840).
Genistein also positively affected levels of insulin-like
growth factor I, a marker of bone growth in postmeno-
pausal women, in a pattern similar to that observed for
bone-specific alkaline phosphatase. Isoflavones have been
shown to upregulate insulin-like growth factor I and trans-
forming growth factor-
!
and inhibit osteoclastogenesis in
bone marrow cells of rats (41). Furthermore, several phy-
tochemicals and synthetic environmental chemicals display
estrogenic properties and mimic estrogens by activating in-
sulin-like growth factor I–signaling events through estro-
gen receptor-
"
binding (42). Klotz and coworkers (42)
observed variability in the lowest dose of genistein that
increased insulin-like growth factor I receptor tyrosine
phosphorylation. This effect may be due to tyrosine kinase
inhibitory activity of genistein (43) or, alternatively, to a
greater affinity for estrogen receptor-
!
(14).
Overall, genistein has multiple effects on bone turn-
over and produces a significant increase in lumbar spine
and femoral neck BMD compared with placebo after 24
months of treatment. This effect was time-dependent, sug-
gesting that long-term intake produces ongoing effects on
bone metabolism.
Table 3. Uterine Safety and Main Adverse Effects*
Variable Placebo Group
(
n
! 191)
Genistein Group
(
n
! 198)
Mean endometrial thickness (SD),
mm
Baseline 3.2 (1.8)† 3.1 (1.5)‡
Year 1 3.0 (1.5)§ 3.0 (1.4)"
Year 2 3.0 (1.1)¶ 3.2 (1.4)**
Adverse events,
n (%)
Abdominal pain 3 (2) 6 (3)
Epigastric pain 0 (0) 5 (3)
Dyspepsia 3 (2) 9 (5)
Vomiting 3 (2) 4 (2)
Constipation 6 (3) 13 (7)
Participants who discontinued treatment
because of adverse events,
n (%)
15 (8) 37 (19)
* Values are based on participants for whom no data were missing. Women with
endometrial thickness greater than 8 mm at baseline, polyps, or hyperplasia and
those who had had a hysterectomy were excluded from this analysis.
Based on 183 measurements.
Based on 186 measurements.
§ Based on 164 measurements.
" Based on 166 measurements.
Based on 154 measurements.
** Based on 150 measurements.
Table 2—Continued
Placebo Group Genistein Group vs. Placebo Group
Change in Value
at 1 Year (95% CI)
Change in Value
at 2 Years (95% CI)
Adjusted Mean
Difference in Change
at 1 Year (95% CI)
P
Value Adjusted Mean
Difference in Change
at 2 Years (95% CI)
P
Value
"0.016 ("0.023 to 0.007) "0.037 ("0.044 to "0.027) 0.023 (0.011 to 0.034) #0.001 0.062 (0.049 to 0.073) #0.001
"0.027 ("0.038 to 0.015) "0.053 ("0.058 to "0.035) 0.055 (0.036 to 0.073) #0.001 0.10 (0.08 to 0.12) #0.001
"0.003 ("0.011 to 0.004) "0.004 ("0.011 to 0.004) 0.586 (0.576 to 0.596) #0.001 0.61 (0.60 to 0.62) #0.001
0.04 (0.023 to 0.058) 0.043 (0.023 to 0.06) 0.013 ("0.01 to 0.033) 0.016 (0.015 to 0.046)
0.16 (0.09 to 0.23) 0.17 (0.09 to 0.24) 0.05 ("0.04 to 0.13) 0.01 0.062 ("0.059 to 0.183) 0.31
0.003 (–0.023 to 0.029) 0.04 (0.01 to 0.064) 0.039 (0.01 to 0.068) 0.013 (–0.019 to 0.042)
0.01 ("0.07 to 0.09) 0.12 (0.03 to 0.20) 0.12 (0.03 to 0.21) 0.01 0.04 ("0.06 to 0.13) 0.45
0.05 ("3.8 to 3.9) "3.5 ("7.5 to 0.4) 2.7 ("1.4 to 6.8) 0.20 4.9 (0.5 to 9.3) 0.03
12.1 (10.0 to 14.3) 12.7 (10.5 to 14.9) 0.33 ("1.95 to 2.61) 0.77 0.14 ("2.31 to 2.59) 0.91
"0.10 ("0.3 to 0.1) "0.11 ("0.3 to 0.1) 3.7 (3.2 to 4.0) #0.001 4.2 (3.8 to 4.6) #0.001
"2.8 ("8.9 to 3.2) 1.8 ("4.5 to 8.0) 20.7 (13.8 to 27.6) #0.001 13.1 (5.7 to 20.5) #0.001
