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Estrogen deficiency and low calcium diet increased bone loss and urinary calcium
excretion, but did not alter arterial stiffness in young female rats.
Jong-Hoon Park, Naomi Omi, Toshiya Nosaka, Ayako Kitajima, Ikuko Ezawa
Jong-Hoon Park, Ayako Kitajima, Naomi Omi ( )
Department of Exercise Nutrition, Graduate School of Comprehensive Human Sciences,
University of Tsukuba, 1-1-1 Tennôdai, Tsukuba, 305-8574, Ibaraki, Japan
Tel. +81-298-53-6319; Fax +81-298-53-6507
e-mail: ominaomi@taiiku.tsukuba.ac.jp
Toshiya Nosaka
Department of Nursing, Nagano College of Nursing 1694, Akaho, Komagane, 399-4117,
Nagano, Japan
Ikuko Ezawa
Department of Food and Nutrition, School of Home-Economics, Japan Women's
University, 2-8-1 Mejirodai, Bunkyo-ku, 112-8681, Tokyo, Japan
Toita Women’s College, 2-21-17, Shiba, Minato-ku, 105-0014, Tokyo, Japan
Key words: estrogen deficiency; dietary Ca restriction; bone loss; urinary Ca excretion;
arterial stiffness
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Abstract Many epidemiological studies have reported that the severity of arterial
diseases such as arterial calcification and stiffness is inversely related to bone loss, i.e.
osteoporosis. However, the nature of this relationship is unclear. The purpose of the
present study was to examine the influences of estrogen deficiency and/or low calcium
diet (0.1% Ca) on bone metabolism and calcium balance, as well as aortic wall
composition and stiffness in young female rats. Twenty eight 6 week-old female rats
were randomized into four groups: OVX-Low calcium (OL) and OVX-Normal calcium
groups (ON) were ovariectomized, and Sham-Low calcium (SL) and Sham-Normal
calcium groups (SN) were sham-operated. After 12 weeks, the bone mineral density of
the lumbar spine and tibial proximal metaphysis were significantly lower in ON than in
SN, and also significantly lower in OL than in ON. Additionally, OL rats had significant
higher (vs. SN and SL) urinary deoxypyridinoline, but not urinary calcium, excretion at
4 weeks after ovariectomy. However, at 12 weeks after ovariectomy, urinary calcium
excretion was significantly higher in OL than in SL, with corresponding increases in
two bone turnover markers, bone type alkaline phosphatase and tartrate-resistant acid
phosphatase. Neither estrogen deficiency nor low calcium diet affected aortic stiffness
or elastin degeneration and calcium deposition over the course of the present study,
although changes of bone metabolism occurred rapidly. Taken together, these results
show that bone loss and arterial stiffness did not progress simultaneously in the present
experimental protocol.
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Introduction
A rapid demographic shift toward aging societies has resulted in the increased
prevalence of age-related diseases such as osteoporosis and cardiovascular disease, in
addition to morbidity and disability in elderly patients. Such chronic diseases have
increased healthcare costs not only in Japan but also in other developed countries [1-4].
The most common type of osteoporosis is the postmenopausal bone loss
associated with ovarian hormone deficiency and dietary low calcium intake [5-7]. Rapid
bone loss is associated with a rise in plasma calcium levels, and many studies have
suggested that calcium loss via urinary excretion may predispose patients to
osteoporosis [8, 9]. Although the influence of ovariectomy on urinary calcium excretion
has been extensively studied [10, 11], the combined influence of estrogen deficiency
and low calcium diet have not been well examined. Therefore, we were interested in
investigating the combined influence of estrogen deficiency and low dietary calcium on
bone loss and urinary calcium excretion, given the assumption that postmenopausal
women tend to have a fairly low calcium intake [12, 13] and are in negative calcium
balance [11,14].
Many epidemiological studies have reported that the severity of arterial diseases
such as arterial calcification and stiffness is inversely related to bone loss, osteoporosis
[15-18]. Although osteoporosis and arterial disease seem to be associated, the nature of
this relationship is controversial. Women lose about 5% of their trabecular bone every
year and about 15% of their total bone in the first 5 years after menopause [19, 20]. The
incidence of cardiovascular disease in women before menopause is significantly lower
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than in males [21]. However, within a few years after menopause, the incidence of
cardiovascular disease in women equals that of males in the same age range [22, 23].
