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,**,
*
National Institute for Food and Nutrition Research, 00178 Rome, Italy; Departments of
Medical Physiopathology and
**
Histology Medical Embryology, University "La Sapienza," 00161 Rome, Italy;
Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy and
Laboratory of Pediatric Endocrinology, University "Tor Vergata," 00133 Rome, Italy
2To whom correspondence should be addressed at National Institute for Food and Nutrition Research, via Ardeatina 546, 00178 Rome, Italy. E-mail: F.Branca{at}agora.stm.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • zinc deficiency • rats • histomorphometry • growth cartilage
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Zinc is the most abundant trace element in bone, being present at a
concentration of up to 300 µg/g (Grynpas et al. 1987
),
and it has been considered an important factor in bone metabolism. Not
surprisingly, skeletal changes, including delayed maturation
(Sandstead et al. 1967
), reduced alkaline phosphatase
activity (Oner et al. 1984
), reduced premenopausal bone
mass (Angus et al. 1988
) and postmenopausal osteoporosis
(Hertzberg et al. 1990
), have been associated with zinc
deficiency. Follis and his collaborators (1941)
demonstrated a decreased thickness of epiphyseal cartilage in long
bones of zinc-deficient rats; long bones were smaller and had
reduced zinc content. A similar impairment of the chondrocyte activity
at the level of the growth plate was described by Wallwork and Sandstead (1990
). A reduction in bone trabeculae has been
described in many experimental models of dietary induced zinc
deficiency (Angus et al. 1988
, Coble et al. 1971
, Eberle et al. 1999
, Hurley et al. 1969
, Murray and Messer 1984
, Oner et al. 1984
). Zinc deficiency has also been reported to affect both
the biosynthesis and degradation of all types of collagen
(Bremner et al. 1995
, McClain et al. 1973
, Wallwork and Sandstead 1990
). A role of
zinc in bone metabolism is also shown by its stimulatory effect on bone
formation in tissue culture (Hurley et al. 1969
), bone
growth and mineralization in weaning rats (Becker and Hoekstra 1966
) and potent inhibitory effect in vitro on bone resorption
(Holloway et al. 1996
, Moonga and Dempster 1995
). However, the specific effects of zinc deficiency on
skeletal metabolism and turnover have not been shown in vivo. We thus
compared rats fed a very low zinc diet with rats fed a normal diet
(adequate intake of zinc) and with rats fed a normal diet in the same
quantity as that fed to the zinc-deficient rats. This experimental
design allows discrimination between the effects of zinc deficiency and
those due to inanition. We hypothesized that zinc deficiency is
responsible for a reduction in bone mass as a result of both impaired
bone growth and increased bone resorption.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was conducted on 30 male Sprague-Dawley albino rats
(Charles River, Como, Italy) soon after weaning (21 d). The care and
use of the rats strictly followed "The Guiding Principles for the
Care and Use of Animals," in accordance with the principles of the
Declaration of Helsinki. The rats were divided into three groups by
individual weight-matching. The first group (n
= 10) had free access to a purified diet (Altromin-Rieper, Trento,
Italy) containing up to 1 mg zinc/kg [zinc deficient
(ZD)3
]. The second group had free access to a diet containing 50 mg zinc/kg but
otherwise was similar to that fed the ZD group [ad libitum (AL)]. The
pair-fed (PF) group was also fed a diet containing 50 mg zinc/kg
but were offered the same quantity consumed by the ZD group. The
composition of each diet is based on the American Institute of
Nutrition 76A formulation (American Institute of Nutrition 1977
) and is shown in Table 1
. The rats were housed individually in stainless steel cages and
maintained at 23°C with a 12-h light/dark cycle. They were allowed
free access to double-distilled water. Food intake and body weight
were measured at the start of the experiment, every 72 h and at
the end of the experiment. Features of zinc deficiency, including
cutaneous lesions and alopecia, were observed in all ZD rats. Except
for one ZD rat that died 1 d before the end of the experimental
period, all of the rats were killed by cardiac puncture after
anesthesia with sodic Tiopenthal at age 48 d. Blood was
removed directly from the heart. Long bones of the limbs were excised
and, after removal of the adherent soft tissues and measurement of the
length and weight, used for different tests (see later). The care, use
and killing of the rats were in accordance with the institutional
guidelines.