"2.8 ("6.8 to 1.2) "0.4 ("4.5 to 3.8) "10.0 ("14.5 to 5.6) #0.001 "12.2 ("17.0 to 7.5) #0.001
"0.28 ("1.2 to 0.67) "0.17 ("1.1 to 0.81) "1.8 ("2.9 to 0.8) 0.001 "1.7 ("2.8 to "0.6) 0.002
ArticleGenistein and Bone Metabolism in Osteopenic Postmenopausal Women
www.annals.org 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 845
Page 7
Finally, 2 years of genistein therapy at 54 mg/d was
not associated with clinically significant adverse effects on
the uterus. This isoflavone dosage is similar to that in veg-
etarian Asian diets. However, caution is needed when ad-
ministering genistein, especially in patients at high risk for
endometrial or breast cancer. Moreover, whether the safety
profile demonstrated in our study extends to isoflavones as
a class remains unclear and warrants further study.
We observed a moderate number of gastrointestinal
side effects in women who received genistein. Some pla-
cebo recipients also experienced gastrointestinal adverse ef-
fects, which may have been related to calcium carbonate.
However, significantly more gastrointestinal adverse effects
occurred in the genistein group than in the placebo group;
these effects were most likely attributable to the isoflavone
(44, 45).
In conclusion, we found that 2 years of treatment with
genistein improved BMD and markers of bone turnover in a
cohort of osteopenic postmenopausal women. On the basis of
these data, future studies in osteoporotic women are war-
ranted to determine whether genistein also significantly de-
creases fracture risk in this group. In addition, studies are
needed to determine whether genistein positively affects bone
loss not related to postmenopausal ovarian hormone loss, such
as glucocorticoid-induced osteoporosis.
From Azienda Ospedaliera Universitaria Policlinico “G. Martino,” Uni-
versity of Messina, Messina, Italy.
Grant Support: By the Italian Ministry of Education, University, and
Research and the University of Messina.
Potential Financial Conflicts of Interest: None disclosed.
Requests for Single Reprints: Francesco Squadrito, MD, Department
of Experimental and Clinical Medicine and Pharmacology, Section of
Pharmacology, Torre Biologica, 5th Floor, Azienda Ospedaliera Univer-
sitaria Policlinico “G. Martino,” Via C. Valeria, 98125 Messina, Italy;
e-mail, Francesco.Squadrito@unime.it.
Current author addresses and author contributions are available at www
.annals.org.
Figure 3. Changes in biochemical variables over time.
Estimates are expected means from mixed-effects models. ALP ! alkaline phosphatase. *P ! 0.001 vs. placebo. P # 0.001 vs. placebo. P ! 0.002 vs. placebo.
Article Genistein and Bone Metabolism in Osteopenic Postmenopausal Women
846 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 www.annals.org
Page 8
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ArticleGenistein and Bone Metabolism in Osteopenic Postmenopausal Women
www.annals.org 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 847
Page 9
Current Author Addresses: Drs. Marini and Adamo: Department of
Biochemical, Physiological and Nutritional Sciences, Section of Physiol-
ogy and Human Nutrition, Azienda Ospedaliera Universitaria Poli-
clinico “G. Martino,” Via C. Valeria, 98125 Messina, Italy.
Drs. Minutoli, Polito, Bitto, Altavilla, and Squadrito: Department of
Clinical and Experimental Medicine and Pharmacology, Section of Phar-
macology, Azienda Ospedaliera Universitaria Policlinico “G. Martino,”
Via C. Valeria, 98125 Messina, Italy.
