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2
Departments of
*
Pediatric Dentistry,
Pharmacology and
**
Oral Anatomy, School of Dentistry, Showa University, Shinagawa-ku 142-8555, Japan and
Bioscience Laboratories, Meiji Seika Kaisha, Ltd., Sakado 350-0289, Japan
2To 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: • calcium • magnesium • bone • fructooligosaccharides • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Magnesium is not only a critical ion in mammals as a cofactor for many
enzymes of the energy extraction system and protein synthesis pathways,
but it is also necessary for bone formation (Rude 1996
).
Thus, Mg might also play an important role in bone structure or the
hardness of bone.
The stimulatory effects of fructooligosaccharides
(FOS),3
which are low-molecular-weight indigestible sugars, on intestinal
calcium and magnesium absorption have been well studied
(Morohashi et al. 1998
, Ohta et al. 1993
, 1994b
, 1995
and 1998c
). The enhancement not only of apparent calcium
and magnesium absorptions, but also of the ratio of Ca to Mg
resulting from FOS consumption has been reported in growing rats
(Ohta et al. 1994a
, 1996
and 1997
). These findings would
lead us to expect an increase in bone mass in animals fed FOS. Previous
studies have relied on the mineral concentrations in ashed bone or bone
mineral density to investigate the effects of FOS on bone (Ohta et al. 1993
and 1998d
and 1998e
). However, these methods cannot
be used to examine bone structure or measure mineral concentrations in
a local area in rats fed a diet containing FOS. The aim of this
experiment was to examine whether the enhanced mineral absorption
resulting from FOS consumption affected femoral bone structure and
mineral concentration in bone, using histomorphometrical and X-ray
microanalysis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The powdered diets were mixed with an equal
amount of purified water. The respective wet diets were dried for
1 d (80°C) to calculate the wet/dry ratio, and calcium
concentrations in the dry diets were then measured to determine the
amount supplied in the wet diet in each group for pair-feeding of
calcium (Morohashi et al. 1998
). The control and FOS
diets contained 5.21 and 5.18 g/kg dry diet of calcium. All of the rats
were fed a constant amount of calcium (95 mg/d) in their respective
diets throughout the experiment, beginning when they were 45 d
old. These respective wet diets corresponded to either 18.23 g dry diet
in the control group or 18.34 g in the FOS group. A mineral metabolic
study was performed to determine apparent calcium and magnesium
absorptions in the intestine. This study was performed 4 d before
killing (at 60 d of age) over a 3-d period. Feces were harvested
on a sheet of decalcified filter paper, ashed (640°C, 3 d) and
then dissolved in 2.0 mol/L HCl. Final body weight and food consumption
during the metabolic study were recorded. The calcium and magnesium in
feces were determined using an atomic absorption spectrophotometer
(Perkin Elmer, Norwalk, CT). Apparent intestinal calcium and magnesium
absorptions were calculated. The treatment of the experimental animals
was approved by the Experimental Animal Committee of Showa University.
FOS consisted of 34% 1-ketose, 53% nystose and 10%
1F-b-fructofuranosyl nystose (Meioligo-P, Meiji Seika Kaisha, Tokyo,
Japan). FOS was manufactured from sucrose using fructosyltransferase
(Hidaka et al. 1988
). Other dietary components were
purchased from Oriental Yeast (Tokyo, Japan). All other reagents were
of analytical grade and purchased from Wako Pure Chemical (Tokyo,
Japan).
After rats were killed, the right femur from each rat was removed
immediately and fixed in 70% ethanol. The bones were embedded in
polyester resin (Rigolac, Nissin EM, Tokyo, Japan). The middle of the
diaphysis and neck of the femoral head were cross-sectioned, and
the distal metaphysis was sagittal-sectioned. These sections were
polished with alumina particles on a polishing cloth. Backscattered
electron images were taken and X-ray microanalysis was conducted
with a scanning electron microscope (S-2500CX, Hitachi, Tokyo, Japan)
to calculate morphometrical variables and to measure mineral
concentrations in local bone areas (Roschger et al. 1995
). Morphometrical variables were defined as follows:
1) % BV, percentage of bone volume in the diaphysis and
femoral neck, and 2) % TBV, percentage of trabecular bone
volume within a 3.7-mm wide window on the secondary spongiosa, 1.0 and
1.9 mm from the centered epiphyseal growth plate. The mineral
concentrations (Ca, Mg, P) were measured at five random points and mean
values were calculated. The scanning area (7.5 x 10
µm) was close to the surface of trabecular bone in the
metaphysis and femoral neck or an ossification of the endosteum in the
front-lateral diaphysis, inner basic lamellae. Results are
expressed as g/100 g.
Data are expressed as means and SD. Statistical analyses were performed using the SPSS statistical software package (SPSS version 6.0, SPSS, Chicago, IL). An unpaired Student’s t test was used to identify differences between the control and FOS groups. Pearson’s correlation coefficient was calculated to analyze the relationship between apparent calcium absorption and calcium concentration in the bone surface. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The fractional absorption of these minerals in the FOS group was also
significantly higher than that in the control group (Table 1)
. The
differences between groups in the minerals absorbed were 10 mg/d for
calcium and 1 mg/d for magnesium.
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). Either augmented trabecular bone or an expanded epiphyseal cartilage
plate is found when % TBV is enhanced (Morohashi et al. 1993
, Wronski et al. 1993
). However, in this
study, no abnormalities in bone structure were found in rats fed the
FOS diet (Fig. 1
).