Estrogen replacement therapies reduce the risk of rapid bone loss [24] and arterial
disease [25] in postmenopausal women. Therefore, estrogen deficiency seems to relate
to the parallel progression of bone loss and aortic disease.
Although benefits of long-term estrogen therapy for age-related decreases in
systemic arterial compliance have been described in postmenopausal women [26, 27],
the effects of estrogen on arterial stiffness are now contradictory [26-28]. Elastin
degeneration and calcium deposition increase arterial stiffness [29] and induce the
migration of vascular smooth muscle cells into the endothelium [30], which is
considered to be a risk factor for atherosclerosis [31]. Estrogen has been reported to
inhibit the proliferation of vascular smooth muscle cells [32] and may also exert a Ca2+
channel blocker-like effect, suggesting that estrogen treatment could inhibit progression
of arterial calcification [33]. However, it is unclear whether rapidly induced estrogen
deficiency affects aortic degeneration and elastin calcium deposition. In the present
study, we postulated that increased elastin calcium content and rapid degenerative
changes in ovariectomized rats might induce an increase in aortic stiffness.
The purposes of this study were: 1) To investigate how estrogen deficiency and/or
low calcium diet affects bone loss and urinary calcium excretion. 2) To assess the
alteration of elastin components and arterial stiffness induced by estrogen deficiency. 3)
To determine whether the degree of bone loss due to estrogen deficiency and low
calcium diet is related to the extent of arterial degeneration, elastin calcium deposition,
5
and aortic stiffening.
Materials and methods
Experimental animals and feeding protocol
Twenty-eight female Sprague-Dawley rats, 6 week-old, were randomized into
four groups: the OVX-Low calcium (0.1% dietary Ca, 0.6% dietary P) group (OL, n =
6) and OVX-Normal calcium (0.6% dietary Ca, 0.6% dietary P) group (ON, n = 6) were
ovariectomized via the dorsal route, and the Sham-Low calcium group (SL, n = 8) and
the Sham-Normal calcium group (SN, n = 8) were operated on without the ovaries being
removed. The experimental period was 12 weeks. The rats were kept in individual cages
(15×25×19.5cm) and allowed access to food and distilled water ad libitum. Food
consumption and body weight gain were measured every second day. Room temperature
was kept at 24±1℃, humidity at 50±5%. Fluorescent lights were on from 8:00 a.m. to
8:00 p.m.. Animal care and experimental procedure were approved by the Animal
Experimental Committee of the University of Tsukuba.
Serum calcium, phosphorus, bone turnover markers, and 1,25-(OH)2D3
At the end of the experimental period, all the rats were deprived of food overnight.
Under ether anesthesia, animals were killed by exsanguinations from the abdominal
aorta. The blood samples were centrifuged at 2,500 rpm for 15 min to extract the serum.
The level of serum Ca was measured by the Inductively Coupled Plasma Atomic
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Emission Spectroscopy (ICAP−AES – 575 v Nippon Jarrell-Ash) and phosphorus was
determined by the Fiske-Subarrow method [34]. The Bone type Alkaline Phosphatase
(BAP) and the Tartrate-resistant Acid phosphatase (TRAP) activities were measured as
previously reported [35]. Serum 1,25-(OH)2D3 was detected using 1,25-(OH)2D3 Radio
Immunoassay (RIA) kit (Immunodiagnostic Systems, Inc., USA).
Measurement of bone mineral density
The lumbar spine, and left and right tibiae of each rat were isolated by dissection,
and freed from any muscle and connective tissue. Thereafter, bone mineral density
(BMD) values for the L3-L6 lumbar spine and the whole tibiae were measured by Dual
- energy X-ray Absorptiometry (DXA; Aloka DCS-600R instrument). The analysis of
the tibial BMD was carried out as previously reported [36]. Briefly, proximal one-fifth
of the tibia, including the epimetaphyseal region representing the trabecular sites, and
middle one-fifth of the tibia representing the cortical diaphyseal region.
