Clinical Endocrinology (2002) 56, 693–701
Isoflavone supplementation and endothelial function
Blackwell Science, Ltd
in menopausal women
Georgina Hale*, Maura Paul-Labrador*,
James H. Dwyer† and C. Noel Bairey Merz*
*Division of Cardiology, Department of Medicine,
Cedars-Sinai Research Institute, Cedars-Sinai Medical
Center, UCLA School of Medicine, Los Angeles and
†Department of Preventive Medicine, Keck School of
Medicine, University of Southern California,
Los Angeles, CA, USA
(Received 22 June 2001; returned for revision 7 August 2001;
finally revised 13 November 2001; accepted 7 February 2002)
Summary
OBJECTIVE Despite strong observational evidence for
a beneficial role of oestrogen in cardiovascular disease,
recent trial results suggest that hormone replacement
therapy (HRT) may have adverse effects in menopausal
women with established coronary heart disease. Iso-
flavones are oestrogen analogues found in plants with
oestrogen-like properties and, because of a favourable
side-effect profile, may be ideal alternatives to HRT
with respect to cardiovascular benefits. Endothelial
function is a marker of cardiovascular health. We aimed
to determine the effect of isoflavones on endothelial
function using the brachial artery reactivity test.
DESIGN AND MEASUREMENTS Twenty-nine healthy
menopausal women underwent entry and exit brachial
artery reactivity testing following randomization to
2 weeks of an oral soy isoflavone concentrate contain-
ing 80 mg of soy isoflavones (Archer Daniel Midland
Inc., IL, USA) or placebo.
RESULTS At study exit, there was no difference between
placebo and isoflavone groups with respect to flow-
mediated dilation (%FMDmax), change (entry to exit) in
%FMDmax or response to nitroglycerine (%TNG). Sub-
group analyses assessing lipid and oestrogen effects
did not produce any significant results.
CONCLUSIONS These results suggest that short-term
oral isoflavone supplements do not improve endothe-
lial function in healthy menopausal women.
Correspondence: Dr G.E. Hale, Department of Obstetrics and
Gynaecology, University of Sydney, Building DO2, Camperdown,
NSW 2006, Australia. E-mail: ghale@med.usyd.edu.au
© 2002 Blackwell Science Ltd
Large observational studies consistently demonstrate a beneficial
association between hormone replacement therapy (HRT) and
lower rates of coronary heart disease in menopausal women (Hu
et al., 2000). Two randomized trials, however, suggest that HRT
may not be useful in the secondary prevention of coronary deaths
(Hulley et al., 1998) or the progression of previously established
coronary plaques (Herrington et al., 2000). The Heart and Estrogen/
progestin Replacement Study (HERS) trial, conducted in women
with established coronary heart disease, found more coronary events
in the women randomized to HRT within the first year of use
relative to women taking placebo (Hulley et al., 1998). At trial end,
there was no difference in rates of coronary events in women with
established coronary disease taking HRT compared to placebo
(Hulley et al., 1998). These uncertainties regarding the safety and
efficacy of HRT in menopausal women, and the possible asso-
ciation with an increased risk of breast cancer (Schairer et al.,
2000) largely underlie the ongoing search for alternatives to HRT.
Isoflavones are naturally occurring plant compounds found in
high amounts in plants such as soy and red clover. The phenolic
ring within the structure of isoflavones resembles steroidal oes-
trogen and confers the ability to bind to the oestrogen receptor
(ER), although with less affinity than oestrogen (Kuiper et al.,
1997). Being naturally occurring ER-ligands, they have been
studied enthusiastically with respect to their oestrogen-like prop-
erties and oestrogen-like effects in menopausal women. This
enthusiasm was sparked by findings from a number of clinical
studies showing positive effects of high soy protein diets on
serum cholesterol. In a meta-analysis of 38 studies, an average
intake of 47 g soy protein in place of animal protein, significantly
decreased serum concentrations of total cholesterol, low density
lipoprotein-cholesterol (LDL-C) and triglycerides in individuals
with moderate to severe hypercholesterolemia (Anderson et al.,
1995). These findings were consistent with the observation of a
lower incidence of cardiovascular disease in populations ingest-
ing isoflavone-rich diets (Colditz et al., 1987). Following these
findings, a few studies have investigated the cholesterol-lowering
effects of soy protein specifically in healthy menopausal women.
In one study of 42 menopausal women, subjects consumed three
daily servings of whole soy foods containing approximately
60 mg/day of isoflavones for 12 consecutive weeks. There was a
significant increase in mean levels of high density lipoprotein-
cholesterol (HDL-C) (3·7%) and a decrease in the total cholesterol :
HDL-C ratio (5·5%) (Scheiber et al., 2001). Similar effects were
also seen in another study of 50 symptomatic perimenopausal
women given 20 g of soy protein daily (34 mg isoflavones) over
a total of 12 weeks. There was a significant decline in mean total
693
694 G. Hale et al.
cholesterol (6%) and mean LDL-C (7%) but no increase in mean
HDL-C or triglycerides (Washburn et al., 1999).
While high soy-protein diets seem to have positive effects on
the serum lipids in menopausal women, isoflavones extracted
from soy or red clover administered in tablet form do not appear
to have the same affect. In five short duration studies using soy
(three studies) or red clover (two studies) extracts in menopausal
and perimenopausal women, there was no significant change in
any of the serum lipid parameters (Nestel et al., 1997, 1999; Simons
et al., 2000; Upmalis et al., 2000). Daily isoflavone doses in these
five studies were in the range 40 – 80 mg and treatment duration
was 8 –12 weeks. In a longer study of 46 menopausal women over
6 months, however, administration of red clover isoflavones did
result in a significant rise in HDL-C levels and a significant
decrease in lipoprotein B levels (Clifton-Bligh et al., 2001). At
6 months, the serum HDL-C increased by 15·7– 28·6% and serum
apolipoprotein B levels fell by 11·5 –17%. These changes were not
evident at 3 months. The magnitude of the response at 6 months,
however, was independent of the three doses used (28·5 mg,
57 mg or 85·5 mg) (Clifton-Bligh et al., 2001). These results
suggest that longer treatment courses are necessary to obtain
measurable changes in lipoproteins in the case of isoflavone
supplements compared to soy-protein. It is possible that lipid
changes are slower in these studies than in studies using soy pro-
tein because of the lack of other possible cholesterol-lowering
compounds, such as saponins, amino acids and phytic acid, found
in soy protein (Greaves et al., 2000) and the lack of dietary modi-
fications, such as a reduction in animal fat and protein.
Although treatment with isoflavone extracts alone appears
to have a weak effect on blood lipids, they may have beneficial
effects on other cardiovascular parameters such as endothelial
function. In menopausal women, both soy and red clover isolates
have been shown to have a positive effect on arterial compliance
(Nestel et al., 1997, 1999). In animal models, they have been
found to have a beneficial effect on vascular reactivity and tone
(Honore et al., 1995, 1997; Figtree et al., 2000; Mishra et al.,
2000; Karamsetty et al., 2001). Recently, endothelial function, as
assessed by noninvasive brachial artery reactivity testing (BART),
has been used by numerous investigators to measure the cardio-
vascular benefits of interventions. This measure is believed to
be a good indicator of cardiovascular health (Celermajer et al.,
1992; Sorensen et al., 1995) and has been shown to be responsive
to interventions such cholesterol-lowering (Schechter et al., 2000)
magnesium supplementation (Schechter et al., 2000), folic acid
supplementation in hyperhomocysteinuria (Woo et al., 1999) and
vitamin C therapy in smokers. Endothelial function using BART
has also been shown to be impaired in states associated with high
cardiovascular risk (Clarkson et al., 1996; de Roos et al., 2001).
By measuring the change in ultrasound-determined brachial
artery diameter in response to blood pressure cuff-induced ischaemia,
interventions designed to improve cardiovascular health can
be tested expeditiously and economically. Oestrogen therapy in
menopausal women has been shown to increase flow-mediated
diameter (FMD) measured by BART (Lieberman et al., 1994;
Bush et al., 1998; Gerhard et al., 1998; Higashi et al., 2001). This
effect is thought to be mediated primarily via the ER and its influ-
ence on nitric oxide release by endothelial cells (Haynes et al.,
2000; Hisamoto et al., 2001). It is possible therefore that, being
ER-ligands, isoflavones can also directly affect vascular function
and positively influence FMD in menopausal women. We studied
the effect of a high dose soy isoflavone concentrate (ADM) on
endothelial function in healthy postmenopausal women. In order
to exclude any effects on the blood lipids, we used a short treatment
course of 2 weeks. In addition, to maximize the chance of detecting
an effect on FMD, we used upper arm occlusion as the stimulus
for hyperaemia instead of the frequently used forearm occlusion.
Methods
Study design and population
Women between the ages of 45 and 70 years were recruited from
newspaper advertisements at the Cedars-Sinai Medical Center
to participate in a randomized, double-blind, placebo-controlled
study. Inclusion criteria included: last menstrual period at least
12 months previously; no hormone replacement therapy for at
least 6 months; no regular isoflavone supplementation for at
least 6 weeks; no antibiotic therapy within the last 6 months; no
cigarette smoking within the last 12 months; a body mass index
of 35 kg/m2 or less; consumption of no more than 14 standard
alcoholic drinks per week; a recent fasting total cholesterol of
< 7.758 mmol/ l and no history of cardiovascular disease. All
participants agreed to consume an isoflavone-free diet at least
4 weeks before and during the study. In addition, all vitamin,
mineral and herbal supplements were discontinued at least 2 days
before each test visit. Participants could not be taking any of
selective oestrogen receptor modulators, β-blockers, α-blockers
or calcium channel blockers. The study was approved by the
Cedars-Sinai Institutional Review Board and all participants gave
their written informed consent.
Study protocol
Eligible study respondents were screened with a short clinical
history and demographic measurements and subjects who quali-
fied and signed an informed consent, arranged two appointments,
2 weeks apart, for each of the brachial reactivity tests (BART).
Subjects fasted for at least 8 h before each visit. If the blood test
(lipid profile, FSH and oestradiol levels) and BART were suc-
cessfully completed at the first visit, the subject was randomized
to either a placebo or active soy isoflavone concentrate (Archer
Daniel Midland Inc., IL, USA) containing 80 mg isoflavones with
© 2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 693 –701