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), were enhanced by FOS
consumption (Table 3
). Similar effects were found in other regions that were close to the
surface of trabecular bone. In addition, the calcium/phosphorus ratio
did not differ between groups. There was a significant relationship
between absorbed calcium in the intestine and calcium concentration in
bone (r = 0.722, P < 0.001), and a
similar relationship was found for magnesium (r = 0.720, P < 0.001).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Dietary FOS also stimulates magnesium absorption and enhances its
balance (Ohta et al. 1994a
, 1996
and 1997
). These
stimulatory effects of FOS have been explained by the fermentation of
FOS in the large intestine to produce short-chain fatty acids,
which in turn reduce luminal pH (Demigné et al. 1989
, Ohta et al. 1994a
, Schulz et al. 1993
, Younes et al. 1996
) and induce the
proliferation of epithelial cells (Sakata 1987
).
Moreover, FOS consumption markedly increases mucosal calbindin-D9k in
the large intestine (Ohta et al. 1998a
and 1998b
). In
this study, FOS had similar effects on mineral absorption. Considering
previous reports and our results, the extra calcium and magnesium
absorbed in the intestine as a result of FOS consumption is likely to
be retained in bone.
The spongiosa of the metaphysis in long bone consists of primary and
secondary parts. Most of the primary spongiosa is eventually converted
into secondary spongiosa by the simultaneous removal of woven bone and
calcified cartilage cores, and the addition of secondary bone. The
secondary spongiosa comprises a zone of slow tissue turnover, compared
with primary spongiosa, and there is a net loss of bone due to
osteoclastic resorption (Jee et al. 1997
). In this
experiment, we measured the volume of secondary spongiosa (% TBV) in
the metaphysis, which was slightly but significantly enhanced in rats
fed FOS. We also observed an enhancement of bone volume in the femoral
neck containing cortex and marrow trabeculae. An increased % TBV is
usually observed in rats when bone resorption is therapeutically or
toxically inhibited by substances such as bisphosphonates or strontium
(Morohashi et al. 1993
, Mühlbauer et al. 1991
). In addition, the stimulated intestinal calcium
absorption in rats fed various doses of calcium is inversely
proportional to bone resorption without any change in bone formation
(Yamada 1994
). Although there was no evidence of bone
turnover in this study, it is likely that bone resorption is slightly
suppressed by the enhancement of calcium absorption that results from
FOS consumption. However, we cannot exclude the possibility that FOS
consumption enhances bone formation. In either case, we can conclude
that FOS consumption influences local bone structure.
An interaction between calcium and magnesium has been reported.
Briefly, reduced magnesium absorption occurs due to a high calcium
intake (Watkins et al. 1992
). In addition, calcium
suppresses magnesium solubility in the ileal lumen and lowers magnesium
absorption in vitro (Brink et al. 1992
). However, in
this study, calcium and magnesium absorptions were enhanced
simultaneously in FOS-fed rats. Similar effects on mineral
absorption have been reported by other investigators (Ohta et al. 1994a
). Considering the effect of FOS on mineral
absorption, we would expect that calcium and magnesium are used for
calcification. In fact, the weight percents of these minerals were
enhanced, as calculated from a small area (7.5 x 10
µm) on the cortex or trabecular bone. Calcium in bone is
usually characterized as hydroxyapatite
[(Ca10(PO4)6(OH)2)]
(Heaney 1996
). Magnesium has been shown to bind to the
surface of hydroxyapatite crystals and to retard the nucleation and
growth of hydroxyapatite in vitro (Bigi et al. 1992
). In
fact, rats fed excess magnesium have smaller mineral crystals in their
bone than control, pair-fed rats. In contrast, the hydroxyapatite
crystals in magnesium-deficient rats are significantly augmented
(Burnell et al. 1986
, Boskey et al. 1992
). Thus, the enhanced weight percents of calcium and
magnesium might be associated with hydroxyapatite crystal size. On the
other hand, Fountos et al. (1999)
suggested that in vivo
measurements of the calcium/phosphorus ratio of bone may be useful for
assessing skeletal aging or some bone diseases. However, in this study,
there were no differences in this ratio in the regions examined. Thus,
FOS consumption might slightly enhance mineral concentrations under
physiologic conditions.
In summary, this is the first report to address the effect of FOS on
bone structure and local mineral concentrations in growing rats. The
loss of both cortical and trabecular bone is believed to contribute to
decreased bone strength (Søgaard et al. 1994
). In
particular, the femoral neck is thought to be an important site for
osteoporotic bone loss in humans (Boyce and Bloebaum 1993
). Peak bone mass in humans is achieved after sexual
maturity and is then maintained for two decades. Thereafter, the mass
of virtually all bones declines until death. Thus, it has been
established that calcium deposition in bone in the growing stage
contributes to the prevention of age-related bone diseases. The
observed effects of FOS, i.e., enhanced calcium and magnesium
absorptions, might be associated in part with local bone structure and
the mineral density without any changes in the skeletal
calcium/phosphorus ratio because a significant relationship was found
between absorbed minerals and mineral concentrations in bone in this
experiment (Fig. 2
). If similar effects are found in humans, treatment with FOS might help
improve bone structure and mineral concentrations in various femoral
osteopenias. Unfortunately, it is not yet clear that FOS consumption
enhances bone formation or suppresses bone resorption, or both, at the
cellular level. Further studies are required to evaluate the effects on
bone turnover.
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FOOTNOTES |
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3 Abbreviation used: BV, bone volume; FOS, fructooligosaccharides; TBV, trabecular bone volume.
Manuscript received December 2, 1999. Revision accepted March 22, 2000.
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