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*
Groupe Ostéoporose, Laboratoire des Maladies Métaboliques et Micronutriments, Institut National de la Recherche Agronomigue Clermont-Ferrand/Theix, 63122 Saint Genès Champanelle, France and
Ecole Nationale des Ingénieurs des Travaux Agricoles de Bordeaux, 33175 Gradignan Cedex, France
2To whom correspondence should be addressed: E-mail: picherit{at}clermont.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • soybean isoflavones • curative effects • bone • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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remains the mainstay for the
prevention of postmenopausal osteoporosis, because the biggest culprit
in the process of bone loss is estrogen deficiency (2)
.
However, hormone replacement therapy is not accepted universally due to
the contraindications in some patients, low compliance and reluctance
of many women because of the fear and dislike of possible side effects
and long-term risks (1)
. On the other hand, the human
diet contains a complex array of naturally occurring bioactive
molecules, the phytochemicals, that may confer important long-term
health benefits (3)
. Specifically, phytoestrogens, which
are well represented in Leguminosae seeds (where they occur mainly as
glycosides) and described as compounds with a weak estrogen-like
activity associated with their ability to bind to the estrogen receptor
(4)
, have attracted the most attention.
Soybeans (Glycine max), which were traditionally used for
fermented and unfermented soy food preparations, are rich in flavonoids
(the most common and widely distributed group of polyphenols) and, in
particular, in the isoflavones
(IF)3
genistin and daidzin. Although extensive data on ipriflavone (a
synthetic IF derivative) suggest that it is a useful and safe
alternative to estrogen therapy in the treatment of existing low bone
mass or osteoporosis in postmenopausal women, data on naturally
occurring IF are very limited but suggest that including them in the
diet results in a reduction in bone resorption caused by estrogen
deficiency (5)
. Indeed, dietary supplementation with
soybean IF might maintain bone mass in postmenopausal women
(6
,7)
.
Recently, the bone-sparing effects of soybean IF, either
administered orally (8
9
10
11
12
13)
or injected subcutaneously
(14)
, have been widely examined in a preventive approach
in the most commonly used animal model for postmenopausal osteoporosis,
the ovariectomized rat (15
16
17
18)
. Little is known about
their curative effects; only one study investigated the impact of the
short-term consumption of soy proteins (with normal or reduced IF
content) in reversing an established bone loss in the young
ovariectomized rat, and that study demonstrated that soy diets were
somewhat effective (19)
. Nevertheless, the
dose-dependent bone-curative effects of IF were never assessed for
a long period in adult rats. Therefore, we investigated the ability of
long-term daily intake of soybean IF to reverse established
osteopenia in the adult ovariectomized rat, according to the IF
ingestion level.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was conducted in accordance with current legislation on
animal experiments in France. Female Wistar rats (n
= 60; 195 d old) were purchased from I.N.R.A.
(Clermont-Ferrand/Theix, France) and housed individually at 21°C with
a 12-h light/dark cycle in metallic cages. Throughout the experimental
period, rats had free access to water and were fed a daily humidified
(1 mL water/g food) soy protein–free powdered semipurified diet
(I.N.R.A., Jouy en Josas, France; Table 1
). At 210 d old (
299 g), five rats designated as initial
controls (IC) were killed by cervical dislocation; simultaneously (on d
0), the 55 remaining rats were anesthetized intraperitoneally with
chloral hydrate (Fluka Chemie AG, Buchs, Switzerland; 80 g/L in saline
solution; 0.4 mL/100 g body) and either sham-operated (SH;
n = 14) or ovariectomized (OVX; n = 41). On d 80, rats designated as intermediate controls (SHi:
n = 5; OVXi: n = 5) were killed to
confirm the ovariectomy-induced bone loss. Then, the remaining OVX