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The mechanical properties of the humeri were measured with
dynametrically with an Instron UTM instrument (model
1140; Instron Ltd., Buckinghamshire, U.K.) by a flexural test
(Baker and Haugh 1979
). A three-point bending device
was used that was adapted for the small dimensions of the bones tested
and consisted of two beams spaced a known distance apart, supporting
the lower bone. In this procedure, another beam is brought down from
above to contact the bone equidistant from either support beams. A
downward movement of the top beam is continued, causing a degree of
deformation of the bone before it breaks. The break stress and the
modulus of deformability were the parameters measured. The use of these
parameters gives a correction for differences in the bone geometry.
Endocrine determinations in serum.
Insulin-like growth factor (IGF)-I was determined by radioimmunoassay
(Nichols Institute, San Juan Capistrano, CA) after
acid-ethanol extraction, with a sensitivity limit of 1.73 nmol/L.
Insulin was measured by radioimmunoassay (Diagnostic Products
Corporation, Los Angeles, CA), and the sensitivity limit was 10.8
pmol/L. Western blot analysis of IGF-binding proteins (IGFBP) was
performed according to a previously reported procedure
(Cianfarani et al. 1996
). Briefly, after the addition of
nonreducing sodium dodecyl sulfate sample buffer, serum samples (3
µl) were processed by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (12.5% gel) and electroblotted, after an incubation
step with [125I]IGF-I (Amersham, Little Chalfont, U.K.).
The radiolabeled IGFBP were visualized by autoradiography.
Densitometric analysis of bands was performed using a Bio-Rad GS
700 imaging densitometer (Richmond, CA).
Bone histology and histomorphometry.
The left femora were fixed in 4% formaldehyde, buffered at pH 7.2 with
0.1 mol phosphate buffer/L. The distal third was then longitudinally
halved dehydrated in acetone and embedded undecalcified in
glycol-methacrylate at a low temperature (Bianco et al. 1988
). Sections were obtained with a Reichert Jung Autocut
microtome equipped with a tungsten carbide knife. For histology, four
2-µm-thick sections were taken at intervals of 
20 µm and stained
with May-Grunwald-Giemsa or Azur II–methylene blue. Other sections
were treated to reveal osteoclasts through a cytochemical reaction for
tartrate-resistant acid phosphatase activity as previously
described (Bianco et al. 1988
). Morphometric analysis
was performed with a semiautomatic image analyzer (IAS 2000; Delta System, Rome, Italy). Different variables, for which nomenclature and
abbreviations were as previously established (Parfitt et al. 1987
), were measured with a x10 objective. To determine
whether cartilaginous growth plate activity and longitudinal bone
growth were altered, the growth plate width (distance in µm
separating the epiphyseal and metaphyseal growth plate junctions) and
the hypertrophic zone width (distance in µm separating the lower
limit of the proliferative zone from the upper limit of the
mineralizing zone) were established with the measurement of five
equally spaced distances (Liu and Kalu 1990
). Bone
volume (percent of whole tissue occupied by bone trabeculae) was
measured in the substantia spongiosa ossium of the distal
metaphysis, within a rectangular zone (0.878 mm high and 2.155 mm wide;
total area, 1.1892 mm2), 1 mm from the central point of the
plate metaphyseal junction. Bone resorption was established with
measurement of the osteoclast surface/bone surface (percent of
trabecular bone surface covered by osteoclasts) and osteoclast
number/bone surface (number of osteoclasts/mm2 of
trabecular surface) and evaluation of tartrate-resistant acid
phosphatase–positive cells in a less extended (0.878 mm high and 0.967
mm wide; total area, 0.849 mm2) and more central zone.
Collagen, calcium and zinc concentrations in bone.
Collagen, calcium and zinc concentrations were determined in bone
tissue hydrolysates. Bones obtained from right femora after completion
of the 7-d organ culture were washed in saline and freeze-dried.
Diaphyses were crushed, and 20-mg aliquots were weighed with an
analytical balance (±0.0001 g accuracy). Hydrolysates were prepared by
suspension in 1 mL HCl (12 mol/L) and treated for 22 h at 108°C.