approximately equal amounts of aglycone genistein and daidzein.
All investigators and participants were blinded to the study code.
Clinical and laboratory measurements
Standard anthropometric measurements and a medical history
were performed at the screening visit and updated on the day of
the BART procedure. Once randomized, all participants were
dispensed exactly 2 weeks of tablets to take and any remaining
tablets were returned for a tablet-count. Adverse effects and
changes in health status or medications were checked at the end
of the 2-week course. Blood samples were immediately centri-
fuged and stored at −70 °C until the completion of the study.
Reproductive hormone assays (oestradiol and follicle stimulat-
ing hormone) were performed at a reproductive hormone core
laboratory by an experienced technician in a single batch using
validated steroid and protein assay methods (Anderson et al.,
1976). Previous work from this laboratory has demonstrated
between-assay coefficients of variation for oestradiol and FSH
of 8% and 8·1%, respectively (Barrett-Connor & Goodman-Gruen,
1995). Lipoprotein determinations were performed in a single
batch at a lipid core laboratory enrolled in the Centers for Disease
Control and Prevention lipid standardization program, previously
used in multiple NHLBI-sponsored lipid-lowering interven-
tion trials. Total plasma cholesterol (TC), triglyceride (TG) and
HDL-C were determined by enzymatic assay and LDL-C was
calculated using the Friedewald formula, as previously described
(Program, 1974). The coefficients of variation for TC, HDL-C
and TG were 1·80%, 1·23% and 3·93%, respectively.
BART protocol
Endothelial function testing by BART has been demonstrated
in our laboratory to have good reproducibility (coefficient of
variation, CV, for repeated flow mediated diameter measures of
1·4%) and to be responsive to lipid lowering (Schechter et al.,
2001) and magnesium interventions (Schechter et al., 2000). Before
testing, the subject is rested in the supine position with her right
upper arm cleared for the pneumatic tourniquet and ultrasound
procedure. Using a 10-MHz linear array (CL 10-5 ATL; Advanced
Technology Laboratories, Bothell, WA, USA) ultrasound apparatus
(ATL HDI 3000cv system), the brachial artery is longitudinally
imaged approximately 5 cm proximal to the antecubital crease,
where the clearest image can be obtained. Baseline imaging is
performed for 3 minutes before the cuff is inflated to at least
50 mmHg above systolic blood pressure (Rosenthal & Chin, 1999).
Successful occlusion is monitored and maintained using the
colour flow Doppler function. Care is taken to keep the trans-
ducer in the same position throughout the test. After the post cuff-
readings are completed, the subject rests for 10 minutes. With the
assistance of a photograph of the baseline reading, the technician
© 2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 693–701
Isoflavones and endothelial function 695
re-establishes as closely as possible, the same position along
the brachial artery. Baseline images are again performed for
3 minutes, followed by administration of 0·4 mg sublingual nitro-
glycerine (NTG) tablet (Nitrostat, 0·4 mg, Park-Davis, NY, USA).
The readings are continued for an additional 4 minutes. Electrocar-
diogram (ECG) monitoring is performed continuously throughout
the study and blood pressure measurements are taken before base-
line, at completion of the cuff and at completion of the NTG phases.
BART data analysis
All the ultrasound images are recorded on S-VHS videotape with
a SLV-RS7 videocassette recorder (Sony, CA, USA). The diameter
of the brachial artery is measured from the anterior to the
posterior interface between the media and adventitia (‘m line’). The
mean diameter of the artery is then calculated from four frames
(cardiac cycles) synchronized with the R-wave peaks on the ECG.
Consecutive frames are read at 1 minute and 11/2 minutes after the
cuff release and then at 1, 2, 3 and 4 minutes after the NTG phase.
All measurements are made at end-diastole to avoid possible errors
resulting from variable arterial compliance. The external diameter
is calculated with PC Prosound software (USC, Los Angeles, CA,
USA) using a Horita Data Translation Image Processing board
(DT2862–60 Hz) (CA, USA). The diameter change caused by
endothelium-dependent flow-mediated vasodilatation (%FMD) and
endothelium-independent (%NTG) vasodilation is expressed as the
percent change relative to that at the initial resting scan (baseline).
Statistical analysis
Group data are expressed as mean ± SD. FMD is calculated by
summing the average baseline artery diameter subtracted from
the maximum artery diameter (post-cuff deflation), then divided
by the average baseline diameter. This is subsequently multiplied
by 100% and denoted as %FMD. Because the FMD is calculated
at both 1 and 11/2 minutes post-cuff release, the higher of these
two (%FMDmax) was used in all statistical analyses. Comparison
of treatment groups before and after treatment was performed
using paired and unpaired Student’s t-test and chi-squared. Asso-
ciations between variables were determined using Pearson’s
correlation coefficient analysis. The Wilcoxon signed-rank test
and Spearman’s correlation coefficients were used to analyse data
that were not normally distributed. To control for the effect that
baseline diameter may exert on FMD, covariate analysis was per-
formed using baseline diameter as the covariant on comparing
FMD differences among the two treatment groups. P < 0·05 was
considered statististically significant.
Reproducibility of %FMDmax in the current study was cal-
culated using baseline and follow-up measurements from the
placebo group (n = 13). The CV was calculated as the SD of the
change in scores (follow-up − baseline) divided by the square root
696 G. Hale et al.

