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Endocrinology Vol. 139, No. 10 4319-4328
Copyright © 1998 by The Endocrine Society

ARTICLES

Therapeutic Efficacy of 1{alpha},25-Dihydroxyvitamin D3 and Calcium in Osteopenic Ovariectomized Rats: Evidence for a Direct Anabolic Effect of 1{alpha},25-Dihydroxyvitamin D3 on Bone1

Reinhold G. Erben, Silke Bromm and Manfred Stangassinger

Institute of Physiology, Physiological Chemistry, and Animal Nutrition, Ludwig Maximilians University, Munich 80539, Germany

Address all correspondence and requests for reprints to: Dr. Reinhold G. Erben, Institute of Animal Physiology, University of Munich, Veterinaerstrasse 13, D-80539 Munich, Germany. E-mail: r.erben{at}lrz.uni-muenchen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is an important question for clinical therapy of osteoporosis with vitamin D metabolites whether these compounds exert their beneficial effects on the skeleton indirectly through an increase in intestinal calcium absorption or whether there is also a major direct component of action on bone. In this study, female 6-month-old Fischer rats were either ovariectomized (OVX) or sham operated. One month before surgery, all rats were placed on a diet containing 0.25% calcium and were kept on this diet throughout the study. Beginning 3 months post-OVX, groups of OVX rats orally received vehicle, a calcium supplement, low dose (0.025 µg/kg·day) or high dose (0.1 µg/kg·day) 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3], or combinations of low and high dose 1,25-(OH)2D3 with the calcium supplement. By 3 months postsurgery, pretreatment OVX controls had lost 74% and 37% of tibial and vertebral cancellous bone, respectively. Two-way factorial ANOVA showed that a 3-month treatment of osteopenic OVX rats with 1,25-(OH)2D3 dose dependently increased vertebral and tibial cancellous bone mass (P < 0.001 and P = 0.021, respectively) and trabecular width (P < 0.001). Furthermore, 1,25-(OH)2D3 increased serum calcium (P = 0.028) and urinary calcium excretion (P < 0.001) and reduced serum PTH levels (P < 0.001), osteoclast numbers (P < 0.001), and urinary collagen cross-links excretion (P < 0.001). Calcium supplementation alone was without therapeutic effect, and there was no significant two-way interaction between the individual treatment effects of 1,25-(OH)2D3 and calcium on bone mass. These data indicate that the anabolic effects of 1,25-(OH)2D3 in osteopenic OVX rats are mediated through a direct activity on bone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH several studies in patients with postmenopausal or senile osteoporosis have shown positive effects on bone mass and a reduction in fracture rate in response to therapy with 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3] or 1{alpha}-hydroxyvitamin D3 (1, 2, 3), the role of active vitamin D metabolites in the therapy of osteoporosis is still controversial. An important unresolved issue in the therapy of osteoporosis with vitamin D metabolites is that it is not known whether these compounds exert their beneficial effects on the skeleton indirectly through an increase in intestinal calcium absorption or whether there is also a major direct component of action on bone (including the bone marrow compartment). Osteoporosis therapy with vitamin D metabolites seems to be generally more effective in countries with a traditionally low calcium intake, such as Japan or Italy, than in countries with a higher dietary calcium intake, such as Northern Europe or the United States (4). One possible explanation for this phenomenon could be that especially in low calcium states, vitamin D metabolites may act to improve calcium balance, thereby indirectly improving bone mineral. Alternatively, however, this finding may also support the idea that a direct action of vitamin D metabolites on bone is an important part of their therapeutic efficacy. Because the major side-effects of therapy with active vitamin D metabolites are hypercalcemia and hypercalciuria due to a stimulation of intestinal calcium absorption, patients with a low calcium intake tolerate higher doses of active vitamin D metabolites than patients on a high calcium diet before a reduction of the dose is required due to the development of untoward side-effects (5). As a result of this, the doses of 1,25-(OH)2D3 used for osteoporosis therapy are usually higher in countries with a traditionally low calcium intake than in countries with a higher calcium intake, although the degree of hypercalciuria may be similar. Therefore, the positive skeletal effects of 1,25-(OH)2D3 in osteoporotic patients may involve direct pharmacological effects of higher doses of 1,25-(OH)2D3 on bone. In line with this argument, it has been suggested that a threshold dose of about 0.5 µg 1,25-(OH)2D3/day exists for therapeutic effectiveness of 1,25-(OH)2D3 in postmenopausal osteoporosis (4).

