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*
Nutritional Science Laboratory, Snow Brand Milk Products Co., Ltd., Kawagoe, Saitama, 350-1165, Japan
Department of Nutritional Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • magnesium • calcium • bone • ovariectomy • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Riggs and Melton 1986
). Bone loss caused by estrogen deficiency is due,
in humans and in experimental animals, primarily to an increase in
osteoclastic bone resorption (Kalu 1991
).
Magnesium is the second most abundant intracellular cation in
vertebrates. About half the total magnesium in the body of a normal
adult human is present intracellularly in soft tissues, and the other
half is in bone. Interactions between calcium and magnesium absorption
in the intestine have been reported. Based on in vitro experiments
using the everted gut sac technique (Hendrix et al. 1963
, Schachter and Rosen 1959
), in vivo studies
to compare magnesium-deficient rats with adequate-magnesium
rats (Alcock and MacIntyre 1962
) and in vivo balance
studies that use old rats (McElroy et al. 1991
), some
researchers have suggested that magnesium and calcium compete with each
other for intestinal absorption. Other studies indicated that
increasing magnesium level results in an increase in intestinal calcium
absorption by in vivo experiments using the technique of balance
studies (Clark 1965
, 1968
; Clark and Belanger 1967
). Previously, we also reported that magnesium
supplementation had an inhibitory effect on calcium absorption in
growing male rats (Toba et al. 1999
). However, our
results indicated that magnesium supplementation increased the dynamic
strength of bone in these rats, suggesting that magnesium
supplementation affects bone metabolism.
Recently, some researchers examined the effect of magnesium
supplementation on bone loss in postmenopausal women (Abraham 1991
, Abraham and Grewal 1990
, Seelig 1990
, Stendig-Lindberg et al. 1993
).
Stendig-Lindberg et al. (1993)
reported that magnesium
supplementation significantly increased bone density or arrested bone
loss in postmenopausal osteoporosis. Abraham and Grewal (1990)
also
reported that a significant increase in density of the calcaneus bone
was observed when postmenopausal women on hormonal replacement therapy
were supplemented with magnesium. However, because their trials were
combined with calcium supplementation or hormonal therapy, it is
unclear whether magnesium itself prevents postmenopausal bone loss.
Moreover, they did not examine the effect of magnesium supplementation
on biochemical markers of bone turnover, but rather analyzed bone
density. Therefore the purpose of this study was to evaluate
the effect of magnesium supplementation itself on calcium and bone
metabolism in aged ovariectomized
(OVX)2
rats as a suitable model of postmenopausal women (Kalu et al. 1989
, Wronski and Yen 1991
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Twenty-four 6-mo-old female Wistar rats (Japan SLC, Shizuoka, Japan)
were housed in individual stainless steel wire-mesh cages in a
temperature- and humidity-controlled room (23°C and 60 ± 5% relative humidity) with a 12-h light/dark cycle. Rats were given
free access to a commercial diet (CE-2; Clea Japan, Tokyo, Japan) and
deionized water for a 1 wk adaptation period. All rats were treated in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals (NRC 1985
). After the 1-wk adaptation period, 16
of the rats were OVX, and 8 were sham-operated (Sham). The rats
were given free access to the CE-2 diet for a 3-d recovery period after
the operation. After recovery, the OVX rats were separated into two
experimental groups of 8. The two groups were transferred to either
0.05% Mg diet or 0.15% Mg diet, as described in Table 1
. The magnesium level of AIN-76 diet is 0.05%, which is mainly based on
growth promotion (AIN 1977
, NRC 1978
). We
chose a 0.15% Mg diet, which does not affect growth and does not cause
symptoms of magnesium excess, as the magnesium- supplemented diet. The
Sham rats were fed the 0.05% Mg diet. All rats were housed
individually in metabolic cages. Since ovariectomy results increase
food intake and concomitantly body weight (Wade 1975
),
the two OVX groups were pair-fed the diets at a level equal to the
mean intake of the Sham group, and all rats were given free access to
deionized water for 42 d. Body weight was recorded once each week,
and food intake was monitored daily. We collected feces and urine on
all rats for 3-d periods starting at d 3, 10, 17, 24, 31 and 38 of the
feeding experiment for calcium and magnesium balance studies. For each
rat, all feces and urine were pooled for each period. We collected
urine for 24 h from d 41 of the feeding experiment to measure the
urinary excretion of deoxypyridinoline (D-Pyr). After the 42-d feeding
period, the rats were deprived of food overnight and killed by
decapitation. Blood was collected and centrifuged to separate the
serum. Femurs were excised from rats, and the muscles and connective
tissues were removed.