Collagen was estimated from the hydroxyproline present in the tissue
hydrolysates, calculating 1 mol of collagen/300 mol hydroxyproline.
Hydroxyproline was measured colorimetrically according to the
chloramine-T method (Prockop and Undenfriend 1960
).
Calcium was analyzed by ion exchange chromatography with a
Dionex-Biolc Ion system equipped with a Dionex CS12 Ion Pac column
(cationic exchange) and a suppressed conductivity detector. Cations
were separated by an isocratic 20 mmol/L methane sulfonic mobile phase
at 1 mL/min flow rate. The chromatographic analysis was completed in 12
min. Zinc was determined by flame atomic absorption spectrometry
(Perkin-Elmer 5000). The absorbance was measured at 213.9 nm. The
calibration line for zinc and calcium was obtained with a BDH standard
solution (Milan, Italy). The precision and accuracy for the two methods
were determined using a certified sample (International Atomic Energy
Agency and National Bureau of Standards).
Statistical analysis.
Statistical analyses were performed with the program STATISTICA for Windows 4.5 StatSoft 1995. The results were reported as means ± SD. Comparisons between groups were performed by one-way ANOVA; post hoc comparison were carried out with Scheffé’s test. A probability level of 5% was used to establish significance of differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Energy protein, calcium and copper intakes were not significantly
different between ZD rats and PF rats but were significantly lower in
both groups compared with AL rats (Table 2
). In contrast, significant differences in zinc intake were observed in
all groups.
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Rats of the different groups had similar initial weights but grew at
different rates. The weight growth rate of the ZD rats (0.9 ± 0.3
g/d) was significantly slower than that of both PF (2.0 ± 0.4
g/d) and AL (8.0 ± 0.5 g/d) rats. The size of long bones was
significantly different among the three groups, with the lowest values
in ZD rats, intermediate values in PF rats and the greatest values in
AL rats (Table 3
).
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The humeri of ZD rats had a significantly greater "elasticity" than
the other groups; the lowest break stress and modulus deformability
values were observed in this group (Table 3)
.
Endocrine control of growth.
IGF-I levels were significantly lower in ZD rats than in AL and PF
rats, whereas insulin and IGFBP-3 levels were lower in ZD rats than in
AL rats but not different from ZD and PF rats (Fig. 1
). No significant differences in IGFBP-1 or -4 were observed among the
three groups of rats. Overall IGF-I levels were closely related to
weight gain (r = 0.78, P < 0.0001),
femur length (r = 0.80, P < 0.0001)
and weight (r = 0.78, P < 0.0001) and
tibia weight (r = 0.76, P < 0.0001).
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The cartilage maturation-bone formation sequence was normally
recognizable in all groups, even though chondrocyte serration and
columnization were minimally distorted in ZD rats. The overall width of
hypertrophic cartilage (Fig. 2A
) and the width of growth cartilage (Fig. 2D
)
were significantly different in all groups, with the lowest values in
ZD rats. Bone trabeculae were substantially reduced and bone volume was
significantly lower in the ZD and PF rats than in the AL rats in the
absence of a significant difference between ZD and PF rats (Fig. 2B
). Compared with the AL group, significantly greater
trabecular bone surface covered by osteoclasts (Fig. 2B
) and
osteoclasts/trabecular surface (Fig. 2C
) values were
observed in PF rats; in contrast, no significance different was
observed between ZD and AL rats.
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In femoral diaphysis, collagen concentrations (ZD rats, 3.25 ± 0.64 mmol/g; PF rats, 3.75 ± 0.53 mmol/g; AL rats, 3.24 ± 0.53 mmol/g) and calcium concentrations (ZD rats, 4.89 ± 0.22 mmol/g; PF rats, 4.79 ± 0.17 mmol/g; AL rats, 4.79 ± 0.12 mmol/g) did not different among the three groups. In contrast, zinc concentrations differed significantly among the three groups, with the lowest values in ZD rats (0.83 ± 0.21 µmol/g). The PF rats (3.81 ± 0.38 µmol/g) had a femoral diaphysis zinc level intermediate to those of the ZD and AL rats (4.65 ± 0.38 µmol/g).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our histological and histomorphometric data confirm the role of zinc in
bone growth and metabolism. As previous established (Angus et al. 1988
, Coble et al. 1971
, Eberle et al. 1999
, Hurley et al. 1969
, Murray and Messer 1984, Oner et al. 1984
), zinc deficiency
is associated with skeletal growth retardation and reduction in bone
mass. In our model, the morphometric abnormalities of the growth
plates, likely linked to the role of zinc in cell division,
differentiation and apoptosis (Lohmann and Beyersmann 1993
, Zalewski et al. 1991
), explain the
skeletal longitudinal growth retardation and the greater
"deformability" of the long bones in ZD rats than in the AL and PF
rats. The reduction in the circulating level of IGF-1 could represent
the mechanism by which growth plate activity is impaired in these rats;
this view is supported by the well known effects of IGF-1 on
proliferation and differentiation of many different cell types,
including chondrocytes and osteoblast-precursor cells
(Schmid et al. 1984
, van Wyk 1984
).
