Int. J. Exp. Path. (1990) 7I, 63I-638
Rickets induced by calcium or phosphate depletion Salem Abugassa* and Olle Svenssont Departments of *General Surgery, tOrthopaedics and Research Center, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden
Received for publication I4 November I989 Accepted for publication 3 April I990
Summary. We studied the effects of calciopenia and phosphopenia on longitudinal growth, skeletal mineralization, and development of rickets in young Sprague-Dawley rats. At an age of 2I days, two experimental groups were given diets containing 0.02% calcium or 0.02% phosphorus; otherwise the diets were nutritionally adequate. After 7, I4, and 2I days, five animals from each group were randomly chosen. The animals were anaesthetized and blood samples were drawn for analysis of calcium, phosphorus, and immunoreactive parathyroid hormone, whereupon the animals were killed. Length, weight, and specific weight of the left femur were measured. After 28 days on the respective diet the remaining animals were killed and one proximal tibia from each animal was processed for light microscopy and subjected to stereological analysis. Both experimental groups developed progressive growth retardation, more so the phosphate-depleted group. The calciopenic animals developed severe hypocalcaemia and secondary hyperparathyroidism, whereas the phosphate-depleted animals, in spite of marked secondary hypercalcaemia, had unaltered levels of immunoreactive parathyroid hormone. By 28 days both experimental groups displayed rachitic changes, more pronounced in the phosphate-depleted animals. This paper provides quantitative data demonstrating that calciopenia per se may cause rickets in young rats, but that the rachitic changes in this condition are less severe, and the growth pattern different from those in phosphate depletion. Keywords: Sprague-Dawley rats, phosphate depletion, calcium depletion, rickets
Rickets primarily affects the skeleton and is usually manifested during periods of rapid growth. The clinical signs include growth retardation, enlargement of the metaphyseal regions, and various deformities associated with decreased formation of bone and deficient skeletal mineralization. Rickets also causes an increase in the height of the physes, mainly because of an increase of its hypertrophic zone. At the lowest part of the physis there is also impairment of calcifica-
tion, deficient cartilage resorption and disorganized ingrowth of capillaries. In rats, rickets and rickets-like lesions can be induced by various agents: in addition to the classical low-phosphate, vitamin D deficiency rickets, rickets-like conditions can be induced by the administration of various metal ions and agents like diphosphonates (Follis I958; Reinholt et al. i984b). Experimentally induced rickets-like conditions have been extensively used to study the
Correspondence: Dr Salem Abugassa, Department of Surgery, Huddinge University Hospital, S-I4I 86 Huddinge, Sweden. 63I
632
S. Abugassa & 0. Svensson
complex process of mineralization. However, there are still divergent opinions as to whether calcium depletion per se causes rickets (Bisaz et al. 1975). One difficulty when interpreting such studies and assessing the degree of the rachitic changes in the physes, is their irregular shape and large biologic variability. In recent years however improved sampling techniques and stereological methods have made possible unbiased estimations of structures with great variability. In this paper we use a stereologic model to make a quantitative comparison between the rachitic effects of calcium depletion and phosphate depletion. Materials and methods
Animals At an age of 2 I days male Sprague-Dawley rats were randomly allocated into three groups. Controls were fed commercially available rat food containing 1.2% w/w Ca, I.0% w/w P, and I 500 IU vitamin D3 per kg (R3, Ewos AB, Sodertalje, Sweden). One experimental group was given a semi-synthetic diet containing 0.02% Ca, 0.72% P, and I5ooIUvitaminD3 perkg (R359, Ewos AB), and another group a diet containing 0.92% w/w Ca, 0.02% w/w P, and I500 IU vitamin D3 per kg (R358, Ewos AB); otherwise these diets were nutritionally adequate. The animals were kept in stainless steel cages under standard laboratory conditions with free access to food and distilled water.