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Feces were dried and micropulverized. Micropulverized fecal samples and
urine were ashed at 550°C for 48 h. The ashed samples were
extracted for analysis by HCl solution (1 mol/L). The amounts of
calcium and magnesium in the feces and urine were determined by atomic
absorption spectrophotometry (AA-640–13; Shimadzu, Kyoto, Japan). The
absorption and retention of calcium and magnesium were determined by
following these equations:
 |
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Measurement of bone mineral density (BMD) of the femur.
BMD and bone mineral content (BMC) for the excised femur were measured by dual-energy X-ray absorptiometry by using a Dichroma Scan DCS-600A (Aloka, Tokyo, Japan) adapted for measuring small animals with beam energies of 22 and 53 keV. The scanning speed was 10 mm/s, and each scanning step was 1 mm.
Determination of bone strength of the femur.
The breaking force and breaking energy of the femoral diaphysis (the
center of the femur) were determined by using a three-point bending
rheolometer (RX-1600; Aitechno, Tokyo, Japan) as in the methods of
Ezawa et al. (1979)
and Takada et al. (1997)
. The measurement
conditions were as follows: sample space, 1.0 cm; plunger speed, 20
mm/min; and load range, 40.0 kg.
Serum analysis.
Serum calcium and magnesium were analyzed by the atomic absorption
spectrophotometry. Serum 1,25-dihydroxycholecalciferol
[1,25(OH)2D3] was purified by passing it
through a reverse-phase chromatography column C18
(Sep-Pac; Waters Associates, Milford, MA). Lipid extracts of the serum
samples were applied to the C18 column and eluted by water,
70% (v/v) methanol in water, 10% (v/v) methylene chloride in
n-hexane, 1% (v/v) isopropanol in
n-hexane, and 5% (v/v) isopropanol in
n-hexane. Serum 1,25(OH)2D3
fractions were eluted by 5% (v/v) isopropanol in
n-hexane. The active metabolite was quantified by a
radioimmunoassay (Reinhard et al. 1984
) that was carried
out with tritiated 1,25(OH)2D3 (Radiochemical
Center, Amersham, United Kingdom) as the radioactive tracer and calf
thymus receptor for 1,25(OH)2D3 (Yamasa Shoyu,
Tokyo, Japan) as the binding protein. Serum parathyroid hormone (PTH)
was measured with a commercial kit (Rat PTH IRMA Kit; Immutopics, San
Clemente, CA).
Measurement of biochemical markers of bone turnover.
Serum osteocalcin was measured with a commercial kit (Rat Osteocalcin
IRMA Kit; Immutopics). Urinary excretion of D-pyr was measured with
a commercial kit (Pyrilinks-D; Metra Biosystems, Mountain View, CA).
Urinary excretion of creatinine (Cr) was analyzed by the Jaffe
reaction, as described by Lustgarten and Wenk (1972)
. Urinary
D-Pyr was normalized by the urinary Cr.
Statistical analysis.
The data are the mean values with SD. Statistical analysis was done by one-way ANOVA. Results of calcium and magnesium balance studies were analyzed by repeated measurement ANOVA to evaluate differences throughout the experimental period among experimental groups. Tukey’s test was used to determine the significantly different groups when the ANOVA indicated a significant effect. All calculations were performed by using Super ANOVA software (Abacus Concepts, Berkeley, CA). Significance was assigned at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The initial body weight, final body weight, weight gain and food intake
were not significantly different among the three groups (Table 2
).