Previous studies noting changes in the growth plate as a result of zinc deficiency were somewhat flawed by the interference of general nutritional imbalances. Indeed, the specific role of zinc deficiency on the growth plate can be assessed only with an experimental design that includes the use of pair-fed animals. Using this approach, we obtained evidence for a specific effect of zinc deficiency on growth plate activity. The growth plate of ZD rats was significantly more atrophic than those of both PF and AL rats. In contrast, we observed an increase in bone resorption as an effect of inanition but did not demonstrate a specific effect of zinc deficiency on bone resorption. PF rats showed significantly increased histomorphometric parameters of bone resorption compared with controls. In view of the similar reduction in bone mass observed in both ZD and PF rats, we postulate that the same ultimate net bone mass is achieved in the two groups via different mechanisms. The low bone mass of ZD rats reflects essentially a severe reduction in bone growth, whereas the low bone mass of PF rats reflects a less severe reduction of bone growth coupled with an increase in bone remodeling. We conclude that in our model the specific effects of zinc deficiency on bone mass are essentially mediated by growth plate dysfunction leading to reduced bone growth, rather than by changes in bone remodeling per se. The potential underlying mechanism for growth plate failure is possibly the direct link between reduced IGF-1 production and quantitative changes in the growth plate structure.
Even though direct extrapolation of these results to humans likely is not valid, they support the hypothesis that insufficient zinc intake during growth may impair the achievement of an optimal skeletal mass and play a role as a risk factor in the development of osteoporosis. Indeed, a key risk factor for human osteoporosis (perhaps the most important one) is peak bone mass, which is achieved as a result of the overall osteogenic activity unfolding during skeletal growth. Any factor that leads to reduced anabolic activity in the skeleton during growth would result in a lower peak bone mass, in turn resulting in overt osteoporosis on interplay with additional determinants (e.g., hormonal imbalances). We have not been able to demonstrate an effect of zinc on bone mass via increased bone resorption. However, the specific role of zinc deficiency should be further clarified in experimental models in which other nutrients are provided in sufficient amounts, possibly by designing diets with higher nutrient contents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AL, group with free access (ad libitum consumption) to the zinc-adequate diet; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; PF, group that was pair-fed the zinc-adequate
diet; ZD, group with free access to zinc-deficient diet.
Manuscript received August 21, 2000. Initial review completed November 28, 2000. Revision accepted December 13, 2000.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. American Institute of Nutrition Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 1977;107:1340-1348
2. Angus R. M., Sambrook P. N., Pocock N. A., Eisman J. A. Dietary intake and bone mineral density. Bone Miner 1988;4:265-277[Medline]
3. Baker J. L., Haugh C. G. Mechanical properties of bone: a review. Trans. A.S.A.E. 1979;22:678-687
4. Becker W. M., Hoekstra W. G. Effect of vitamin D on Zn-65 absorption and turnover in rats. J. Nutr. 1966;90:301-309
5. Bianco P., Ballanti P., Bonucci E. Tartrate-resistant acid phosphatase activity in rat osteoblasts and osteocytes. Calcif. Tissue Int. 1988;43:167-171[Medline]
6. Bremner I., Beattie J. H. Copper and zinc metabolism in health and disease: speciation and interactions. Proc. Nutr. Soc. 1995;54:489-499[Medline]
7.