Drs. Atteritano, Gaudio, Mazzaferro, A. Frisina, N. Frisina, and Bon-
aiuto: Department of Internal Medicine, Azienda Ospedaliera Universi-
taria Policlinico “G. Martino,” Via C. Valeria, 98125 Messina, Italy.
Dr. Lubrano: Department of Medical Physiopathology, “La Sapienza”
University, Rome, Italy.
Drs. D’Anna, Cannata, and Corrado: Department of Obstetrical and
Gynecological Sciences, Azienda Ospedaliera Universitaria Policlinico
“G. Martino,” Via C. Valeria, 98125 Messina, Italy.
Dr. Wilson: Department of Health Initiatives, National Jewish Medical
and Research Center, Denver, CO 80206.
Author Contributions: Conception and design: H. Marini, R. D’Anna,
F. Squadrito.
Analysis and interpretation of the data: H. Marini, L. Minutoli, F.
Polito, R. D’Anna, S. Wilson, F. Squadrito.
Drafting of the article: H. Marini, A. Bitto, D. Altavilla, R. D’Anna, F.
Squadrito.
Critical revision of the article for important intellectual content: H.
Marini, R. D’Anna, F. Squadrito.
Provision of study materials or patients: M. Atteritano, A. Gaudio, S.
Mazzaferro, A. Frisina, N. Frisina, C. Lubrano, M. Bonaiuto, R.
D’Anna, M.L. Cannata, F. Corrado, E.B. Adamo.
Obtaining of funding: N. Frisina, C. Lubrano, R. D’Anna.
Administrative, technical or logistic support: N. Frisina, R. D’Anna, F.
Squadrito.
Collection and assembly of data: H. Marini, L. Minutoli, F. Polito.
Annals of Internal Medicine
W-192 19 June 2007 Annals of Internal Medicine Volume 146 Number 12 www.annals.org
Page 10
Page 11
  • Source
    • "Moreover, some studies showed that the aglycone forms of isoflavones had a faster and more efficient absorption than the glycoside and mixed forms [20] [21]. Weaver et al. reported that genistein and daidzein (the two main components of soy isoflavones) may be resistant to each other [22], and previous intervention studies suggested that isolated genistein or daidzein was more effective in the improvement of bone health than mixed isoflavones [22] [23] [24]. Therefore, the effect of soy foods or isoflavones on diabetes remains inconclusive, and further studies are needed to determine the effects of isolated genistein and daidzein on the risk of diabetes or glycemic control in humans. "
    [Show abstract] [Hide abstract] ABSTRACT: ScopeThis randomized, double-blind, and placebo-controlled trial evaluated the effect of isolated daidzein and genistein on glycemic control and insulin sensitivity in 165 Chinese women aged 30-70 with impaired glucose regulation (IGR).Methods and resultsParticipants were randomly assigned to one of three groups with a daily dose of 10 g of soy protein plus (i) no addition, (ii) 50 mg of daidzein, or (iii) 50 mg of genistein for 24 wk. Fasting glucose (FG), insulin, and glycosylated hemoglobin (HbA1c), and glucose concentrations at 30, 60, 120, and 180 min and insulin concentrations at 30, 60, and 120 min after an oral 75-g glucose tolerance test were assessed at baseline and at 12 and 24 wk postintervention. a total of 158 and 151 subjects completed the measures at wk 12 and 24, respectively. There were no significant differences in the changes (%) of FG and the 2-h glucose, HbA1c, fasting, and 2-h insulin or the area under the curve of glucose and insulin between the three treatment groups at wk 12 or 24 (all p > 0.05).Conclusion Neither isolated daidzein nor genistein has a significant effect on glycemic control and insulin sensitivity in Chinese women with IGR over a 6-month supplementation period.
