ARTICLE
Auteur(s) :, Hiroshi Matsuzaki1,*, Shin-Ichi
Katsumata2, Mariko Uehara2, Kazuharu
Suzuki2, Kahoru Nakamura1
1Department of Nutrition, Junior College of Tokyo
University of Agriculture, 1-1-1 Sakuragaoka,Setagaya-ku, Tokyo
156-8502, Japan
2Department of Nutritional Science, Faculty of Applied
Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo
156-8502, Japan
Approximately half of the total magnesium (Mg) in the body exists
in bone and plays an important role in bone metabolism. Mg
deficiency is one of risk factor for osteoporosis. Previous studies
[1-3] have reported that Mg intake was correlated with bone mineral
content and/or bone mineral density. Furthermore, experimental
animals fed a Mg-deficient diet showed impaired bone growth and
increased skeletal fragility [4-6].On the other hand, calcium (Ca)
is an essential mineral that plays a very important role in the
maintenance of bone mass and preventing fractures. Although low Ca
intake induced an increase in bone resorption and a decrease in
bone mineral density [7-10], high Ca intake enhanced bone mineral
content and bone mineral density [11-13]. From these observations,
we hypothesize that increased Ca intake may prevent bone impairment
in Mg-deficient rats. However, few studies have examined the
relationship between Ca intake and bone metabolism in Mg-deficient
rats. Accordingly, this study examined the effects of high Ca
intake on bone metabolism in Mg-deficient rats.
Materials and methods
Animals and diets
Experimental animals were 4-week-old male Wistar rats obtained from
Clea Japan (Tokyo, Japan). The rats were housed in individual
stainless-steel wire-mesh cages. During the experiment, cages were
located in a room with controlled lighting under a 12-h light:dark
cycle (light, 0800-2000h), a temperature of 22 ± 1°C and relative
humidity of 60-65%. The study protocols were approved by the Animal
Use Committee at Tokyo University of Agriculture, and animals were
maintained in accordance with the university’s guidelines for the
care and use of laboratory animals.
The compositions of the experimental diets are shown in
table 1( Table 1 ).
Experimental diets were based on an AIN-93G diet [14]. Magnesium
oxide was excluded from the AIN-93G mineral mix in the two
Mg-deficient diets. Mg and Ca concentrations in the experimental
diets were as follows: control diet, 0.05% Mg and 0.5% Ca;
Mg-deficient diet, Mg-free and 0.5% Ca; Mg-deficient
Ca-supplemented diet, Mg-free and 2.0% Ca. The Mg and Ca
concentrations as measured from an analysis of the experimental
diets is shown in table 1. All experimental diets were stored
at 4°C until used.
Table 1 Composition of experimental diets.
|
Control diet
|
Mg-deficient diet
|
- Mg-deficient
- Ca-supplemented diet
|
|
Ingredient (g/kg of diet)
|
|
Cornstarch
|
528.657
|
529.486
|
492.028
|
|
Casein
|
200.0
|
200.0
|
200.0
|
|
Sucrose
|
100.0
|
100.0
|
100.0
|
|
Soybeanoil
|
70.0
|
70.0
|
70.0
|
|
Cellulosepowder
|
50.0
|
50.0
|
50.0
|
|
Mineralmixa
|
35.0
|
35.0
|
35.0
|
|
Vitaminmixb
|
10.0
|
10.0
|
10.0
|
|
L-Cystine
|
3.0
|
3.0
|
3.0
|
|
Cholinebitartrate
|
2.5
|
2.5
|
2.5
|
|
Tert-butylhydroquinone
|
0.014
|
0.014
|
0.014
|
|
MgO
|
0.829
|
–
|
–
|
|
CaCO3
|
–
|
–
|
37.458
|
|
Chemical analysis (%)
|
|
Mg
|
0.047
|
0.004
|
0.004
|
|
Ca
|
0.49
|
0.48
|
1.94
|
aThe mineral mix is a modification of the AIN-93G
mineral mix without magnesium oxide.
bAIN-93 vitamin mix.