Stereological design After 28 days on the respective diet the animals were given 2500 IU heparin (Lbvens AB, Malmo, Sweden) and anaesthetized by intraperitoneal injections of fentanylfluanison, 0.2-0.8 ml per kg body weight (Hypnorm, Leo, Helsingborg, Sweden). The animals were perfused transcardially as previously described (Svensson et al. I985): first with buffered Ringer's solution containing 2% Dextran T40 (Pharmacia, Uppsala, Sweden) and 0.2% procaine (Astra, Sbder-
talje, Sweden), pH 7.4, 370C, for 30 s, then with 2% glutaraldehyde in o.07 M cacodylate buffer containing 3% Dextran T40, 0.02% procaine, and 0.05 M sucrose, pH 7.4, at 3 70C for 5 min. The osmolarity of the perfusate was 320 mOsmol. The upper part of the tibia was dissected free and was cut longitudinally by hand into Io regions, as previously described (Reinholt I982). A random piece from each one of these regions was fixed overnight in the same fixative as above. After rinsing in buffer the pieces were dehydrated in graded ethanol and embedded in an epoxy resin (JB-4, BioRad). The blocks were coded and microscopy and reading of micrographs were performed blindly. For light microscopy, approximately I . -gum thick sections were cut from each block with a Sorvall Porter-Blum microtome. The sections were stained according to a slightly modified von Kossa procedure, using toluidine blue in water as counterstain. For estimation of volume density of each zone, one micrograph covering the four zones was taken from each section at a final magnification of x 525. The resting zone was defined as the zone between the upper border of the epiphyseal growth cartilage and the top of the first cell in the cell columns. The proliferative zone is the zone of flat chondrocytes, and the border between the proliferative and hypertrophic zones was drawn where the ratio between cell height and cell width is less than 2. The calcifying zone was defined as the zone between the mineralization front, as seen with the modified von Kossa procedure, and the top of the first opened lacuna (Engfeldt & Hjertquist I969). In the rachitic animals where the calcifying zone was virtually absent, the hypertrophic zone was divided into an upper (Hi) and a lower (H2) part; the border between these two was drawn at the level of the highest-reaching vascular pouches from the metaphysis. Physical parameters The femoral length was measured with a
Rickets induced by Ca or P depletion 5r micrometer; the bones were subsequently ashed in a furnace at 700°C for 24 h and the ash weight was recorded under constant conditions.
633 IP
q,
E 4 CE.0 3
Assays
0
From the anaesthetized animals blood samples were taken from the inferior caval vein. The samples were centrifuged to yield serum which was immediately frozen and kept at - 70°C until analysis. To reduce the effect of circadian fluctuations, all samples were taken between ogoo and I 500 h. Immunoreactive parathyroid hormone (iPTH) was measured by a radioimmunoassay (RatPTH-MM, INC, MN, USA) detecting the midpart of the molecule. The concentrations of total calcium and phosphate were determined by standard colorimetric methods (Gindler & King I972; Bartels & Roijers
E 0 c)
2
I
I0
, 2 3 Time (weeks)
4
Fig. 2. Serum calcium in 0, controls; *, phosphate and 0, calcium depletion.
300 r 0 .
1975).
200 o .C
-0
Results The experimental animals were subjected to severe deprivation of calcium and phosphate, respectively, during the period when normally they have their most rapid growth. This regimen resulted in growth retardation of both experimental groups (Fig. i). Their
C
C
o
0E
100 H
0
2 3 Time (weeks)
4
Fig. 3. Immunoreactive parathyroid hormone in 0, controls; *, phosphate and 0, calcium depletion. 30
28 E
-
071 C
-
somewhat
26 24 22
IT
curves
also
cor-
general condition, although at the end of the
c
0
diverging weight
respond to differences in general condition: the calcium-depleted rats had fairly good
20
I8
2
16 0
2
3
Time (weeks) Fig. I. Fe-moral growth in El, controls; phate ancI 0, calcium depletion.
'
4 4
experimental period they developed shabby fur. The phosphate-depleted group, on the other hand, displayed failure to thrive by about a week, and their condition deteriorated during the rest of the experimental period. Moreover, in both experimental groups
U,
phos-
longitudinal growth decelerated, but
it is interesting to note that growth completely abolished (Fig. i).
was
not
S. Abugassa & 0. Svensson The calcium-depleted animals developed a progressive hypocalcaemia, which at the end of the experimental period was severe (Fig. 2)
634 4 E -
a)
2
Q 0
0
£!