Cells of the osteoblastic lineage have been shown to contain intracellular 1,25-(OH)2D3 receptors (6), and high doses of 1,25-(OH)2D3 up-regulate tibial osteocalcin messenger RNA levels (7) and increase the number of osteoblast precursor cells in bone marrow (8) in vivo in the rat. Moreover, numerous in vitro studies have shown that 1,25-(OH)2D3 can modulate osteoblast proliferation and osteoblast production of type I collagen, alkaline phosphatase, and osteocalcin (9). Therefore, it is a distinct possibility that the actions of vitamin D metabolites on bone are mediated through a direct effect on cells of the osteoblastic lineage. However, the relevance of these mechanisms for chronic treatment of osteopenic humans or animals with 1,25-(OH)2D3 is unclear at present.

It was the aim of the present study to further elucidate this unresolved issue. Using osteopenic ovariectomized (OVX) rats as an experimental model of estrogen depletion-induced bone loss, we examined the therapeutic efficacy of orally administered 1,25-(OH)2D3 at different dosages and in combination with different dietary calcium contents. The idea behind this study was that if the skeletal effects of 1,25-(OH)2D3 are mainly mediated through positive effects on intestinal calcium absorption and calcium balance, calcium supplementation should enhance the anabolic effects of 1,25-(OH)2D3 on bone. However, a factorial ANOVA of the data provided by the current investigation clearly showed that although the dose of 1,25-(OH)2D3 was significantly associated with the improvement of histomorphometric indexes of vertebral and tibial cancellous bone mass and structure in osteopenic OVX rats, calcium supplementation was without influence. Furthermore, with few exceptions, we found no interaction between the treatment effects of calcium and 1,25-(OH)2D3 on bone. Thus, our study suggests that a direct action of 1,25-(OH)2D3 on bone is the major component of the bone anabolic effect of 1,25-(OH)2D3 in osteopenic OVX rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal procedures
Seventy-four 6-month-old female Fischer-344 rats (Charles River, Sulzfeld, Germany), weighing about 180 g, were used for this experiment. Six rats served as baseline controls. Sixty-eight rats were either bilaterally ovariectomized by the dorsal approach under ether anesthesia or sham operated (SHAM). The animals were kept in pairs at 24 C with a 12-h light, 12-h dark cycle. One month before surgery, all rats were switched from a standard rat chow containing 0.9% calcium and 0.7% phosphorus (Altromin, Lage, Germany) to a diet containing 0.25% calcium, 0.6% phosphorus, and 600 IU/kg vitamin D3 (Altromin) and were kept on this diet throughout the study. From the beginning of the experiment until 2 weeks before the start of therapy with 1,25-(OH)2D3 and calcium at 3 months post-OVX, the rats received food pellets. Thereafter, the diets of all rats were given in powdered form. Food was available ad libitum to the SHAM rats. The food consumption of the OVX rats was restricted to that of the SHAM group (pair-feeding).

Three months post-OVX, six SHAM and six OVX rats were killed as pretreatment controls. Beginning 3 months post-OVX, groups of eight OVX rats each orally received vehicle, a calcium supplement, low dose (0.025 µg/kg BW·day) or high dose (0.1 µg/kg·day) 1,25-(OH)2D3, or combinations of low and high dose 1,25-(OH)2D3 with the calcium supplement. 1,25-(OH)2D3 was dissolved in ethanol-1,2-propandiol (1:10) and added to the powdered diet. 1,25-(OH)2D3 was provided by Dr. M. Haberey (Schering, Berlin, Germany). The concentration of 1,25-(OH)2D3 in the diet for each group of rats was calculated on the basis of the amount of food they received according to the pair-feeding protocol, the dose of 1,25-(OH)2D3, and the mean body weight of the respective group. The concentration of 1,25-(OH)2D3 in the diet was regularly adjusted to reflect changes in the amount of food allotted by the pair-feeding protocol and/or changes in body weight for each group of rats. The pair-fed OVX rats regularly consumed all food given to them. There were no differences in food intake between the different OVX groups. Because the variability of body weight is low in the inbred strain of rats we used for this experiment, the maximum dosage error in individual animals introduced by this dosing regimen is about 10%.