Experimental design
Before the study period began, there was a 7-d acclimatization
period during which all rats were given free access to the control
diet and demineralized water. After the acclimatization period,
rats were divided into three groups of 6 rats with each group
having a similar mean body weight. One of the experimental diets
was assigned to each group and rats were given free access to the
assigned experimental diet as well as demineralized water
throughout the experimental period. Body weight and food intake
were recorded daily. From days 10 to 13 of the experiment, rats
were housed individually in stainless-steel metabolic cages, and
feces were collected from each rat. Subsequently, urine was
collected for 24h from each rat. At the end of the 14-d
experimental period, all rats were killed by exsanguination from
the carotid artery. Blood was collected in tubes at the time of
exsanguination, and was centrifuged to obtain serum. The femur was
removed and cleaned of the muscles, and connective tissues were
discarded. Samples were stored at -40°C until needed for analysis.
Chemical analysis
Samples of the experimental diets, feces and femur were ashed at
550 °C for 48h in a muffle furnace, and minerals were
extracted in 1 mol/L of HCl for analysis. Ca and Mg were determined
by atomic absorption spectrometry (Hitachi A-2000) [15].
Osteocalcin in serum was measured with an Osteocalcin rat ELISA
system (Amersham Biosciences K.K., Tokyo, Japan). Deoxypyridinoline
in urine was measured with a Pyrinks-D (Quidel Corp., USA).
Creatinine in urine was measured with a Creatinine-Test Wako (Wako
Pure Chemical Industries, Osaka, Japan). The apparent absorption of
minerals was calculated as the intake–fecal excretion.
Statistical analysis
Data are expressed as mean values with SD. Data were analyzed by
one-way ANOVA. Tukey’s test was used to evaluate the significance
of differences in multiple comparisons among groups, with
differences being considered significant at p < 0.05. All
statistical analyses were performed using the SPSS package program
Ver. 11.0 J.
Results
Body weight and food intake
Final body weight and food intake were significantly lower in the
two Mg-deficient groups than in the control group, and were
significantly lower in the Mg-deficient Ca-supplemented group than
in the Mg-deficient group (table 2)( Table
2 ).
Table 2 Body weight and food intake in the control,
Mg-deficient or Mg-deficient Ca-supplemented groupsd.
|
Control group
|
Mg-deficient group
|
- Mg-deficient
- Ca-supplemented group
|
|
Initial body weight (g)
|
80.5 ± 3.1
|
80.6 ± 2.3
|
80.6 ± 2.3
|
|
Final body weight (g)
|
185.6 ± 5.7a
|
137.7 ± 3.0b
|
117.5 ± 3.3c
|
|
Food intake (g/d)
|
14.7 ± 0.5a
|
11.3 ± 0.2b
|
10.1 ± 0.5c
|
a,b,cValues with different superscript letters are
significantly different (p < 0.05).
dValues are means ± SD, n = 6 per group.
Femoral mineral content and biochemical markers of bone
turnover
Femoral dry weight was significantly lower in the Mg-deficient
Ca-supplemented group than in the other two groups (table 3)(
Table 3 ). Femoral Ca content was
significantly lower in the Mg-deficient Ca-supplemented group than
in the other two groups. Femoral Mg content was significantly lower
in the two Mg-deficient groups than in the control group. Femoral
Mg content also was significantly lower in the Mg-deficient
Ca-supplemented group than in the Mg-deficient group. Serum
osteocalcin levels were significantly lower in the Mg-deficient
group and Mg-deficient Ca-supplemented group than in the control
group. Urinary deoxypyridinoline excretion was significantly higher
in the Mg-deficient Ca-supplemented group than in the control group
and Mg-deficient group.
Table 3 Femoral mineral content, serum osteocalcin
levels and urinary deoxypyridinoline excretion in the control,
Mg-deficient or Mg-deficient Ca-supplemented groupsd.
|
Control group
|
Mg-deficient group
|
- Mg-deficient
- Ca-supplemented group
|
|
Femur
|
|
|
|
|
Dry weight (g)
|
0.243 ± 0.010a
|
0.246 ± 0.011a
|
0.220 ± 0.012b
|
|
Ca (mg/g dry weight)
|
192.9 ± 9.1a
|
194.3 ± 4.6a
|
178.4 ± 5.6b
|
|
Mg (mg/g dry weight)
|
3.91 ± 0.12a
|
1.23 ± 0.09b
|
1.01 ± 0.06c
|
|
Osteocalcin in serum (ng/mL)
|
142.2 ± 12.8a
|
83.8 ± 13.9b
|
71.0 ± 16.0b
|
|
Deoxypyridinoline in urine (μmol/mmol creatinine)
|
0.76 ± 0.16a
|
0.81 ± 0.15a
|
1.05 ± 0.14b
|
a,b,cValues with different superscript letters are
significantly different (p < 0.05).
dValues are means ± SD, n = 6 per group.