E CI)
I 0
I
X
2
3
I 4
Ti me (weeks) Fig. 4 Serum phosphate in 0, controls;
,
phospilate and 0, calcium depletion. 2C)o -
Discussion
0-
._
3
IC
)O
0
0
E
IL
,
TI0
JY
The calcium-depleted animals developed a secondary hyperparathyroidism, not sufficient however to maintain the serum calcium at a normal level. The present situation involved an unphysiological low-calcium stress, and it is known that the parathyroid gland has a limited capability to respond to prolonged stimuli (Cohn et al. I986). The of calcium from the skeleton , , I,mobilization 4 was monitored by femoral ash weight, and
3 2 Time (weeks) Fig. 5. Femoral ash weight in 0, controls; o, phospheate and calcium depletion (overlapping
o
and accompanied by a reciprocal increase of serum iPTH (Fig. 3). Phosphate deprivation resulted in successive decrement of the serum phosphate (Fig. 4) with a compensatory hypercalcaemia (Fig. 2); these animals, however, had unaltered levels of iPTH (Fig. 3). Figure 5 shows the femoral ash weight. The stereological findings are given in Table i. Both experimental groups thus developed rachitic changes, mainly due to an increased height of the hypertrophic zones (Figs 6-8).
values).
apparently there was no net calcium accretion during the experimental period (Fig. 5). It may seem puzzling that the phospho-
penic animals had unaltered levels of iPTH
Table i. Results of the stereological examination
Hypertrophic or Total height
(mm)
Group
Controls
382
Low phosphate 792 rickets Low calcium 5I4
Resting zone (Vv)t:
Proliferative zone (Vv)
(i6) 0.079 (0.004) 0.383 (O.OI2) (40)* 0.052 (0.007)* 0.298 (o.oI6)*
hypertrophic i zone (Vv) 0.375 0.530
Calcifying or hypertrophic 2 zone (Vv)
(0.0I3) O.I62 (O.OI) (O.I4)* O.I20 (0.005)*
(32)*t o.o6o (o.oo6)* 0.296 (0.009)* 0.525 (O.OIO)* O.II7 (o.oo8)*
rickets *
Different from the corresponding control value at a level of significance of P
Rickets induced by calcium or phosphate depletion Salem Abugassa* and Olle Svenssont Departments of *General Surgery, tOrthopaedics and Research Center, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden
Received for publication I4 November I989 Accepted for publication 3 April I990
Summary. We studied the effects of calciopenia and phosphopenia on longitudinal growth, skeletal mineralization, and development of rickets in young Sprague-Dawley rats. At an age of 2I days, two experimental groups were given diets containing 0.02% calcium or 0.02% phosphorus; otherwise the diets were nutritionally adequate. After 7, I4, and 2I days, five animals from each group were randomly chosen. The animals were anaesthetized and blood samples were drawn for analysis of calcium, phosphorus, and immunoreactive parathyroid hormone, whereupon the animals were killed. Length, weight, and specific weight of the left femur were measured. After 28 days on the respective diet the remaining animals were killed and one proximal tibia from each animal was processed for light microscopy and subjected to stereological analysis. Both experimental groups developed progressive growth retardation, more so the phosphate-depleted group. The calciopenic animals developed severe hypocalcaemia and secondary hyperparathyroidism, whereas the phosphate-depleted animals, in spite of marked secondary hypercalcaemia, had unaltered levels of immunoreactive parathyroid hormone. By 28 days both experimental groups displayed rachitic changes, more pronounced in the phosphate-depleted animals. This paper provides quantitative data demonstrating that calciopenia per se may cause rickets in young rats, but that the rachitic changes in this condition are less severe, and the growth pattern different from those in phosphate depletion. Keywords: Sprague-Dawley rats, phosphate depletion, calcium depletion, rickets
Rickets primarily affects the skeleton and is usually manifested during periods of rapid growth. The clinical signs include growth retardation, enlargement of the metaphyseal regions, and various deformities associated with decreased formation of bone and deficient skeletal mineralization. Rickets also causes an increase in the height of the physes, mainly because of an increase of its hypertrophic zone. At the lowest part of the physis there is also impairment of calcifica-