of 2 [CV = SD(∆)/√2]. This estimate of CV is equal to the square

root of the residual mean square from a repeated measures analysis
of variance. The CV for %FMDmax in the control group was 3·7%
at 1 minute, 5·3% at 1·5 minutes and 5·0% at maximum. The CV
for brachial artery baseline diameter was 1·2% and for response
to nitroglycerine, 2·5% at 1 minute and 2·3% at 11/2 minutes.

Results

Among the 32 women randomized, 29 successfully completed both

baseline and post-treatment tests (one subject became ill between
visit 1 and 2, and two subjects had unsuccessful second visit BART
studies). All subjects took the prescribed number of doses of either
the soy tablet or the placebo as assessed by a tablet count at visit 2.
There were no complaints of adverse effects. The baseline charac-
teristics of the placebo and treatment groups were similar (Table 1).

Baseline diameter and response to nitroglycerine

There were no group differences in brachial artery diameter,

%FMDmax or NTG response (Table 1). All but two subjects had
at least a 10% response to sublingual NTG. These two subjects
had an average response of 8·7% and 7·5% and were included
in the analyses because they responded consistently at both visits. Fig. 1 (a) Individual %FMDmax measurements before and after
treatment in the placebo group. (b) Individual %FMDmax measurements
before and after treatment in the soy tablet group.
Change in %FMD according to intervention
Changes in blood pressure and lipids with treatment
There was no difference between the placebo or soy tablet groups
with respect to the change in %FMDmax (Fig. 1a,b), absolute There were no changes in either systolic or diastolic blood
%FMDmax at study exit (Fig. 2) or response to the nitroglycerine pressure (data not shown) or blood lipid profile with treatment
(data not shown). (Table 2).