rats (n = 36; 366 g) were randomly assigned
into one of four groups of nine rats each, fed IF at 0 (OVX), 20
(IF20), 40 (IF40) or 80 (IF80) mg/(kg body · d) for 84 d and
killed on d 164. IF were fed as a powdered soy IF concentrate (Novasoy
Isoflavone compound 152-400; Archer Daniels Midland Company, Decatur,
IL) containing 348 mg/g as total IF (genistin, 159; daidzin, 156;
glycitin, 33) and mixed with the semipurified diet. Simultaneously, SH
and OVX rats were fed the semipurified diet without any additional
compounds. Throughout the experiment, the quantity of food distributed
to each rat each day was adjusted to the mean level consumed by SH rats
the previous day, to prevent ovariectomy-induced hyperphagia. Rats
were weighed weekly to allow adjustment of IF doses to body weight
during the second part of the experiment. At 48 h before the rats
were killed, body composition was estimated with dual-energy x-ray
absorptiometry (DEXA) (20)
. On d 0, 40, 80, 122 and 164, a
24-h urine sample was collected from rats that were housed individually
for 24 h in metabolic cages, to measure the urinary excretion of
calcium and/or deoxypyridinoline (DPD), a marker of bone resorption
(21)
. Simultaneously, blood samples were harvested at
0900 h into ice-cooled heparinized plastic tubes containing
200 peptidase inhibitory units of aprotinin (Iniprol; Choay, Paris,
France) per mL blood and centrifuged immediately (3500 g for
5 min at 4°C). Then, plasma was frozen at -20°C until measurements
were made of phytoestrogens, calcium and/or osteocalcin (OC), a marker
of osteoblast activity (22)
. On d 80 or 164, uterine horns
were removed from each rat and weighed. Right or left femurs were
cleaned from adjacent tissues and used for bone mineral density (BMD)
measurements or mechanical testing, followed by image analysis,
respectively.
|
Genistein, daidzein and equol were measured with enzyme-linked
immunosorbent assays (23
,24)
. The sensitivity was 35, 40,
and 10 nmol/L for genistein, daidzein and equol, respectively. The
intra-assay variation was 4.8, 5 and 5% for genistein, daidzein
and equol, respectively, whereas the interassay variation was 13.1,
12.8 and 13.6% for genistein, daidzein and equol, respectively.
Plasma OC concentrations.
The OC concentrations were assessed by radioimmunoassays with rat 125I-labeled OC, goat anti-rat OC antibody and donkey anti-goat second antibody (Biochemical Technologies, Stoughton, MA). The sensitivity was 0.01 nmol/L. The intra- and interassay variations were 7 and 9%, respectively.
Urinary DPD excretion.
DPD excretion was determined by competitive radioimmunoassay with rat
monoclonal anti-DPD antibody coated to the inner surface of a
polystyrene tube and 125I-labeled DPD (Pyrilinks-D RIA kit;
Metra Biosystems, Mountain View, CA). The sensitivity was 2 nmol/L. The
intra- and interassay variations were 4 and 6%, respectively. Results
were expressed as nmol DPD/mmol creatinine (21)
. The
urinary creatinine assay, based on a modified Jaffés method in
which picric acid forms a colored solution in the presence of
creatinine, was used to adjust DPD values for variation in urine
volume.
Plasma and urinary calcium concentrations.
These concentrations were measured by atomic absorption spectrophotometry with a Perkin-Elmer 400 spectrophotometer (Perkin-Elmer Cetus, Norwalk, CT). Samples were previously diluted with lanthanum oxide solution (1 g/L; Carlo Erba Reagenti, Val de Reuil, France).
BMD.
BMD was assessed with DEXA with a Hologic QDR-4500 A x-ray bone
densitometer (Hologic, Massy, France). The total femur BMD (T-BMD), as
well as the BMD of two subregions, one corresponding to the diaphysis
(D-BMD) (rich in cortical bone) and the other corresponding to the
distal femur metaphyseal zone (M-BMD) (mainly cancellous bone), were
determined (25)
.
Mechanical testing.
Immediately after collection, the femoral length and the mean
diaphyseal diameter were measured with precision calipers (Mitutoyo,
Shropshire, U.K.). Then, the femoral failure load was determined with a
Universal Testing Machine (Instron 4501; Instron, Canton, MA),
according to a three-point bending test (26)
.
Image analysis.