tion, deficient cartilage resorption and disorganized ingrowth of capillaries. In rats, rickets and rickets-like lesions can be induced by various agents: in addition to the classical low-phosphate, vitamin D deficiency rickets, rickets-like conditions can be induced by the administration of various metal ions and agents like diphosphonates (Follis I958; Reinholt et al. i984b). Experimentally induced rickets-like conditions have been extensively used to study the
Correspondence: Dr Salem Abugassa, Department of Surgery, Huddinge University Hospital, S-I4I 86 Huddinge, Sweden. 63I
632
S. Abugassa & 0. Svensson
complex process of mineralization. However, there are still divergent opinions as to whether calcium depletion per se causes rickets (Bisaz et al. 1975). One difficulty when interpreting such studies and assessing the degree of the rachitic changes in the physes, is their irregular shape and large biologic variability. In recent years however improved sampling techniques and stereological methods have made possible unbiased estimations of structures with great variability. In this paper we use a stereologic model to make a quantitative comparison between the rachitic effects of calcium depletion and phosphate depletion. Materials and methods
Animals At an age of 2 I days male Sprague-Dawley rats were randomly allocated into three groups. Controls were fed commercially available rat food containing 1.2% w/w Ca, I.0% w/w P, and I 500 IU vitamin D3 per kg (R3, Ewos AB, Sodertalje, Sweden). One experimental group was given a semi-synthetic diet containing 0.02% Ca, 0.72% P, and I5ooIUvitaminD3 perkg (R359, Ewos AB), and another group a diet containing 0.92% w/w Ca, 0.02% w/w P, and I500 IU vitamin D3 per kg (R358, Ewos AB); otherwise these diets were nutritionally adequate. The animals were kept in stainless steel cages under standard laboratory conditions with free access to food and distilled water.
Stereological design After 28 days on the respective diet the animals were given 2500 IU heparin (Lbvens AB, Malmo, Sweden) and anaesthetized by intraperitoneal injections of fentanylfluanison, 0.2-0.8 ml per kg body weight (Hypnorm, Leo, Helsingborg, Sweden). The animals were perfused transcardially as previously described (Svensson et al. I985): first with buffered Ringer's solution containing 2% Dextran T40 (Pharmacia, Uppsala, Sweden) and 0.2% procaine (Astra, Sbder-
talje, Sweden), pH 7.4, 370C, for 30 s, then with 2% glutaraldehyde in o.07 M cacodylate buffer containing 3% Dextran T40, 0.02% procaine, and 0.05 M sucrose, pH 7.4, at 3 70C for 5 min. The osmolarity of the perfusate was 320 mOsmol. The upper part of the tibia was dissected free and was cut longitudinally by hand into Io regions, as previously described (Reinholt I982). A random piece from each one of these regions was fixed overnight in the same fixative as above. After rinsing in buffer the pieces were dehydrated in graded ethanol and embedded in an epoxy resin (JB-4, BioRad). The blocks were coded and microscopy and reading of micrographs were performed blindly. For light microscopy, approximately I . -gum thick sections were cut from each block with a Sorvall Porter-Blum microtome. The sections were stained according to a slightly modified von Kossa procedure, using toluidine blue in water as counterstain. For estimation of volume density of each zone, one micrograph covering the four zones was taken from each section at a final magnification of x 525. The resting zone was defined as the zone between the upper border of the epiphyseal growth cartilage and the top of the first cell in the cell columns. The proliferative zone is the zone of flat chondrocytes, and the border between the proliferative and hypertrophic zones was drawn where the ratio between cell height and cell width is less than 2. The calcifying zone was defined as the zone between the mineralization front, as seen with the modified von Kossa procedure, and the top of the first opened lacuna (Engfeldt & Hjertquist I969). In the rachitic animals where the calcifying zone was virtually absent, the hypertrophic zone was divided into an upper (Hi) and a lower (H2) part; the border between these two was drawn at the level of the highest-reaching vascular pouches from the metaphysis. Physical parameters The femoral length was measured with a
Rickets induced by Ca or P depletion 5r micrometer; the bones were subsequently ashed in a furnace at 700°C for 24 h and the ash weight was recorded under constant conditions.