Table 1 Baseline characteristics in placebo and

Placebo (mean ± SD) Soy tablet (mean ± SD) soy groups
(n = 13) (n = 16) P

Age (years) 58 ± 7·2 56·5 ± 4·5 0·47

FMP (years) 8·3 ± 9·5 5·9 ± 4·9 0·42
BMI (kg/m2) 24·5 ± 2·8 25·3 ± 4·9 0·62
Systolic blood pressure (mmHg) 117·8 ± 25·1 127·5 ± 18·3 0·24
Diastolic blood pressure (mmHg) 72·7 ± 11·7 78·1 ± 10·3 0·19
Oestradiol (pg/ml) 18 ± 14·14 13·8 ± 5·2 0·33
FSH (IU/ml) 64·1 ± 18·1 63·4 ± 25·1 0·93
Lipids (mg/dl)
Total cholesterol 198·3 ± 28 193·6 ± 38 0·71
LDL-C 156·7 ± 39 151·75 ± 48·6 0·77
Triglycerides 109 ± 64 108 ± 76 0·98
HDL-C 63·2 ± 10·3 63·5 ± 17·5 0·94
Baseline diameter (mm) 3·56 ± 0·72 3·83 ± 0·44 0·23
%FMDmax 9·5 ± 6·1 8·4 ± 5·4 0·6
% NTG response 17·7 ± 6 17·4 ± 5·9 0·89

FMP, Final menstrual period; BMI, body mass index; LDL-C, low density lipoprotein-
cholesterol; HDL-C, high density lipoprotein-cholesterol; NTG, nitroglycerine.

© 2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 693 –701


Fig. 2 Average %FMDmax at study exit according to treatment group.
Bars indicate SD.
Fig. 3 Relationship between %FMDmax and diastolic blood pressure at
baseline in all subjects.
%FMD correlations
Baseline brachial artery diameter was inversely correlated with
%FMDmax (r = −0·516, P = 0·0042) and, when analysed as a
group at baseline, was also inversely correlated with diastolic
blood pressure (r = −0·48, P = 0·008) (Fig. 3). The negative
correlation between systolic blood pressure and %FMDmax
did not reach significance (r = −0·33, P = 0·08). There was no
relationship between oestradiol level and %FMDmax at baseline
(Spearman rank, r = −0·12, P = 0·54) or oestradiol level and
baseline brachial artery diameter. There was also no correlation
between %FMDmax and any of the blood lipids, meditation, exer-
cise or reported prior vitamin intake (multivitamins, vitamin E,
fish oil).
Table 2 Change in blood lipids with treatment
according to group
Total cholesterol (mg/dl)
LDL-C (mg/dl)
HDL-C (mg/dl)
Triglycerides (mg/dl)
LDL-C, Low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol.
© 2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 693–701
Isoflavones and endothelial function 697
Because of the observed relationship between baseline
brachial artery diameter and %FMDmax, we performed covari-
ate analyses by intervention group. When using baseline dia-
meter as a covariate, %FMDmax was not significantly different
in the placebo and soy tablet groups (F = 1·92; P = 0·166,
trend: the larger the baseline diameter, the smaller the
%FMDmax).
Discussion
In this randomized, blinded, placebo-controlled study of 29
menopausal women, we were unable to detect an improvement
in endothelial function after a 2-week course of an 80-mg soy
isoflavone concentrate given daily. Similar negative findings were
seen in a previous study of 20 menopausal women (using forearm
cuff occlusion in the BART) using an 8-week course of an 80-
mg/day soy isoflavone supplement (Simons et al., 2000). Because
intravenous oestrogen administration has been shown to have an
immediate positive effect on endothelial function in menopausal
women (Al-Khalili et al., 1998), we were interested in investig-
ating any similar immediate effects by isoflavones. Thus, we chose
a short 2-week course of oral isoflavone administration. Such a
short course would also have excluded any effects on the blood lipids.
The immediate endothelial effects of oestrogen are largely mediated
via ER interaction (Hodges et al., 2000) and the resultant increase
in endothelial cell production of nitric oxide (NO) (Weiner et al.,
1994). Other less important ER-related mechanisms may include
arterial smooth muscle cell production of relaxing factors, such
as prostacyclin (Gisclard et al., 1988; Clarkson, 1998), inhibition
of superoxide formation (Arnal et al., 1996), inhibition of angio-
tensin II production (Kuroski de Bold, 1999), calcium channel
antagonism (Jiang et al., 1992) and endothelin receptor blockade
(Vogel & Corretti, 1998). Although the intravenous administra-
tion of genistein and daidzein has been shown to cause vascular
relaxation in animal models (Mishra, Abbot et al., 2000; Figtree
et al., 2000), these effects have not been studied in humans.
Our negative results are perhaps not unexpected given that
isoflavones have a low affinity for the ER compared to oestrogen
and therefore may not influence nitric oxide release to the same
degree. Isoflavones have an affinity for the ER-α subtype that is
at least 100-fold less than oestradiol (Hopert et al., 1998; Hunter
Placebo (mean ± SD) Soy tablet (mean ± SD)
(n = 13) (n = 16) P
8·4 ± 23·9 14·9 ± 27·4 0·51
4·6 ± 25·5 −1·2 ± 38·2 0·35
0·54 ± 7·95 0·38 ± 8·28 0·96
−17·0 ± 39·4 −1·2 ± 38·2 0·28
698 G. Hale et al.
et al., 1999). The affinity for the ER-β is much higher, and in
the case of genistein, it is almost as high as oestrogen (Kuiper
et al., 1997, 1998; Makela et al., 1999). Although both subtypes
of the ER (ER-α and ER-β) are found in human endothelium and
vascular smooth muscle (Hodges et al., 2000; Critchley et al.,
2001; Lecce et al., 2001), it is not clear what role each of the
subtypes plays. Isoflavones are specific ER-β ligands and how
their interaction with the ER-β influences endothelial function
is unclear. The negative findings from our study and the study by
Simons et al. (2000), however, suggest that the ER-β does not
play a major in NO release and endothelial function. The ER-β
may play a role in longer-term cardiovascular health, for example,
by positively affecting blood lipids and inhibiting smooth muscle