Apparent mineral absorption and serum mineral levels
Apparent Ca absorption was significantly higher in the Mg-deficient
Ca-supplemented group than in the other two groups (table 4)(
Table 4 ). Apparent Mg absorption was
significantly lower in the two Mg-deficient groups than in the
control group, and that was significantly lower in the Mg-deficient
Ca-supplemented group than in the Mg-deficient group. Serum Ca
levels were significantly higher in the two Mg-deficient groups
than in the control group, and the levels were significantly higher
in the Mg-deficient Ca-supplemented group than in the Mg-deficient
group. Serum Mg levels were significantly lower in the two
Mg-deficient groups than in the control group, and the Mg levels
were significantly lower in the Mg-deficient Ca-supplemented group
than in the Mg-deficient group.
Table 4 Apparent mineral absorption and serum mineral
levels in the control, Mg-deficient or Mg-deficient Ca-supplemented
groupsd.
|
Control group
|
Mg-deficient group
|
- Mg-deficient
- Ca-supplemented group
|
|
Apparent absorption
|
|
Ca (mg/d)
|
60.2 ± 6.1a
|
47.1 ± 6.8a
|
83.0 ± 14.7b
|
|
Mg (mg/d)
|
6.17 ± 0.36a
|
0.31 ± 0.04b
|
0.14 ± 0.02c
|
|
Serum
|
|
Ca (mg/dL)
|
11.3 ± 0.2a
|
12.0 ± 0.3b
|
12.8 ± 0.7c
|
|
Mg (mg/dL)
|
2.07 ± 0.12a
|
0.46 ± 0.03b
|
0.34 ± 0.05c
|
a,b,cValues with different superscript letters are
significantly different (p < 0.05).
dValues are means ± SD, n = 6 per group.
Discussion
Mg deficiency reduced final body weight in the present study. This
finding may relate to food intake. In the present study, the rats
were given free access to the experimental diet, and consequently
food intake was decreased in rats fed the Mg-deficient diet. On the
other hand, it has been reported that despite the pair-feeding
method used, body weight was decreased in rats fed the Mg-deficient
diet [16, 17]. From the results of the present study and previous
studies, we speculate that reduced body weight in rats fed the
Mg-deficient diet is not attributable to low food intake only. It
is also suggested that body weight in rats fed the Mg-deficient
diet may be influenced by Mg consumption rather than food
consumption. Furthermore, with regard to the effects of
pair-feeding on bone Ca content, rats, which were treated by ad
libitum or pair-feeding with an Mg-restricted diet for 3 weeks,
showed a similar femoral Ca content between the ad libitum group
and a pair-fed group [16]. This finding indicates that pair-feeding
has no effect on femoral Ca content. Therefore, we believe that
pair-feeding did not need to be done in the present study.
Probably, the femoral Ca content in Mg deficiency would be
unchanged, in spite of the pair-feeding method being used in the
present study.
It has been reported that bone Ca content in rats fed
Mg-deficient diets was unchanged, while bone Mg content was
decreased by a Mg-deficient diet [18-20]. In the present study,
although the Mg-deficient diet had no effects on femoral Ca
content, femoral Mg content was reduced in rats fed the
Mg-deficient diet. The present study also found that femoral Mg
content was lower in the Mg-deficient Ca-supplemented group than in
the control group and Mg-deficient group. Reduction of femoral Mg
content by the Mg-deficient Ca-supplemented diet may be related to
Mg absorption in the intestine. We observed that apparent Mg
absorption was lower in rats fed the Mg-deficient Ca-supplemented
diet than in rats fed the control diet and Mg-deficient diets. A
previous study has reported that a high Ca intake induced a
decrease in apparent Mg absorption [21], and indicated that high Ca
intake has an inhibitory effect on Mg absorption. In other words,
we suggest that a Mg-deficient Ca-supplemented diet-induced
reduction of femoral Mg content is due to a decrease in apparent Mg
absorption by dietary Ca supplementation.
Bone Ca content was not affected by the Mg-deficient diet
[18-20], however femoral Ca content in the Mg-deficient
Ca-supplemented group was decreased in the present study. It was
very interesting that despite the general belief that bone Ca
content is enhanced by a high Ca intake, dietary Ca supplementation
reduced femoral Ca content in rats fed the Mg-deficient diet. The
mechanism responsible for the decreased femoral Ca contents in
Mg-deficient Ca-supplemented group, cannot be ascertained from the
results of the present study. However, the present study observed
that apparent absorption and serum levels of Mg in the Mg-deficient
Ca-supplemented group were lower than in the Mg-deficient group,
and indicated that Mg availability was reduced by high Ca intake.