    Full-text · Article · Feb 2015 · Molecular Nutrition & Food Research
  • Source
    • "Inositol is a polyol which may be considered a second messenger of insulin [12], and myo-inositol is one of its nine isomers, capable of reducing insulin resistance, blood pressure, and improving lipid profile in a small cohort of postmenopausal women affected by metabolic syndrome [10] [11]. The soy-derived isoflavone genistein acting as a natural selective estrogen receptor modulator (SERM) has a proven efficacy on markers of CVD risk [9] and in reducing bone loss in postmenopausal women [13]. Very recently, genistein has shown to reduce insulin resistance, blood pressure, and homocysteine and it improved lipid profile in a cohort of women with metabolic syndrome [14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background and Aim. Cardiovascular risk is increased in women with menopause and metabolic syndrome. Aim of this study was to test the effect of a new supplement formula, combining cocoa polyphenols, myo-inositol, and soy isoflavones, on some biomarkers of cardiovascular risk in postmenopausal women with metabolic syndrome. Methods and Results. A total of 60 women were enrolled and randomly assigned (n = 30 per group) to receive the supplement (NRT: 30 mg of cocoa polyphenols, 80 mg of soy isoflavones, and 2 gr of myo-inositol), or placebo for 6 months. The study protocol included three visits (baseline, 6, and 12 months) for the evaluation of glucose, triglycerides, and HDL-cholesterol (HDL-C), adiponectin, visfatin, resistin, and bone-specific alkaline phosphatase (bone-ALP). At 6 months, a significant difference between NRT and placebo was found for glucose (96 ± 7 versus 108 ± 10 mg/dL), triglycerides (145 ± 14 versus 165 ± 18 mg/dL), visfatin (2.8 ± 0.8 versus 3.7 ± 1.1 ng/mL), resistin (27 ± 7 versus 32 ± 8 µg/L), and b-ALP (19 ± 7 versus 15 ± 5 µg/mL). No difference in HDL-C concentrations nor in adiponectin levels between groups was reported at 6 months. Conclusions. The supplement used in this study improves most of the biomarkers linked to metabolic syndrome. This Trial is registered with NCT01400724.
    Full-text · Article · Aug 2014 · International Journal of Endocrinology
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    • "Furthermore, because genistein has been demonstrated to act as a bone-sparing and antiresorptive agent, it has been considered to be a phytoestrogen [15]. This preventive action on bone loss has been reported in various animal and epidemiological studies [16,17]. According to previous in vitro studies, genistein stimulates osteoblastic differentiation and mineralization and inhibits osteoclast formation from pre-osteoclast cells and the bone resorption activity of osteoclasts [18,19]. "
    [Show abstract] [Hide abstract] ABSTRACT: Genistein, a phytoestrogen, has been demonstrated to have a bone-sparing and antiresorptive effect. Genistein can inhibit the osteoclast formation of receptor activator of nuclear factor-κB ligand (RANKL)-induced RAW 264.7 cells by preventing the translocation of nuclear factor-κB (NF-κB), a redox-sensitive factor, to the nucleus. Therefore, the suppressive effect of genistein on the reactive oxygen species (ROS) level during osteoclast differentiation and the mechanism associated with the control of ROS levels by genistein were investigated. The cellular antioxidant capacity and inhibitory effect of genistein were confirmed. The translation and activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1), as well as the disruption of the mitochondrial electron transport chain system were obviously suppressed by genistein in a dose-dependent manner. The induction of phase II antioxidant enzymes, such as superoxide dismutase 1 (SOD1) and heme oxygenase-1 (HO-1), was enhanced by genistein. In addition, the translational induction of nuclear factor erythroid 2-related factor 2 (Nrf2) was notably increased by genistein. These results provide that the inhibitory effects of genistein on RANKL-stimulated osteoclast differentiation is likely to be attributed to the control of ROS generation through suppressing the translation and activation of Nox1 and the disruption of the mitochondrial electron transport chain system, as well as ROS scavenging through the Nrf2-mediated induction of phase II antioxidant enzymes, such as SOD1 and HO-1.
    Full-text · Article · Jun 2014 · International Journal of Molecular Sciences
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