cell migration and proliferation in response to endothelial injury
(Finking et al., 1999; Makela et al., 1999). This is supported by
the fact that ER-β is the more predominant ER subtype in human
vascular smooth muscle (Hodges et al., 2000) and has stronger
transcriptional activity that ER-α (Hodges et al., 2000).
Our results were undoubtedly hampered by an unexpectedly
high CV that decreased our ability to detect an effect. The average
group %FMD in the study by Simons et al. (2000) was 3·3–4·1,
which is consistent with three other BART studies in meno-
pausal women (Al-Khalili et al., 1998; Perregaux et al., 1999;
McCrohon et al., 2000). The average %FMD in our study was
8·4 and 9·4 in the soy tablet and placebo groups, respectively.
This is a reflection of the use of upper arm occlusion, which is a
more potent hyperaemic stimulus (Vogel et al., 2000), and one
that we performed in order to increase the ability to detect a
response. Although it has not been our experience in this labo-
ratory, other investigators have found increased %FMD variabil-
ity with the use of upper arm cuff occlusion compared to forearm
cuff occlusion (Vogel et al., 2000). The CV for %FMD in this
study was between 3·7 and 5·3, which is approximately three- to
four-fold higher than our average of 1·4% from at least seven
previous studies in this same laboratory using upper arm cuff
occlusion. Given this higher than expected observed variability,
we would have needed 25 in each group to detect an average
change in FMD of 5–12% (power 0·8), similar to the effect seen
with steroidal oestrogens. We would have needed at least 130 in
each group, however, to detect a weaker response of between 3%
and 4%.
There are a number of possible factors that could have con-
tributed to our high CV. Although we did not have any reports
of illness throughout the course of the brief study period, inad-
vertent infection may have transiently increased the FMD meas-
urement at either test visit (Hingorani et al., 2000). Second, some
of our subjects were hypertensive. Eight subjects (three in the
placebo group) had a resting systolic blood pressure of greater
than 135 mmHg at both visits and six subjects (two in the placebo
group) had a diastolic blood pressure greater than 85 mmHg at
both visits. Only two of these subjects (both in the soy group) were
receiving treatment for hypertension (chlorothiazide, atenolol,
diovan and lopressor). It has been found that the use of upper
arm occlusion in BART measurements may increase variability
when coronary risk factors are present ( Vogel et al., 2000).
Finally another possible source of variability in this group are
differences in estradiol levels between and within individuals at
each visit. Two women in the placebo group had oestradiol levels
of 180 and 172 pmol/ l at the first visit, despite being more than
16 months from their final menstrual period. One of these women
had a level of 40 pmol/ l at the second visit and the other,
187 pmol/ l. The subject who had a decrease in serum oestradiol
from the first to the second visits had a decrease in %FMD from
24 to 12. The subject in whom the oestradiol level was the same
between visits had a %FMD of approximately 12 at both visits.
Although, in our analysis, there was little relation between oestra-
diol levels and %FMD, numbers were small. It thus remains
possible that even small fluctuations in oestradiol levels influence
%FMD in some individuals. Perimenopausal and recently meno-
pausal women are reported to have highly variable oestradiol
levels (Shideler et al., 1989; Santoro et al., 1996) and are some-
times found to have follicular phase levels of oestradiol up to
24 months after the final menstrual period (Longcope et al., 1986;
Longcope, 2001). Neither the CV for the %FMD measurements
or oestradiol levels were reported in the study by (Simons et al.,
2000). In the studies by Al-Khalili et al. (1998)., Perregaux et al.
(1999) and McCrohon et al. (2000) using forearm cuff occlu-
sion, average untreated %FMD values in Caucasian menopausal
women were 1·8 ± 2% (including six women with coronary heart
disease), 2·82 (confidence interval 1·32–3·28) and 2·7 ± 2·9%,
respectively. There have been no other BART studies using upper
arm cuff occlusion specifically in either perimenopausal or
menopausal women.
An increase in vessel diameter is thought to be a marker of
endothelial dysfunction and a predictor of the development of
lumen encroachment by atheroma (Sorensen et al., 1995). We
found a significant inverse association between %FMD and
diastolic blood pressure (Celermajer et al., 1994) supportive of
this concept. Although we did not measure either blood or urine
isoflavones, given the 100% compliance reported by our subjects
and short study duration, we think it unlikely that our lack of
response was due to poor compliance with tablets or diet. We
cannot exclude variability in absorption; however, prior work in
animals (Honore et al., 1995, 1997) and humans (Xu et al., 1995;
King & Bursill, 1998) has demonstrated that isoflavone supple-
ments are effectively absorbed from the gastrointestinal tract. The
80-mg dose used in our study compares with the doses used in
the two prior isoflavone and BART studies (Nestel et al., 1997;
Simons et al., 2000), and is 11/2 to two-fold higher than the iso-
flavone intake in the typical Japanese diet (Kimira et al., 1998).
In conclusion, oral isoflavone supplementation in the form of
soy tablets does not appear to improve endothelial function in
© 2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 693 –701