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The apparent absorption and retention of calcium in the OVX rats fed
the 0.15% Mg diet were significantly lower than in the Sham and OVX
rats fed the 0.05% Mg diet (Table 3
). The urinary calcium excretion in the OVX rats fed the 0.15% Mg diet
was significantly higher than in OVX rats had 0.05% Mg but did not
differ from Sham rats. The apparent absorption and retention of
magnesium in the OVX rats fed the 0.15% Mg diet were significantly
higher than in rats of the other two groups. The urinary magnesium
excretion in the Sham rats was significantly higher than in the OVX
rats fed the 0.05% Mg diet, and the excretion of magnesium by the OVX
rats fed 0.15% Mg was significantly higher than in both other groups.
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The BMD and BMC of the femur in rats of the two OVX groups did not
differ and were significantly lower than those in the Sham rats fed the
0.05% Mg diet (Table 4
). The breaking force of the excised femur in the Sham rats fed the
0.05% Mg diet and in OVX rats fed the 0.15% Mg diet was significantly
higher than in the OVX rats fed the 0.05% Mg diet. The breaking energy
of the excised femur in OVX rats fed the 0.15% Mg diet was
significantly higher than in the OVX rats fed the 0.05% Mg diet, with
an intermediate value in the Sham rats.
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The serum calcium concentration in the OVX rats fed the 0.05% Mg diet
was significantly lower than in the Sham rats fed that diet with an
intermediate concentration found in OVX rats fed 0.15% Mg.
(Table 5
). The serum magnesium level in rats of the two OVX groups was
significantly lower than in Sham rats fed the 0.05% Mg, and the level
in the OVX rats fed 0.15% Mg was significantly higher than in the OVX
rats fed 0.05% Mg. Serum
1,25(OH)2D3 did not differ
among the three groups. The serum PTH concentration in the Sham rats
and in OVX rats fed the 0.15% Mg diet was significantly lower than in
the OVX rats fed 0.05% Mg.
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The serum osteocalcin concentration in the OVX rats fed 0.05% Mg was
significantly higher than in the Sham rats fed the 0.05% Mg diet but
significantly lower than the level in the OVX rats fed the 0.15% Mg
diet (Table 6
). The urinary excretion of D-Pyr in rats of the two OVX groups was
significantly higher than in the Sham rats, and the excretion in the
OVX rats fed 0.15% Mg was significantly lower than in the OVX rats fed
0.05% Mg.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Kalu and Orhii 1999
). In this study, the
apparent calcium absorption did not differ between the Sham and OVX
rats fed the 0.05% Mg diet. Previously, we reported that the apparent
calcium absorption rate of growing male rats fed diets containing
0.15% magnesium was lower than in rats fed diets containing 0.05%
magnesium (Toba et al. 1999
). Similar results were found
in the present study. A common transport mechanism has been suggested
for the bivalent cations, calcium and magnesium, across the intestinal
wall. The interaction between dietary magnesium and calcium absorption
was also investigated in vitamin D-deficient rats by Levine et al. (1981)
. They proposed not only that high dietary magnesium impaired the
paracellular movement of calcium, but also that calcium and magnesium
might share certain cellular transport sites that are stimulated by
vitamin D. Therefore, in the present study, magnesium and calcium may
have competed with each other for intestinal absorption, even in OVX
rats. Additionally, calcium absorption is reduced with age
(Armbrecht et al. 1979
). In the present study, apparent
calcium absorption ranged from a positive 1.3% (in the OVX rats fed
0.05% Mg) to a negative 5.8% (in the OVX rats fed the 0.15% Mg
diet). We contend that the apparent calcium absorption in our study was
low mainly because we used aged rats. One of the reasons for the low
apparent calcium absorption in aged rats is that much endogenous
calcium is secreted back into their intestines. In this study,
magnesium supplementation may have promoted the secretion of endogenous
calcium into the intestine. Further studies are needed to elucidate why
magnesium supplementation reduces apparent calcium absorption.