Femoral weights and mechanical breaking test
At each dissection, femur samples were collected, freed from adhering connective
tissues. Thereafter, the bone strength at the middle diaphysis of the femur was tested by
measuring the mechanical strength, with an Iio DYN−1255 instrument as previously
reported [37]. The force necessary to produce a break at the center of the femur was
measured under the following conditions: the sample space was 1.0 cm, the plunger
speed was 100.0 mm/min, the load range was 50.0 kg, and the chart speed was 120.0
cm/min. Afterwards, the femurs were dried at 95℃ for 24 hr to measure their dry
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weight. The bones were dry-ashed at temperatures from 150℃ to 550℃, with 50℃
increments at each 4 hr and at 600℃ for 24 hr and then the ash weight was measured.
Calcium and deoxypyridinoline excretions in urine
Animals were placed in individual metabolic cages (24×20×18 cm3). The first
phase was carried out on the 3rd and 4th day after starting the experimental diets period
(Phase Ⅰ). The next phase (Phase Ⅱ) was carried out on the 31st and 32nd day. The
third phase (Phase Ⅲ) was on the 56th and 57th day. The final phase (Phase Ⅳ) took
place on the 80th and 81st day, just before the end of the experimental diets period. At
each phase, urine was collected over two 24 hr periods. Urine was collected under
acidic conditions using 2ml 2N hydrochloric acid. All urine was centrifuged at 2,500
rpm for 15 min to eliminate refuse. The urinary Ca was measured using the same
method as that for the biochemical assay of the serum. The bone resorption marker
deoxypyridinoline (Dpd, bone-specific Type 1 collagen degradation product) was
measured in 24 hr urine samples from all four collection phases, using commercially
available kits (Metra Dpd EIA Kit, Quidel USA).
Aortic biochemical studies
The descending thoracic aorta was divided longitudinally into two parts. Each
sample was dried, and its dry weight was determined. One half of the aorta from each
rat was designated for the measurement of the content of aortic calcium. The other half
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of the aorta was boiled with 0.1 N NaOH for fifty minutes, according to the method of
Lansing et al, for the determinations of elastin content (alkali-resistant elastin
preparation) [38], and calcium content in elastin. The elastin content and calcium
content in elastin were also determined from the aortic arches. The calcium content was
measured using the same method as that for the biochemical assay of the serum.
Aortic biomechanical studies
To obtain a static stress-strain curve, each ring specimen of thoracic aorta was
mounted as an intact loop between two smooth rods attached to an Instron-type tensile
testing machine (Toyo Baldwin) as previously described [39,40]. Briefly, 2 mm ring
specimens for tensile testing were excised from the proximal portion of each descending
thoracic aorta, and were immersed in saline solution at 4℃. The incremental elastic
modulus (a measure of arterial stiffness) of the aorta at an extension ratio of 1.5 was
defined as the ratio of the incremental stress to the incremental strain of 0.1 across the
extension ration of 1.5 which is comparable to the aortic wall distension at an arterial
blood pressure of 100 mmHg. The ultimate tensile stress (tensile strength) and ultimate
tensile strain were obtained from the stress-strain curves as the values at the breaking
point of the specimen.
Statistical analysis
All the data are expressed as the mean ± SE. One-way and two-way analysis of
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variance were used to test for statistically significant differences between groups.
Analysis of effects of ovariectomy, dietary calcium, and interaction between those
factors as grouping variables was performed and the significance between individual
groups was determined using post hoc PLSD test. Statistical comparisons of calcium
and Dpd excretions in urine among four groups over time were performed by a mixed
design two-way ANOVA with repeated measures. After significant interactions,
one-way ANOVA were employed each phase (4 phases) to compare and contrast the
effect of groups. If a significant difference was detected, these were further evaluated by
post hoc PLSD test. Association between bone turnover markers and urinary calcium
excretion was performed by Pearson correlation test. A significant level of p < 0.05 was
used for all comparisons. All statistical treatments were done using the Stat View 5.01
software (SAS Institute Inc. Cary, NC, USA, 2000-2001).