To optimize the design of this experiment, we intended to adjust the dietary calcium supplement in such a way that the urinary excretion of calcium, the most sensitive parameter of the effects of 1,25-(OH)2D3 on calcium homeostasis, would be about the same in the OVX rats receiving high dose 1,25-(OH)2D3 alone as that in the group receiving low dose 1,25-(OH)2D3 plus the calcium supplement. The calcium supplement was given in form of CaCO3 that was added to the powdered diet. In preliminary experiments with OVX rats of the same strain and age, we found that when the total calcium content of the diet was increased to 0.6%, a dose of 0.025 µg 1,25-(OH)2D3/kg·day increased urinary calcium excretion in a similar fashion as the administration of 0.1 µg 1,25-(OH)2D3/kg·day alone. Therefore, the total calcium content of the diet was increased from 0.25% to 0.6% in the groups receiving the dietary calcium supplement.

Urine was sampled at monthly intervals throughout the study, and blood was drawn before death. For urine collection, the rats were deprived of food for 8 h and were then placed in metabolic cages without food and water for a 14-h period overnight. Calcein (Sigma, Deisenhofen, Germany) at a dose of 20 mg/kg BW was injected ip on days 9 and 4 before death. All remaining rats were killed by exsanguination from the abdominal aorta under ketamine/xylazine (50/10 mg/kg, ip) anesthesia 6 months post-OVX, i.e. 3 months after the start of 1,25-(OH)2D3 therapy. The serum and urine samples were stored at -40 C until assayed. The success of ovariectomy was confirmed by failure to detect ovarian tissue and observation of marked atrophy of the uterine horns. All animal procedures were approved by the local government authorities.

Blood and urine analysis
Total calcium in serum and urine was determined by flame photometry (ELEX 6361, Eppendorf, Hamburg, Germany). Phosphorus in serum and urine was analyzed on a Hitachi 717 Autoanalyzer (Boehringer Mannheim, Mannheim, Germany). Serum alkaline phosphatase activity was measured with a commercially available test kit (Sigma). Serum PTH was measured with a rat-specific immunoradiometric assay reacting with both N-terminal and intact PTH (Immutopics, San Clemente, CA). The intra- and interassay variabilities of this assay in our laboratory were 4.5% and 8.3%, respectively. Serum PTH was measured only in the posttreatment groups at the end of the trial. Urinary creatinine was determined with a colorimetric assay (Boehringer Mannheim). Total pyridinoline (PYD) and deoxypyridinoline (DPD) concentrations in urine were determined after acid hydrolysis using a HPLC technique described previously (10). The intra- and interassay variabilities of this method in our laboratory were 2.3% and 5.8% for PYD, and 3.6% and 8.5% for DPD, respectively. Urinary excretion of calcium, phosphorus, and collagen cross-links was expressed as a ratio to creatinine excretion.

Histology
At necropsy, the first lumbar vertebrae and the proximal right tibiae were defleshed and fixed immediately in 40% ethanol at 4 C for 48 h. After fixation, the bones were embedded undecalcified in methylmethacrylate, as described previously (11). Five-micron thick undecalcified sections were prepared with an HM 360 microtome (Microm, Walldorf, Germany). The sections were sampled in the median plane of the vertebrae and the midsagittal plane of the tibiae, and stained with von Kossa/toluidine blue (12) and toluidine blue at acid pH (13).

Histomorphometry
As the structural elements in the cancellous bone of both the lumbar vertebral body and the proximal tibial metaphysis show a markedly anisotropic distribution in the rat, the measurements are generally presented as two-dimensional histomorphometric terms. All parameters were calculated and expressed according to the recommendations made by the American Society for Bone and Mineral Research nomenclature committee (14).