Moreover, to investigate the effect of magnesium supplementation on
bone metabolism in OVX rats, we measured two biochemical markers
(urinary excretion of D-Pyr and serum osteocalcin level) of bone
turnover. Urinary excretion of D-Pyr (a product of collagen
breakdown) is a biochemical marker of bone resorption (Uebelhart et al. 1990
). In humans, menopause induces a two- to threefold
increase in the D-Pyr level, reflecting the postmenopausal increase
in bone turnover with degradation by osteoclasts (Eyre et al. 1988
). In this study, magnesium supplementation suppressed the
increased urinary excretion of D-Pyr induced by ovariectomy. That
is, our results suggest that magnesium supplementation suppressed the
increased bone resorption resulting from ovariectomy. On the other
hand, serum osteocalcin (a protein of bone matrix) is a biochemical
marker of bone formation. Postmenopausal women have a higher level of
serum osteocalcin than premenopausal women do because menopause induces
high bone turnover (Leino et al. 1994
). In the present
study, ovariectomy also increased serum osteocalcin and the
concentration in the OVX rats fed 0.15% Mg was significantly higher
than in the OVX rats fed 0.05% Mg. These results suggest that
magnesium supplementation of OVX rats increases bone formation by
activating osteoblasts and suppresses bone resorption by inactivating
osteoclasts.
We measured two hormones, PTH and
1,25(OH)2D3, that affect
bone and calcium metabolism. It has been reported that elevated serum
magnesium concentrations suppress PTH secretion (Buckle et al. 1968
, Massry et al. 1970
). In the present study,
magnesium supplementation suppressed the increased serum PTH levels due
to ovariectomy. The decreased serum magnesium level due to ovariectomy
also was raised by magnesium supplementation. The target organs of PTH
are bone and kidney. Its major skeletal effect is to increase bone
resorption by stimulating osteoclasts (McSheehy and Chambers 1986
). Therefore, the low level of serum PTH in OVX
rats fed 0.15% Mg also indicates that magnesium supplementation
suppressed bone resorption. On the other hand, the renal effect of PTH
is to synthesize 1,25(OH)2D3 and serum
1,25(OH)2D3 did not differ among the groups in
this study. These results suggest that the decreased serum PTH level
due to magnesium supplementation affects bone metabolism rather than
the synthesis of 1,25(OH)2D3 in OVX rats.
Recently, it was reported that magnesium supplementation had no effect
on biochemical markers (serum PTH, urinary excretion of D-Pyr, and
osteocalcin) in healthy young adult females (Doyle et al. 1999
). The effect of magnesium supplementation on bone
metabolism may depend on the age and/or physical condition.
Because magnesium supplementation reduced apparent calcium absorption, calcium retention in the OVX rats fed the 0.05% Mg diet was significantly greater than in those fed 0.15% Mg. However, magnesium supplementation increased bone formation and suppressed bone resorption. Therefore, the BMC of the femur did not differ between the two OVX groups. That is, we suggest that magnesium supplementation reduces calcium retention because it has an inhibitory effect on calcium absorption in intestine and/or promotes the secretion of endogenous calcium into intestine, but that the absorbed magnesium from the intestine also directly and/or indirectly affects osteoblasts and osteoclasts in bone and has preventive effects on bone loss in OVX rats. Moreover, we measured bone strength (the breaking force and breaking energy) by using a three-point bending method to investigate the dynamic strength of femur. The breaking force and breaking energy of the excised femur in the OVX rats fed the 0.15% Mg diet were significantly higher than in the OVX rats fed the 0.05% Mg diet. Moreover, the bone strength of the excised femur in OVX rats fed 0.15% Mg was as high as in the Sham rats fed 0.05% Mg. Therefore we speculate that magnesium supplementation increases the dynamic strength of bone because it increases bone formation through osteocalcin synthesis and suppresses bone resorption. Further studies are needed to elucidate the effect of magnesium on calcium and bone metabolism and bone strength.
In conclusion, our results indicate that magnesium supplementation reduces apparent calcium absorption, but promotes bone formation and prevents bone resorption in OVX rats. That is, it individually affects intestinal calcium absorption and bone metabolism in OVX rats. Moreover, our results indicate that magnesium supplementation increases the dynamic strength of bone. Therefore, it may be beneficial for the prevention of osteoporosis when the problem of decline in calcium absorption resulting from the intake of magnesium is solved.
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FOOTNOTES |
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Manuscript received June 24, 1999. Initial review completed August 25, 1999. Revision accepted October 11, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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