Results
Body weight, food intake, and food efficiency
Table 1 shows body weight, food intake, and food efficiency. Initial body weight
did not differ among the four groups. Two-way ANOVA analysis showed that
ovariectomy significantly altered the final body weight, body weight gain, food intake,
and food efficiency (p < 0.0001), but dietary calcium did not significantly influence
them in both Sham-operated groups and OVX groups.
BMD of lumbar spine and tibia
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The BMD of the lumbar spine and tibia are shown in Fig 1. The BMD of the
lumbar spine, which contains cancellous-rich region, was significantly lower in OL than
in ON and Sham-operated groups (SN and SL). The BMD of the tibial proximal
metaphysis, which contains cancellous-rich region, showed the same trend as those of
the lumbar spine. Both ovariectomy (p < 0.0001) and dietary calcium (p < 0.0001) had
significant effects on the cancellous BMD of the lumbar spine and tibial proximal
metaphysis, and the interaction was highly significant for the BMD of the tibial
proximal metaphysis (p = 0.0479). Low calcium diet significantly reduced the BMD of
the tibial diaphysis, which contains little or no cancellous bone (cortical-abundant
region), in both Sham-operated groups and OVX groups (p < 0.001), but estrogen
deficiency alone did not reduce the BMD values.
Serum calcium, phosphorus, bone turnover markers, and serum PTH and 1,25 (OH)2D3
levels
Table 2 shows serum calcium, phosphorus, BAP, and TRAP levels. It did not
observe any significant differences in serum calcium and phosphorus among the groups.
Two-way ANOVA analysis showed that the interaction between ovariectomy and
dietary calcium was significant for serum calcium (p = 0.0017), but the values were
physiologically within normal range. The combined influence of estrogen deficiency
and low calcium diet (OL) produced significant increases in BAP levels compared with
the other groups, and also showed significant increases in TRAP levels compared with
SL. BAP and TRAP levels in OVX groups (ON and OL) were correlated with urinary
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calcium excretion at 12 weeks from OVX operation (Pearson r = 0.565, p < 0.05 for
BAP and Pearson r = 0.592, p < 0.05 for TRAP), but those in sham groups (SN and SL)
were not observed. Low calcium diet significantly increased serum 1,25 (OH)2D3 level
in both Sham-operated groups (p < 0.001) and OVX groups (p < 0.01). The serum 1,25
(OH)2D3 level was significantly higher in ON than in SN. Two-way ANOVA analysis
showed that ovariectomy tended to alter the serum 1,25 (OH)2D3 level (p = 0.0643), but
dietary calcium significantly influenced the level (p < 0.001).
Femoral characteristics and biomechanical testing
As shown in table 3, the femoral dry and ash weights were significantly lower in
OL than in the other groups. Ovariectomy (p < 0.0001) and dietary calcium (p < 0.0001)
had significant effects on the dry and ash weights. The femoral breaking force was
reduced by low calcium diet in both Sham-operated groups and OVX groups, and the
femoral breaking energy was significantly lower in OL than in the other groups.
Calcium and Dpd excretions in urine
Calcium and Dpd excretions in urine showed in Fig. 2. Two-way ANOVA with
repeated measures for calcium and Dpd excretions in urine showed significant
group-by-time interactions (p < 0.0001) as well as group (p < 0.0001) and time (p <
0.0001) effects. Urinary Ca excretion in normal calcium diet groups (SN and ON) were
significantly higher than in SL from phaseⅠ to phase Ⅳ and in OL from phaseⅠ to
phase Ⅱ. With time, the urinary Ca excretion in OL increased and was significantly
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higher than in SL from phase Ⅲ to phase Ⅳ. Urinary Ca excretion was significantly
higher in ON than in SN only in phase Ⅲ. Urinary Dpd excretion did not differ among
the groups at phaseⅠ, but from phase Ⅱ to phase Ⅳ, it was significantly higher in SL
and OVX groups (OL and ON) than in SN. Especially, in the phaseⅡ, the urinary Dpd
excretion was significantly in OL higher than in SN and SL. Urinary Dpd excretion in
SL and OVX groups (OL and ON) began with the increase being greatest in earlier
period (phaseⅡ) and it declined with time.