the short term. Long-term benefits may be possible, however, if
used in conjunction with other dietary and lifestyle changes, such
as exercise and lowering dietary animal fat and protein intake.
Future BART studies in menopausal women using upper arm cuff
occlusion should exclude women with any known cardiovascular
risk factors, such as high blood pressure and hyperlipidemia and
also those subjects with oestradiol levels greater than approxim-
ately 70 pmol/l.
Acknowledgements
This work was supported by grants from Archer, Daniel, Mid-
land, Inc., Decatur, Il, the Gustavus and Louis Pfeiffer Research
Foundation, Denville, New Jersey, the Women’s Guild of Cedars-
Sinai Medical Center, Los Angeles, California, and a GCRC
grant MO1-RR00425 from the National Center for Research
Resources.
References
Al-Khalili, F., Eriksson, M., Landgren, B.M. & Schenek-Gustafsson, K.
(1998) Effect of conjugated estrogen on peripheral flow-mediated
vasodilation in postmenopausal women. American Journal of Cardio-
logy, 82, 215 – 218.
Anderson, D.C., Hopper, B.R., Lasley, B.L. & Yen, S.S. (1976) A simple
method for the assay of eight steroids in small volumes of plasma.
Steroids, 28, 179 –196.
Anderson, J.W., Johnstone, B.M. & Cook-Newell, M.E. (1995) Meta-
analysis of the effects of soy protein intake on serum lipids. New
England Journal of Medicine, 333, 276–282.
Arnal, J.F., Clamens, S., Pechet, C., Negre-Salvayre, A., Allera, C.,
Girolami, J.P., Salvayre, R. & Bayard, F.L. (1996) Ethinylestradiol
does not enhance the expression of nitric oxide synthase in bovine
endothelial cells but increases the release of bioactive nitric oxide by
inhibiting superoxide anion production. Proceedings of the National
Academy of Sciences USA, 93, 4108 –4113.
Barrott-Connor, E. & Goodman-Gruen, D. (1995) Prospective study of
endogenous sex hormones and fatal cardiovascular disease in post-
menopausal women. BMJ, 311 (7014), 1193–1196.
Bush, D.E., Jones, C.E., Bass, K.M., Walters, G.K., Bruza, J.M. &
Ouyang, P. (1998) Estrogen replacement reverses endothelial dysfunc-
tion in postmenopausal women. American Journal of Medicine, 104,
552 – 558.
Celermajer, D.S., Sorensen, K.E., Gooch, V.M., Spiegelhalter, D.J.,
Miller, O.I., Sullivan, I.D., Lloyd, J.K. & Deanfield, J.E. (1992) Non-
invasive detection of endothelial dysfunction in children and adults at
risk of atherosclerosis. Lancet, 340, 1111–1115.
Celermajer, D.S., Sorensen, K.E., Spiegelhalter, D.J., Georgakopoulos, D.,
Robinson, J. & Deanfield, J.E. (1994) Aging is associated with
endothelial dysfunction in healthy men years before the age-related decline
in women. Journal of the American College of Cardiology, 24, 471– 476.
Clarkson, P., Celermajer, D.S., Donald, A.E., Sampson, M., Sorensen, K.E.,
Adams, M., Yue, D.K., Betteridge, D.J. & Deanfield, J.E. (1996)
Impaired vascular reactivity in insulin-dependent diabetes mellitus is
related to disease duration and low density lipoprotein cholesterol
levels. Journal of the American College of Cardiology, 28, 573–579.
Clarkson, T.B. (1998) Effects of estrogens, progestins, and androgens on
© 2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 693–701
Isoflavones and endothelial function 699
coronary vasomotion and atherosclerosis. Journal of Reproductive
Medicine, 43 (Suppl. 8), 741–745.
Clifton-Bligh, P.B., Baber, R.J., Fulcher, G.R., Nery, M.L. & Moreton, T.
(2001) The effect of isoflavones extracted from red clover (Rimostil)
on lipid and bone metabolism. Menopause, 8, 259 – 265.
Colditz, G.A., Willett, W.C., Stampfer, M.J., Rosner, B., Speizer, F.E.
& Hennekens, C.H. (1987) Menopause and the risk of coronary heart
disease in women. New England Journal of Medicine, 316, 1105 –
1110.
Critchley, H.O., Brenner, R.M., Henderson, T.A., Williams, K., Nayak,
N.R., Slaydon, O.D., Millar, M.R. & Saunders, P.T. (2001) Estrogen
receptor beta, but not estrogen receptor alpha, is present in the vascular
endothelium of the human and nonhuman primate endometrium. Jour-
nal of Clinical Endocrinology and Metabolism, 86, 1370 –1378.
de Roos, N.M., Bots, M.L. & Katan, M.B. (2001) Replacement of dietary
saturated fatty acids by trans fatty acids lowers serum HDL cholesterol
and impairs endothelial function in healthy men and women. Arteri-
osclerosis Thrombosis and Vascular Biology, 21, 1233 –1237.
Figtree, G.A., Griffiths, H., Lu, Y.Q., Webb, C.M., MacLeod, K. &
Collins, P. (2000) Plant-derived estrogens relax coronary arteries in
vitro by a calcium antagonistic mechanism. Journal of the American
College of Cardiology, 35, 1977–1985.
Finking, G., Wohlfrom, M., Lenz, C., Wolkenhauer, M., Eberle, C. &
Hanke, H. (1999) The phytoestrogens Genistein and Daidzein, and 17
beta-estradiol inhibit development of neointima in aortas from male
and female rabbits in vitro after injury. Coronary Artery Disease, 10,
607–615.
Gerhard, M., Walsh, B.W., Tawakol, A., Haley, E.A., Creager, S.J.,
Seely, E.W., Ganz, P. & Creager, M.A. (1998) Estradiol therapy com-
bined with progesterone and endothelium-dependent vasodilation in
postmenopausal women. Circulation, 98, 1158 –1163.
Gisclard, V., Miller, V.M. & Vanhoulle, P.M. (1988) Effect of 17 beta-
estradiol on endothelium-dependent responses in the rabbit. Journal
of Pharmacology and Experimental Therapeutics, 244, 19 – 22.
Greaves, K.A., Wilson, M.D., Rudel, L.L., Williams, J.K. & Wagner, J.D.
(2000) Consumption of soy protein reduces cholesterol absorption
compared to casein protein alone or supplemented with an isoflavone
extract or conjugated equine estrogen in ovariectomized cynomolgus
monkeys. Journal of Nutrition, 130, 820 – 826.
Haynes, M.P., Sinha, D., Russell, K.S., Collinge, M., Fulton, D., Morales-
Ruiz, M., Sessa, W.C. & Bender, J.R. (2000) Membrane estrogen
receptor engagement activates endothelial nitric oxide synthase via the
PI3-kinase-Akt pathway in human endothelial cells. Circulation
Research, 87, 677–682.
Herrington, D.M., Reboussin, D.M., Brosnihan, K.B., Sharp, P.C.,
Shumaker, S.A., Snyder, T.E., Furberg, C.D., Kowalchuk, G.J.,
Stuckey, T.D., Rogers, W.J., Givens, D.H. & Waters, D. (2000) Effects
of estrogen replacement on the progression of coronary-artery athero-
sclerosis. New England Journal of Medicine, 343, 522 – 529.
Higashi, Y., Sanada, M., Sasaki, S., Nakagawa, K., Goto, C., Matsuura, H.,
Ohama, K., Chayarra, K. & Oshima, T. (2001) Effect of estrogen
replacement therapy on endothelial function in peripheral resistance
arteries in normotensive and hypertensive postmenopausal women.
Hypertension, 37, 651–657.
Hingorani, A.D., Cross, J., Kharbanda, R.K., Mullen, M.J., Bhagat, K.,
Taylor, M., Donald, A.E., Palacios, M., Griffin, G.E., Deanfield, J.E.,
MacAllister, R.J. & Vallance, P. (2000) Acute systemic inflammation
impairs endothelium-dependent dilatation in humans. Circulation, 102,
994–999.
Hisamoto, K., Ohmichi, M., Kurachi, H., Hayakawa, J., Kanda, Y., Nishio, Y.,
Adachi, K., Tasaka, K., Miyoshi, E., Fujiwara, N., Taniguchi, N. &
700 G. Hale et al.
Murata, Y. (2001) Estrogen induces the Akt-dependent activation of
endothelial nitric-oxide synthase in vascular endothelial cells. Journal
of Biological Chemistry, 276, 3459 – 3467.
Hodges, Y.K., Tung, L., Yan, X.D., Graham, J.D., Horwitz, K.B. &