Structural parameters in tibial and vertebral cancellous bone. These measurements were made with an automatic image analysis system (VIDAS, Zeiss, Oberkochen, Germany) connected to a Zeiss Axioskop microscope (Zeiss) via a television camera (Bosch, Stuttgart, Germany). All measurements with the automatic image analysis system were performed with a x2.5 objective on sections stained with von Kossa/toluidine blue. The area within 0.5 mm from the growth plates was excluded from the measurements in the proximal tibial metaphysis and the first lumbar vertebral body. The average measuring area was about 13 mm2 in each section in the tibiae, and about 6–7 mm2 in the vertebrae. One section was analyzed per animal. The image analysis system automatically determined the measuring area (tissue area), bone area, bone perimeter, and number of trabeculae. From these data, the structural parameters bone area (bone area/tissue area), trabecular width, trabecular area, trabecular number per bone area, trabecular number, and trabecular separation were calculated. Trabecular area represents the mean area (in square millimeters) of individual trabeculae. Trabecular number per bone area represents the number of individual trabecular profiles found per mm2 cancellous bone. For the calculation of trabecular number and trabecular separation, the parallel plate model was used (14).

Cellular and fluorochrome-based parameters in vertebral cancellous bone
Histomorphometric measurements of cellular and fluorochrome-based parameters in the cancellous bone of the first lumbar vertebral body were made using a semiautomatic system (Videoplan, Zeiss) and a Zeiss Axioskop microscope with a drawing attachment. In the centrally located cancellous bone of the first lumbar vertebral body, about 3–4 mm2 of tissue area were evaluated in each section, corresponding to about 20–30 mm of trabecular bone surface. The area within 0.5 mm from the cranial and caudal growth plates was excluded from the measurements. Static histomorphometric parameters were measured in sections stained with toluidine blue, and dynamic, fluorochrome-based parameters were measured in unstained sections. Polarized light was used to analyze for woven bone formation in sections stained with toluidine blue.

The following primary parameters were determined at x200: bone area, bone perimeter, osteoid perimeter, number of osteoclasts, and fluorochrome double labeled perimeter. Osteoid width was determined directly at x400, sampling each osteoid seam every 50 µm. Osteoclasts were defined as large, irregularly shaped cells with a foamy, slightly metachromatic cytoplasma containing one or more nuclei and residing within Howship’s lacunae. Typical anucleate osteoclast profiles located within Howship’s lacunae were also counted as osteoclasts. Osteoclast numbers were expressed using the mineralized bone perimeter as referent. The mineral apposition rate (MAR) was measured at x400, sampling each double label every 50 µm. Values for MAR were not corrected for obliquity of the plane of section. The mineralizing perimeter was defined as the percentage of fluorochrome double labeled bone perimeter. The bone formation rate was calculated by multiplying the mineralizing perimeter with the mineral apposition rate. Osteoid maturation time was defined as osteoid width divided by mineral apposition rate and was expressed in days. The mineralizing surface/osteoid surface (MS/OS) ratio was calculated by dividing the mineralizing perimeter by the osteoid perimeter.

Statistical analysis
Statistics were computed using SPSS for Windows 6.1 (SPSS, Chicago, IL). Statistical comparisons between the baseline and pretreatment groups were made using one-way ANOVA. When the one-way ANOVA performed over all groups indicated a significant (P < 0.05) difference among the groups, statistical differences between individual groups were subsequently evaluated with the Student-Newman-Keuls multiple comparison test.