Aortic biochemical results
As shown in table 4, the content of calcium in thoracic aorta did not differ
between the four groups. The content of elastin in the thoracic and arch aortas did not
differ between the four groups and there was also no significant difference in the elastin
calcium content.
Aortic biomechanical results
As shown in table 5, there were no significant differences in incremental elastic
modulus, ultimate tensile stress, and ultimate tensile extension ratio between the four
groups.
Discussion
We observed that the combined influence of estrogen deficiency and low calcium
diet decreased both the BMD of the tibia and the lumbar spine. Additionally, the
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combination of estrogen deficiency and low calcium diet resulted in significantly
increased urinary calcium excretion and bone turnover marker expression at 12 weeks
after ovariectomy. However, estrogen deficiency and/or low calcium diet did not affect
aortic stiffness or elastin degeneration and calcium deposition during periods of rapid
change in bone metabolism.
The BMD in both cortical (tibial diaphysis) and cancellous (tibial proximal
metaphysis and lumbar spine) bones was significantly decreased in OVX rats fed low
calcium diet (Fig 1). These results correspond to the reduced dry and ash femur weights
observed in OVX rats fed low calcium diet (Table 3). Femoral breaking force and
energy were also markedly decreased in OVX rats fed low calcium diet (Table 3),
suggesting the increased risk of fracture. Previous studies using OVX rats fed 0.1% low
calcium diet have shown decreases in bone mineral density as well as increases in
circulating parathyroid hormone and 1,25-(OH)2D3 [7, 52]. In consistence with the
results of the previous studies, the present study showed that serum 1,25-(OH)2D3 in
OVX rats fed low calcium diet clearly increased, but normal calcium diet suppressed the
level.
At 4 weeks after ovariectomy, urinary Dpd excretion in OVX rats fed low calcium
diet was significantly higher than in sham-operated rats fed low calcium diet (Fig 2).
The increased Dpd excretion is suggestive of osteopenia resulting from increased bone
resorption [41]. In this study, it is possible that increased bone resorption in OVX rats
fed low calcium diet led to higher bone loss at the earlier time point of 4 weeks after
ovariectomy. Despite the increase in bone resorption, urinary calcium excretion was
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unchanged at 4 weeks (Fig 2). Estrogen receptors modulate tubular calcium
reabsorption in the kidneys [42], and urinary calcium excretion significantly increases
after menopause, indicating that renal tubular reabsorption of calcium decreases in
estrogen deficiency state [53]. It has been generally considered that higher urine
calcium excretion after menopause reflects an increase in the filtered load of calcium
from kidney, occurring as a result of increased bone resorption [54]. However, in the
present study, it is possible that in 4 weeks after ovariectomy, calcium reabsorption in
the kidneys might still occur through an alternate mechanism.
Urinary calcium excretion in OVX rats fed low calcium diet started to increase 8
weeks after ovariectomy and was significantly higher than that of sham-operated rats
fed low calcium diet from 8 weeks to 12 weeks after ovariectomy (Fig 2). At 12 weeks
after ovariectomy, positive correlations between urinary calcium excretion and the
expression of two bone turnover markers, BAP and TRAP, were observed in the OVX
groups only. Morris et al also showed that OVX rats with higher bone turnover lost
more urinary calcium when compared to those with lower levels of bone turnover [43].
The positive correlation between urinary calcium excretion and expression of bone
turnover markers in the present study was not observed in sham-operated rats regardless
of dietary calcium intake at all time points, suggesting that estrogen might influence the
renal reabsorption of calcium [42]. Estrogen receptors are present in human bone [44],
and increases in urinary calcium excretion during estrogen deficiency are accompanied
by increased bone resorption [43]. Therefore, at 12 weeks after ovariectomy, the
increased urinary calcium excretion in OVX rats fed low calcium diet may reflect not
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only increased bone resorption but also an inhibitory effect on renal tubular calcium
reabsorption. In the present study, although bone formation marker, serum BAP, in
OVX rats fed low calcium diet was significantly higher than in the other groups, bone
resorption marker, serum TRA P, was not so higher. It might be because the bone
turnover markers were analyzed using serum sample at the end point of this experiment
(i.e., 12 weeks after ovariectomy) when much progression of bone loss had already
occurred compared with rapid progression of bone loss at the earlier time point (i.e., 4
weeks after ovariectomy). Actually, at 4 weeks after ovariectomy, urinary Dpd excretion
in OVX rats fed low calcium diet was significantly higher than in both sham-operated
rats fed low calcium diet and sham-operated rats fed normal calcium diet. Taken
together, our results suggest that increased urinary calcium and bone turnover might be
risk factors for osteoporosis in postmenopausal women who have low calcium intake.