We therefore suggest that reduction in Mg availability may, at
least in part, account for the reduction of femur Ca contents in
Mg-deficient rats by high Ca intake, since Mg plays an important
role in bone growth. On the other hand, Creedon and Cashman [8]
found that dietary Ca supplementation (4 times the normal level)
did not enhance bone Ca content, and concluded that increasing
dietary Ca intake above the recommended level had no effect on bone
mineral composition. Dietary Ca concentration in their experiment
was similar to the Mg-deficient Ca-supplemented diet in the present
study.
With regard to the effects of Mg deficiency on bone formation
and bone resorption, Mg deficiency induces a decrease in serum
osteocalcin levels [4]. Rude et al. [20] found that the osteoblast
number of Mg-depleted rats was lower than that of control rats. We
observed that serum osteocalcin levels were decreased in rats fed a
Mg-deficient diet. The osteoclast number was increased in
Mg-depleted rats, as measured by bone histomorphometry, and
indicates that Mg deficiency also enhanced the bone resorption rate
[19, 20]. These findings suggest that Mg deficiency decreases bone
formation rate and increases bone resorption rate, and that these
effects are the major causes of impaired bone growth of
Mg-deficient rats. On the other hand, the present study observed
that although there was no difference in serum osteocalcin levels
between the Mg-deficient group and Mg-deficient Ca-supplemented
group, urinary deoxypyridinoline excretion was higher in the
Mg-deficient Ca-supplemented group than in the Mg-deficient group.
This finding suggests that high Ca intake stimulates the bone
resorption rate of Mg-deficient rats, but has no effect on bone
formation rate. The increased urinary deoxypyridinoline excretion
in the present study may be related to Mg availability. Our
observation of reduced Mg availability in the Mg-deficient
Ca-supplemented group suggests that increased urinary
deoxypyridinoline excretion in the present study is due to the
reduction of Mg availability. Furthermore, we suggest that the
decrease in femoral Ca content in the Mg-deficient Ca-supplemented
group was due to increased bone resorption. In other words, in rats
fed a Mg-deficient diet, the high Ca intake reduced in vivo Mg
availability, thus elevating bone resorption. Subsequently, a
greater decrease in femoral Ca content was observed in the
Mg-deficient Ca-supplemented group than in the Mg-deficient
group.
The details of mechanisms for the changes in bone formation rate
and bone resorption rate by Mg deficiency are still unclear.
However, parathyroid hormone (PTH) and 1,25(OH)2-vitamin
D are important factors in bone formation, since both hormones
stimulate osteoblast activity and synthesis of procollagen and
osteocalcin. Serum PTH and 1,25(OH)2-vitamin D levels
are decreased in rats fed the Mg-deficient diet, and it is
suggested that the decrease in these hormones may contribute to
inhibit bone formation in Mg-deficient rats [17, 20]. Mg deficiency
induces increasing substance P and tumor necrosis factor-α (TNF-α)
[17, 22]. Increases in osteoclast activity and bone resorption in
Mg deficiency may be due to increased substance P and TNF-α
[17].
Conclusion
The effects of high Ca intake on bone metabolism in Mg-deficient
rats were investigated. The rats were fed a control diet (control
group), a Mg-deficient diet (Mg-deficient group) or a Mg-deficient
Ca-supplemented diet (Mg-deficient Ca-supplemented group) for 14 d.
Femoral Ca content in the Mg-deficient group was not changed,
however femoral Ca content in the Mg-deficient Ca-supplemented
group was decreased. Femoral Mg content was decreased in the
Mg-deficient group and Mg-deficient Ca-supplemented group.
Furthermore, femoral Mg content was lower in the Mg-deficient
Ca-supplemented group than in the Mg-deficient group. Serum
osteocalcin levels (a biochemical marker of bone formation) were
decreased in the Mg-deficient group and Mg-deficient
Ca-supplemented group. Urinary deoxypyridinoline excretion (a
biochemical marker of bone resorption) was increased in the
Mg-deficient Ca-supplemented group. These results suggest that a
high Ca intake had no preventive effect on alteration of bone
metabolism in Mg-deficient rats.
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