Ilorowitz, L.D. (2000) Estrogen receptors alpha and beta: prevalence
of estrogen receptor beta mRNA in human vascular smooth muscle
and transcriptional effects. Circulation, 101, 1792–1798.
Honore, E.K., Williams, J.K. & Anthony, M.S. (1995) Enhancement of
coronary vasodila-tion by soy phytoestrogens and genistein. Circula-
tion, 92, 349 – 354.
Honore, E.K., Williams, J.K., Anthony, M.S. & Clarkson, T.B. (1997)
Soy isoflavones enhance coronary vascular reactivity in atherosclerotic
female macaques. Fertility and Sterility, 67, 148–154.
Hopert, A.C., Beyer, A., Frank, K., Strunck, E., Wunsche, W. & Vollmer, G.
(1998) Characterization of estrogenicity of phytoestrogens in an
endometrial-derived experimental model. Environmental Health
Perspectives, 106, 581– 586.
Hu, F.B., Stampfer, M.J., Manson, J.E., Grodstein, F., Colditz, G.A.,
Speizer, F.E. & Willett, W.C. (2000) Trends in the incidence of coronary
heart disease and changes in diet and lifestyle in women. New England
Journal of Medicine, 343, 530 – 537.
Hulley, S., Grady, D., Busch, T., Furberg, C., Herrington, D., Riggs, B.
& Vittinghoff, E. (1998) Randomized trial of estrogen plus progestin
for secondary prevention of coronary heart disease in postmenopausal
women. Heart and Estrogen/progestin Replacement Study (HERS)
Research Group. JAMA, 280, 605 – 613.
Hunter, D.S., Hodges, L.C., Vonier, P.M., Fuchs-Young, R., Gottards, M.M.
& Walker, C.L. (1999) Estrogen receptor activation via activation
function 2 predicts agonism of xenoestrogens in normal and neoplastic
cells of the uterine myometrium. Cancer Research, 59, 3090–3099.
Jiang, C., Sarrel, P.M., Poole-Wilson, P.A. & Collins, P. (1992) Acute effect
of 17 beta-estradiol on rabbit coronary artery contractile responses
to endothelin-1. American Journal of Physiology, 263, H271–H275.
Karamsetty, M.R., Klinger, J.R. & Hill, N.S. (2001) Phytoestrogens
restore nitric oxide-mediated relaxation in isolated pulmonary arteries
from chronically hypoxic rats. Journal of Pharmacology and Experi-
mental Therapeutics, 297, 968 – 974.
Kimira, M., Arai, Y., Shimoi, K. & Watanabe, S. (1998) Japanese intake
of flavonoids and isoflavonoids from foods. Journal of Epidemiology,
8, 168 –175.
King, R.A. & Bursill, D.B. (1998) Plasma and urinary kinetics of the
isoflavones daidzein and genistein after a single soy meal in humans.
American Journal of Clinical Nutrition, 67, 867–872.
Kuiper, G.G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J.,
Nilsson, S. & Gustafsson, J.A. (1997) Comparison of the ligand binding
specificity and transcript tissue distribution of estrogen receptors alpha
and beta. Endocrinology, 138, 863 – 870.
Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H.,
van der Saag, P.T., van der Burg, B. & Gustafsson, J.A. (1998) Inter-
action of estrogenic chemicals and phytoestrogens with estrogen
receptor beta. Endocrinology, 139, 4252 –4263.
Kuroski de Bold, M.L. (1999) Estrogen, natriuretic peptides and the
renin-angiotensin system. Cardiovascular Research, 41, 524–531.
Lecce, G., Meduri, G., Ancelin, M., Bergeron, C. & Perrot-Applanat, M.
(2001) Presence of estrogen receptor beta in the human endometrium
through the cycle: expression in glandular, stromal, and vascular cells.
Journal of Clinical Endocrinology and Metabolism, 86, 1379–1386.
Lieberman, E.H., Gerhard, M.D., Vehata, A., Walsh, B.W., Selwyn, A.P.,
Ganz, P., Yeung, A.C. & Creager, M.A. (1994) Estrogen improves
endothelium-dependent, flow-mediated vasodilation in postmenopausal
women. Annals of Internal Medicine, 121, 936–941.
Longcope, C. (2001) Endocrine function of the postmenopausal
ovary. Journal of the Society for Gynecologic Investigation, 8, S67–
S68.
Longcope, C., Franz, C., Morello, C., Baker, R. & Johnston, Jr., C.C.
(1986) Steroid and gonadotropin levels in women during the peri-
menopausal years. Maturitas, 8, 189–196.
Makela, S., Savolainen, H., Aavik, E., Myllarniemi, M., Strauss, L.,
Taskinen, E., Gustafsson, J.A. & Harry, P. (1999) Differentiation
between vasculoprotective and uterotrophic effects of ligands with dif-
ferent binding affinities to estrogen receptors alpha and beta. Proceed-
ings of the National Academy of Sciences of the United States of
America, 96, 7077–7082.
McCrohon, J.A., Woo, K.S. & Celermajer, D.S. (2000) A comparison of
endothelial function in Caucasian and Chinese women before and after
the menopause. Maturitas, 35, 31–37.
Mishra, S.K., Abbot, S.E., Choudhury, Z., Cheng, M., Khatab, N.,
Maycock, N.J., Zavery, A. & Aaronson, P.I. (2000) Endothelium-
dependent relaxation of rat aorta and main pulmonary artery by the
phytoestrogens genistein and daidzein. Cardiovascular Research,
46, 539–546.
Nestel, P.J., Pomeroy, S., Kay, S., Komesaroff, P., Behrsing, J., Cameron, J.D.
& West, L. (1999) Isoflavones from red clover improve systemic arterial
compliance but not plasma lipids in menopausal women. Journal of
Clinical Endocrinology and Metabolism, 84, 895 – 898.
Nestel, P.J., Yamashita, T., Sasahara, T., Pomeroy, S., Dart, A., Komesaroff, P.,
Owen, A. & Abbey, M. (1997) Soy isoflavones improve systemic
arterial compliance but not plasma lipids in menopausal and peri-
menopausal women. Arteriosclerosis, Thrombosis and Vascular Biology,
17, 3392–3398.
Perregaux, D., Chaudhuri, A., Mohanty, P., Bukhari, L., Wilson, M.F.,
Sung, B.H. & Dandona, P. (1999) Effect of gender differences and
estrogen replacement therapy on vascular reactivity. Metabolism:
Clinical and Experimental, 48, 227–232.
Program, L.R.C. (1974) Lipid and lipoprotein analysis. In The Manual
of Laboratory Operations (ed Editor A.) National Institute of Health,
Bethesda, MD.
Rosenthal, D.N. & Chin, C. (1999) Brachial artery reactivity: a modified
technique with applicability to children. Journal of the American Society
of Echocardiography, 12, 850–852.
Santoro, N., Brown, J.R., Adel, T. & Skurnick, J.H. (1996) Characteri-
zation of reproductive hormonal dynamics in the perimenopause.
Journal of Clinical Endocrinology and Metabolism, 81, 1495 –1501.
Schairer, C., Lubin, J., Troisi, R., Sturgeon, S., Brinton, L. & Hoover, R.
(2000) Menopausal estrogen and estrogen-progestin replacement ther-
apy and breast cancer risk. JAMA, 283, 485 – 491.
Schechter, M.T., Sharir, M., Paul Labrador, M.J., Forrester, J.S., Silver, B.
& Bairey Merz, C.N. (2000a) Oral magnesium therapy improves
endothelial function in patients with coronary artery disease. Circu-
lation, 102, 2353–2358.
Schechter, M.T., Sharir, M., Paul Labrador, M.J., Forrester, J.S. & Bairey
Merz, C.N. (2000b) Is there a benefit to achievement of low-density
lipoprotein cholesterol below 100 mg /dl in patients with coronary
artery disease? American Journal of Cardiology, 86, 1256 – 1259.
Scheiber, M.D., Liu, J.H., Subbiah, M.T.R., Rebar, R.W. & Setchell, K.D.R.
(2001) Dietary inclusion of whole soy foods results in significant
reductions in clinical risk factors for osteoporosis and cardio-
vascular disease in normal postmenopausal women. Menopause, 8,
384–392.
Shideler, S.E., DeVane, G.W., Kalra, P.S., Benirschke, K. & Lasky, B.L.
(1989) Ovarian–pituitary hormone interactions during the perimeno-
pause. Maturitas, 11, 331–339.
© 2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 693 –701