Distal femurs were first dehydrated in a graded series of ethanol
solutions and embedded in methyl metacrylate (Sigma, L’Isle d’Abeau,
France) (27)
. Then, 100- to 200-µm frontal sections were
cut with a low speed saw (Isomet 2000; Buehler,Krautkramer,Champagne-Mont d’Or,France) (with a diamond-tipped cutting
blade), ground to 80-µm sections with a polishing machine (Metaserv 2000; Buehler) and stained according to the Von Kossa silver method
(AgNO3; Sigma). To characterize static cancellous bone,
image analysis was performed in the secondary spongiosa of the distal
femur metaphysis with an image-analysis system with OsteoLab
software (Biocom, Paris, France) that allows an evaluation of
cancellous bone area/measured surface and the number, separation or
thickness of trabeculae.
Statistical methods.
Results were expressed as means ± SEM. All data were analyzed with GraphPad InStat software (Microsoft, San Diego, CA). An ANOVA was first performed to test for any significant differences among groups. When significant, the Student-Newman-Keuls multiple comparison test was used to determine the specific differences between means. Parametric ANOVA was performed when data were sampled from populations with equal variance. Otherwise, nonparametric methods were selected. Thus, a Kruskal-Wallis test was first performed. If it indicated a significant difference among groups, the Mann-Whitney U test was used to determine specific differences. To test for any significant differences among days within a group, repeated measures ANOVA was performed and, when significant, the Student-Newman-Keuls multiple comparison test was used to determine the specific differences between means. The level of significance was set at P < 0.05 for all statistical tests.
|
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although body weight increased between d 0 and 80 in both SH and OVX
rats (d 80; P < 0.0001), the former were lighter than
the latter (P < 0.05) at the end of the first
experimental period (Fig. 1
). As body weight increased between d 80 and 164 in both SH and OVX rats
(P < 0.001), SH rats were still lighter than OVX rats
(P < 0.05) at the end of the experiment. Furthermore,
except in the IF20 group, body weight did not significantly vary
between d 80 and 164 in ovariectomized rats fed IF. As a result, on d
164, rats in the IF80 group had a body weight not different from that
of SH rats and lower than that in OVX rats (P < 0.05),
whereas values in IF20 or IF40 rats were not different from those in SH
or OVX rats. On the other hand, no significant differences were
observed among groups for fat and lean relative masses (28 ± 1
and 69 ± 1 g/100 g body, respectively), except in rats in the IC
group, which had a lower relative fat mass and a higher relative lean
mass than the others (14 ± 2 and 83 ± 2 g/100 g body,
respectively; P < 0.001).
|
It was lower in OVX than in SH rats (P < 0.001), on
both d 80 and d 164 (Table 2
). Moreover, on d 164, uterine weights in ovariectomized rats fed IF
were not different from that of OVX rats.
|
Values on d 164 were lower in IF20 rats than in IF40 or IF80 rats
(P < 0.0001) but did not differ among IF40 and IF80
rats (Table 3
). Simultaneously, very low levels of genistein, daidzein and equol were
detected in the plasma of untreated rats (0.08 ± 0.01, 0.09
± 0.01 and 0.05 ± 0.01 µmol/L, respectively, P
< 0.0001).
|
On each day of measurement, plasma OC values were higher in OVX than in
SH rats (Fig. 2
). Moreover, although plasma OC concentrations in IF20 or IF80 rats were
not different from that in SH rats on d 122, values in the three groups
of rats fed IF also did not differ from that in OVX rats. By contrast,
as circulating OC levels in treated rats decreased between d 122 and
164, although they were not modified in OVX or SH rats, concentrations
at d 164 in IF-fed rats were not different from that in SH rats and
lower than that in OVX rats (except in rats from the IF20 group).
|
Values were significantly higher in OVX than in SH rats at all time
points (Fig. 3
). On the other hand, although urinary DPD excretion at d 122 was higher
in IF-treated rats than in SH rats, concentrations in IF80 rats,
but not in IF20 or IF40 rats, were significantly lower than that in OVX
rats (P < 0.05). Moreover, DPD excretion in IF40 rats
decreased between d 122 and 164 (P < 0.05). As a
result, although on d 164 DPD excretion was greater in all
ovariectomized rats than in SH rats, urinary DPD excretion in IF40 or
IF80 rats (but not in IF20 rats) was significantly lower than that in
OVX rats (P < 0.05).
|
Plasma calcium levels did not differ among groups at any time point (2.58 ± 0.07, 2.53 ± 0.03, 2.61 ± 0.01 and 2.47 ± 0.01 mmol/L on d 0, 80, 122 and 164, respectively). Urinary calcium excretion also did not differ among groups (2.6 ± 0.2, 3.1 ± 0.2, 2.8 ± 0.2 and 3.9 ± 0.2 mg/d on d 0, 80, 122 and 164, respectively).