633 IP
q,
E 4 CE.0 3
Assays
0
From the anaesthetized animals blood samples were taken from the inferior caval vein. The samples were centrifuged to yield serum which was immediately frozen and kept at - 70°C until analysis. To reduce the effect of circadian fluctuations, all samples were taken between ogoo and I 500 h. Immunoreactive parathyroid hormone (iPTH) was measured by a radioimmunoassay (RatPTH-MM, INC, MN, USA) detecting the midpart of the molecule. The concentrations of total calcium and phosphate were determined by standard colorimetric methods (Gindler & King I972; Bartels & Roijers
E 0 c)
2
I
I0
, 2 3 Time (weeks)
4
Fig. 2. Serum calcium in 0, controls; *, phosphate and 0, calcium depletion.
300 r 0 .
1975).
200 o .C
-0
Results The experimental animals were subjected to severe deprivation of calcium and phosphate, respectively, during the period when normally they have their most rapid growth. This regimen resulted in growth retardation of both experimental groups (Fig. i). Their
C
C
o
0E
100 H
0
2 3 Time (weeks)
4
Fig. 3. Immunoreactive parathyroid hormone in 0, controls; *, phosphate and 0, calcium depletion. 30
28 E
-
071 C
-
somewhat
26 24 22
IT
curves
also
cor-
general condition, although at the end of the
c
0
diverging weight
respond to differences in general condition: the calcium-depleted rats had fairly good
20
I8
2
16 0
2
3
Time (weeks) Fig. I. Fe-moral growth in El, controls; phate ancI 0, calcium depletion.
'
4 4
experimental period they developed shabby fur. The phosphate-depleted group, on the other hand, displayed failure to thrive by about a week, and their condition deteriorated during the rest of the experimental period. Moreover, in both experimental groups
U,
phos-
longitudinal growth decelerated, but
it is interesting to note that growth completely abolished (Fig. i).
was
not
S. Abugassa & 0. Svensson The calcium-depleted animals developed a progressive hypocalcaemia, which at the end of the experimental period was severe (Fig. 2)
634 4 E -
a)
2
Q 0
0
£!
E CI)
I 0
I
X
2
3
I 4
Ti me (weeks) Fig. 4 Serum phosphate in 0, controls;
,
phospilate and 0, calcium depletion. 2C)o -
Discussion
0-
._
3
IC
)O
0
0
E
IL
,
TI0
JY
The calcium-depleted animals developed a secondary hyperparathyroidism, not sufficient however to maintain the serum calcium at a normal level. The present situation involved an unphysiological low-calcium stress, and it is known that the parathyroid gland has a limited capability to respond to prolonged stimuli (Cohn et al. I986). The of calcium from the skeleton , , I,mobilization 4 was monitored by femoral ash weight, and
3 2 Time (weeks) Fig. 5. Femoral ash weight in 0, controls; o, phospheate and calcium depletion (overlapping
o
and accompanied by a reciprocal increase of serum iPTH (Fig. 3). Phosphate deprivation resulted in successive decrement of the serum phosphate (Fig. 4) with a compensatory hypercalcaemia (Fig. 2); these animals, however, had unaltered levels of iPTH (Fig. 3). Figure 5 shows the femoral ash weight. The stereological findings are given in Table i. Both experimental groups thus developed rachitic changes, mainly due to an increased height of the hypertrophic zones (Figs 6-8).
values).
apparently there was no net calcium accretion during the experimental period (Fig. 5). It may seem puzzling that the phospho-
penic animals had unaltered levels of iPTH
Table i. Results of the stereological examination
Hypertrophic or Total height
(mm)
Group
Controls
382
Low phosphate 792 rickets Low calcium 5I4
Resting zone (Vv)t:
Proliferative zone (Vv)
(i6) 0.079 (0.004) 0.383 (O.OI2) (40)* 0.052 (0.007)* 0.298 (o.oI6)*
hypertrophic i zone (Vv) 0.375 0.530
Calcifying or hypertrophic 2 zone (Vv)
(0.0I3) O.I62 (O.OI) (O.I4)* O.I20 (0.005)*
(32)*t o.o6o (o.oo6)* 0.296 (0.009)* 0.525 (O.OIO)* O.II7 (o.oo8)*
rickets *
Different from the corresponding control value at a level of significance of P