Cianfarani S., Germani D., Rossi P., Spagnoli A., Mercanti D. Do insulin-like growth factor binding proteins (IGFBPs) modulate the IGF-I growth promoting and differentiating effects in human neuroblastoma cells?. Eur. J. Endocrinol. 1996;135:716-723
8.
Coble Y. D., Jr, Bardin C. W., Ross G. T., Darby W. T. Studies of endocrine function in boys with retarded growth, delayed sexual maturation and zinc deficiency. J. Clin. Endocrinol. Metab. 1971;32:361-367
9. Eberle J., Schmidmayer S., Erben R. G., Stangassinger M., Roth H. P. Skeletal effects of zinc deficiency in growing rats. J. Trace Elem. Med. Biol. 1999;13:21-26[Medline]
10. Follis R. H., Day R. G., McCollum E. V. Histological studies of the tissues of rats fed a diet extremely low in zinc. J. Nutr. 1941;22:223-237
11. Grynpas M. D., Pritzker K.P.H., Hancock R. G. Neutron activation of bulk and selected trace elements in bone using a low flux SLOWPOKE reactor. Biol. Trace Elem. Res. 1987;13:333-344
12. Hertzberg M., Foldes J., Steinberg R., Menczel J. Zinc excretion in osteoporotic women. J. Bone Miner. Res. 1990;5:251-257[Medline]
13. Holloway W. R., Collier F. M., Herbst R. E., Hodge J. M., Nicholson G. C. Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number. Bone 1996;19:137-142[Medline]
14. Hurley L. S., Gowan J., Milhaud G. Calcium metabolism in manganese-deficient and zinc-deficient rats. Proc. Soc. Exp. Biol. Med. 1969;130:856-860[Medline]
15. Liu C. C., Kalu D. N. Human parathyroid hormone-(1–34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats. J. Bone Miner. Res. 1990;5:973-982[Medline]
16. Lohmann R. D., Beyersmann D. Cadmium and zinc mediated changes of the Ca2+-dependent endonuclease in apoptosis. Biochem. Biophys. Res. Commun. 1993;190:1097-1103[Medline]
17. McClain P. E., Wiley E. R., Beecher G. R., Anthony W. L., Hsu J. M. Influence of zinc deficiency on synthesis and cross-linking of rat skin collagen. Biochem. Biophys. Acta 1973;304:457-465[Medline]
18. Moonga B. S., Dempster D. W. Zinc is a potent inhibitor of osteoclastic bone resorption in vitro. J. Bone Miner. Res. 1995;10:453-457[Medline]
19. Murray E. J., Messer H. H. Turnover of bone zinc during normal and accelerated bone loss in rats. J. Nutr. 1981;111:1641-1647
20. National Research Council NCR Nutrient Requirements of Laboratory Animals 3rd ed. 1985 National Academy of Sciences Washington, D.C.
21.
Oner G., Bhaumick B., Bala R. M. Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats. Endocrinology 1984;114:1860-1863
22. Parfitt A. M., Drezner M. K., Glorieux F. H., Kanis J. A., Malluche H., Meunier P. J., Ott S. M., Recker R. R. Bone histomorphometry: standardization of nomenclature, symbols, and units: report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res. 1987;2:595-610[Medline]
23. Prockop D. J., Udenfriend S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal. Biochem. 1960;1:228[Medline]
24. Sandstead H. H., Prasad A. S., Schulert A. S., Farid Z., Miale A., Bassilly S., Darby W. J. Human zinc deficiency, endocrine manifestations and response to treatment. Am. J. Clin. Nutr. 1967;20:422-442[Abstract]
25. Schmid C., Steiner T., Froesch E. R. Insulin-like growth factor-1 supports differentiation of cultured osteoblast-like cells. FEBS Lett 1984;173:48-51[Medline]
26. van Wyk J. J. The somatomedins: biological actions and physiological control mechanisms. Li C. H. eds. Hormonal Proteins and Peptides 1984:81-125 Academic Press New York, NY.
27. Wallwork J. C., Sandstead H. H. Zinc. Simmons D. J. eds. Nutrition and Bone Development 1990 Oxford University Press New York, NY.
28. Zalewski P. D., Forbes I. J., Giannakis C. Physiological role for zinc in prevention of apoptosis (gene-directed death). Biochem. Int. 1991;24:1093-1101[Medline]
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