BMD.
BMD values did not differ among IC, SHi or SH rats but were
significantly lower in ovariectomized rats than in SH rats, on both d
80 and 164 (Fig. 4
). Furthermore, BMD values on d 164 were not greater in IF-treated
rats than in OVX rats.
|
No significant difference among groups was demonstrated for femoral length, diaphyseal diameter or femoral failure load throughout the experiment (37.1 ± 0.1 mm, 3.62 ± 0.04 mm or 115 ± 2 N, respectively).
Image analysis.
Cancellous bone area/measured surface in the distal femur metaphysis
was not different among IC, SHi and SH groups. By contrast, it was
significantly lower in ovariectomized rats than in SH rats on both d 80
and d 164 (Fig. 5
). Furthermore, values on d 164 were not greater in IF-treated rats
than in OVX rats. Similarly, trabecular number was lower in all
ovariectomized rats than in all nonovariectomized rats (1.6 ± 0.1
versus 3.2 ± 0.2 trabeculae/mm, P < 0.0001).
Trabecular separation was higher in all ovariectomized rats than in all
nonovariectomized rats (613 ± 36 versus 242 ± 19 µm,
P < 0.0001). By contrast, although trabecular
thickness (which did not differ among IC, SHi and SH rats) was lower in
OVXi than in SHi rats (65 ± 3 versus 77 ± 3 µm,
P < 0.05), no significant difference was observed
among groups on d 164 (84 ± 2 µm).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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9
10
11
12
13)
. However, only one experiment showed that once
bone loss has occurred, its reversal through daily consumption of soy
proteins (with normal or reduced IF content) from d 35 to d 100 after
surgery was difficult to obtain (19)
. Nevertheless, this
short-term study in young rats was performed with only one dose.
Indeed, several consumption levels associated with a long-term
ingestion should be tested. Furthermore, an experiment in adult rather
than in young rats should be required to assess bone loss. Therefore,
the present study investigated the dose-dependent bone-curative
effects of daily soybean IF intake in rats that underwent ovariectomy
at the age of 7 mo and were fed IF for 84 d from d 80 after
surgery.
As expected, Figure 4
indicates that in intermediate or experimental
rats, ovariectomy (confirmed with uterine atrophy; Table 2
) induced
femoral osteopenia, in both in the distal femur metaphysis (rich in
cancellous bone that is mainly involved in metabolic functions) and the
femoral diaphysis (rich in cortical bone that fulfills essentially
mechanical and protective functions), as shown by M- and D-BMD,
respectively. However, the D-BMD reduction was not associated with
an impairment of mechanical properties, as indicated by the femoral
failure load, which remained constant. On the other hand, the M-BMD
drop was associated with a decrease in cancellous bone area (Fig. 5)
.
Simultaneously, trabeculae number decreased, whereas trabecular
separation increased. By contrast, although trabecular thickness was
lower in OVXi than in SHi rats, no significant difference was observed
between SH and OVX rats on d 164. It might indicate that bone loss has
occurred by lowering the trabeculae number rather than by reducing
them. Moreover, osteopenia probably resulted from an increase in bone
turnover, as shown by the higher plasma OC concentration and urinary
DPD excretion in OVX than in SH rats (Figs. 2
, 3)
.
Unlike Arjmandi et al. (19)
, who reported that soy diets
that provide per os genistin plus daidzin at 25 or 2.5 µg/(g
body · d) during a 65-d period were somewhat effective in reversing
the femoral bone density loss (assessed with the Archimedes principle),
IF consumption in the present experiment did not elicit any curative
effect on femoral BMD (assessed by DEXA), at both the cortical and
cancellous sites (Fig. 4)
. Nevertheless, our results are in accordance
with those of Arjmandi et al. (19)
, who also reported that
ash weight (g/100 g dry bone) in the right femur was lower in
ovariectomized rats whether fed or not fed soy diets than in SH rats.