In the present study, urinary calcium excretion was significantly higher in OVX
rats fed normal calcium diet at 8 weeks after ovariectomy when compared to
sham-operated rats, whereas no difference was observed at 12 weeks (Fig 2). The effect
of ovariectomy on urine calcium excretion is currently controversial, with reports of no
effect [7, 45] and also of an increased excretion [10, 11]. However, the previous studies
only assessed urinary calcium excretion only at one time point. Therefore, the
conflicting findings may have been caused by differences in the age of the rats or the
duration of the experiments.
In the present study, we did not observe any changes in aortic stiffness or elastin
degeneration and calcium deposition during estrogen deficiency (Table 4, 5). Potential
16
protective mechanisms of estrogen relating to arterial stiffness include induction of
endothelium-dependent vasodilative factors [46, 47] and inhibition of vasocontracting
factors [48, 49] (i.e. functional factors). In the present study, to analyze arterial stiffness
we used dissected thoracic aorta, which did not have dynamic functional factors and
might be influenced by structural factors such as the composition of arterial wall elastin
and its calcium content. A previous study showed that the calcification level of the
arterial tunica media was positively correlated with antemortem aortic pulse wave
velocity [50]. In the present study, estrogen deficiency did not induce aortic elastin
degeneration or calcification, which may have in turn caused the observed lack of
change in aortic stiffness. We cannot rule out the possibility of the estrogen
deficiency-induced increase of arterial stiffness in aged OVX rats, being expected that
significant changes in phenotypes of artery relating to arterial stiffness can be observed.
However, to our knowledge, little has been reported whether aged OVX rats induce
increase in arterial stiffness. Furthermore, structural arterial alterations have been
observed to develop within 2 months in a rat hypertension model [51], but there were no
changes in the osteoporotic model of young rat in the present study. Therefore, future
studies using a combination model rat, e.g. atherosclerotic stimuli in ovariectomized
rats or aged ovariectomized rats may help to clarify the epidemiological links between
osteoporosis and aortic diseases.
We also did not observe any changes in aortic stiffness or elastin degeneration and
calcium deposition when estrogen deficiency was combined with low calcium diet
(Table 4, 5). The paradox of bone loss accompanied by aortic calcification, where there
17
might be a calcium shift from bone to arteries (calcium shift theory), has been reported
in previous epidemiological studies. However, in the present study, aortic calcification
and stiffness were unchanged during periods of rapid bone loss caused by either
estrogen deficiency or the combined influence of estrogen deficiency and low calcium
diet. It is possible that the surrounding vascular tissue was insufficient to accept plasma
calcium deposition after bone resorption.
In conclusion, we observed that the combined influence of estrogen deficiency
and low calcium diet decreased the bone mineral density of both cortical and cancellous
bones in the tibia and lumbar spine. The combination of estrogen deficiency and low
calcium diet also caused an increase in urinary calcium corresponding to increased bone
turnover, 12 weeks after ovariectomy. Although rapid changes were observed in bone
metabolism, neither estrogen deficiency nor low calcium diet affected aortic stiffness or
elastin degradation and calcium deposition. These data suggest that rapid bone loss and
biochemical aortic alterations caused by estrogen deficiency and low calcium diet did
not progress simultaneously.
Acknowledgments The authors are grateful to Dr. Yonekura L (National Food Research
Institute, Tsukuba, Ibaraki, Japan) for valuable comments and also to Evan Thomas for
help in the preparation of this manuscript.
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