Simons, L.A., von Konigsmark, M., Simons, J. & Celermajer, D.S.
(2000) Phytoestrogens do not influence lipoprotein levels or endothe-
lial function in healthy, postmenopausal women. American Journal of
Cardiology, 85, 1297–1301.
Sorensen, K.E., Celermajer, D.S., Spiegelhalter, D.J., Georgakopoulos, D.,
Robinson, J., Thomas, O. & Deanfield, J.E. (1995) Non-invasive
measurement of human endothelium dependent arterial responses:
accuracy and reproducibility. British Heart Journal, 74, 247–253.
Upmalis, D.H., Lobo, R., Bradley, L., Warren, M., Cone, F.L. & Lamia, C.A.
(2000) Vasomotor symptom relief by soy isoflavone extract tablets in
postmenopausal women: a multicenter, double-blind, randomized,
placebo-controlled study. Menopause, 7, 236–242.
Vogel, R.A. & Corretti, M.C. (1998) Estrogens, progestins, and heart
disease: can endothelial function divine the benefit?. Circulation,
97, 1223 –1226.
Vogel, R.A., Corretti, M.C. & Plotnick, G.D. (2000) A comparison of
brachial artery flow-mediated vasodilation using upper and lower arm
© 2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 693–701
Isoflavones and endothelial function 701
arterial occlusion in subjects with and without coronary risk factors.
Clinical Cardiology, 23, 571–575.
Washburn, S., Burke, G.L., Morgan, T. & Anthony, M. (1999) Effect
of soy protein supplementation on serum lipoproteins, blood pressure,
and menopausal symptoms in perimenopausal women. Menopause, 6,
7–13.
Weiner, C.P., Lizasoain, I., Baylis, S.A., Knowles, R.G., Charles, I.G. &
Moncada, S. (1994) Induction of calcium-dependent nitric oxide
synthases by sex hormones. Proceedings of the National Academy of
Sciences of the United States of America, 91, 5212 – 5216.
Woo, K.S., Chook, P., Lolin, Y.I., Sanderson, J.E., Metreweli, C. &
Cleremajer, D.S. (1999) Folic acid improves arterial endothelial func-
tion in adults with hyperhomocystinemia. Journal of the American
College of Cardiology, 34, 2002–2006.
Xu, X., Harris, K.S., Wang, H.J., Murphy, P.A. & Hendrich, S. (1995)
Bioavailability of soybean isoflavones depends upon gut microflora in
women. Journal of Nutrition, 125, 2307– 2315.