Moreover, associated with M-BMD data, no curative effect of IF on
changes in cancellous bone area (Fig. 5)
or number and separation of
trabeculae was observed in the present study. By contrast, bone
turnover was lower in IF-fed rats than in OVX rats (Figs. 2
, 3)
.
Although osteocalcinemia in IF20 rats was not different from that in
OVX rats, values at d 164 in the three groups of rats fed IF were
similar to that in SH rats. Furthermore, plasma OC concentrations were
decreased between d 122 and d 164 in IF-fed rats, whereas they
remained constant in OVX rats (Fig. 2)
. Although urinary DPD excretion
was higher in IF-fed ovariectomized rats than in SH rats, values
were lower in IF80 than in OVX rats on both d 122 and 164 (Fig. 3)
.
Moreover, they decreased between d 122 and 164 in the IF40 group,
whereas they remained stable in OVX rats, resulting in a d-164 DPD
excretion lower in IF40 than in OVX rats. These data suggest that the
IF-induced antiosteoclastic activity occurred in a
dose-dependent manner, because only the two highest levels of
consumption reduced bone resorption. However, Arjmandi et al.
(19)
reported that the daily intake of genistin plus
daidzin at 25 or 2.5 µg/(g body · d) did not slow down the
ovariectomy-induced higher rates of bone turnover. Nevertheless,
considering the first soy group in this later study and the IF20 group
in the present experiment, results do not differ completely in that in
both groups, bone resorption was similar to that measured in OVX rats
and higher than that in SH rats.
In parallel with bone turnover parameters, plasma phytoestrogen
concentrations at d 164 were increased in a dose-dependent manner
between IF20 and IF40 groups but not between IF40 and IF80 groups
(Table 3)
. It could be in part explained by a maximal IF level beyond
which IF absorption might not be further increased and by the
possibility of an elimination system of plasma phytoestrogens more
efficient with high levels than with low doses, in 10- to 13-mo-old
ovariectomized rats. Moreover, in the early part of soybean feeding
and, therefore probably in association with a small adaptation period
to the diet, a weak (and nonsignificant) reduction in body weight was
observed in the IF80 group (Fig. 1)
. As a result and because OVX rats
exhibited a greater body weight than SH rats regardless of whether the
pair-feeding to SH rats had minimized the ovariectomy-induced
hyperphagia, body weight at d 164 in IF80 rats was lower than that in
OVX rats and did not differ from that in SH rats. Finally, these
present results also indicate that IF feeding was unable to reverse the
ovariectomy-induced uterine atrophy (Table 2)
. In the same way,
65 d of soy feeding providing per os genistin plus daidzin at
25 µg/(g body · d) did not result in any uterotrophic activity
in young ovariectomized rats (19)
.
In conclusion, the present study demonstrated that a daily soybean IF intake in adult ovariectomized rats reduced bone turnover but did not reverse a previously established bone loss. Furthermore, it appeared that the two highest consumption levels were more effective in depressing the ovariectomy-induced increase in bone turnover (and in bone resorption specifically) than the lowest dose. Therefore, ingestion levels of soybean IF should be considered to improve bone health in a preventive rather than a curative approach of human postmenopausal osteoporosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BMD, bone mineral density; D-BMD bone mineral density in the femoral diaphysis; M-BMD, bone mineral density the distal femur metaphysis; T-BMD, bone mineral density in the total femur; DEXA, dual-energy x-ray
absorptiometry; DPD, deoxypyridinoline; IC, initial control; IF, isoflavones; IF20, IF40 or IF80, ovariectomized rats fed isoflavones at 20, 40 or 80 mg/(kg body · d); OC, osteocalcin; OVX, ovariectomized; OVXi, intermediate ovariectomized; SH, sham-operated; SHi, intermediate sham-operated.
Manuscript received September 20, 2000. Initial review completed November 3, 2000. Revision accepted December 11, 2000.
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Within SH, OVX or IF20 groups, means on d 80
were significantly different from d 164, P < 0.001; means on d 80 or d 164 not sharing a letter differ
significantly, P < 0.05.