Statistical comparisons between the vehicle-treated SHAM and OVX posttreatment groups were made with a two-sided t test. The data for all six OVX posttreatment groups were analyzed using two-way factorial ANOVA, where the two factors were calcium supplementation (presence or absence) and the dosage of 1,25-(OH)2D3 (0, 0.025, and 0.1 µg/kg·day). Two-way factorial ANOVA evaluated whether there were treatment effects of calcium supplementation and 1,25-(OH)2D3 therapy in OVX rats and also determined whether there was a two-way interaction between the individual treatment effects. Thus, this statistical technique allows the determination of whether two different treatment factors mutually influence each other in a nonadditive way. P < 0.05 was considered significant for all statistical analyses. The data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of ovariectomy in pretreatment controls
Three months postovariectomy, the body weights of SHAM and OVX rats did not differ significantly (Table 1Go). Urinary DPD excretion tended to be higher and the PYD/DPD ratio was significantly decreased in OVX rats relative to those in SHAM controls. Relative to baseline control levels, cancellous bone mass was reduced by 74% in the proximal tibiae and by 37% in the vertebrae of pretreatment OVX rats (Tables 2Go and 3Go and Fig. 1Go). The reductions in vertebral and tibial cancellous bone mass in OVX rats were accompanied by structural deterioration of trabecular bone, as reflected in decreased values for trabecular width and trabecular number and in increases in trabecular separation and trabecular number per bone area. Histomorphometric indexes of bone formation (osteoid perimeter, mineralizing perimeter, and bone formation rate) and bone resorption (osteoclast number) were elevated in OVX compared to SHAM animals 3 months postovariectomy (Table 2Go).


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  		Table 1. Body weight and biochemical data in serum and urine, 3 months postovariectomy (pretreatment control)

 

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  		Table 2. Histomorphometric data in the cancellous bone of the first lumbar vertebra, 3 months postovariectomy (pretreatment control)

 

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  		Table 3. Histomorphometric data in the cancellous bone of the proximal tibial metaphysis, 3 months postovariectomy (pretreatment control)

 


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  		Figure 1. Effects of ovariectomy in pretreatment control animals, 3 months post-OVX. Compared with the SHAM rat (A), there is a pronounced reduction in vertebral cancellous bone mass and also increased discontinuity of trabecular bone structure in the OVX rat (B). Undecalcified, median sections of the first lumbar vertebrae are shown. Von Kossa/toluidine blue stain was used. Magnification, x13.

 
Compared with baseline control levels, SHAM animals showed a small reduction in vertebral, but not in tibial cancellous bone mass (Tables 2Go and 3Go). Further, osteoid width and osteoid maturation time were significantly increased, and the portion of osteoid actively mineralizing (MS/OS ratio) was nonsignificantly decreased in pretreatment SHAM and OVX rats relative to that in baseline controls. It is likely that these changes were caused by the reduced amount of calcium and/or the low calcium/phosphorus ratio in the basal diet (0.25% calcium/0.6% phosphorus) that was used in this experiment.

Effects of treatment with 1,25-(OH)2D3 and calcium in osteopenic OVX rats
Body weight and biochemical findings. Six months postovariectomy, the body weights did not differ between vehicle-treated SHAM and OVX rats, and there were no significant effects of calcium or 1,25-(OH)2D3 treatment on body weight in OVX animals (data not shown). Vehicle-treated OVX rats had higher serum phosphorus levels (2.30 ± 0.09 vs. 1.79 ± 0.22 mmol/liter; P < 0.05), a higher serum alkaline phosphatase activity (P < 0.001; Fig. 2BGo), and a lower ratio of PYD/DPD in urine (P < 0.01; Fig. 2EGo) than SHAM controls. Urinary phosphorus did not differ in vehicle-treated OVX rats and SHAM animals (data not shown).



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  		Figure 2. Serum calcium (A), serum alkaline phosphatase (B), serum PTH (C), and urinary excretion of the collagen cross-link DPD (D) as well as PYD/DPD ratio (E) in SHAM and OVX rats 6 months post-OVX. Beginning 3 months post-OVX, OVX rats orally received vehicle (Veh), a calcium supplement (Ca), low dose (0.025 µg/kg·day) or high dose (0.1 µg/kg·day) 1,25-(OH)2D3 (1,25D), or combinations of low dose and high dose 1,25-(OH)2D3 with the calcium supplement. Statistical comparisons between SHAM and vehicle-treated OVX rats were performed using a two-sided t test. The treatment effects of 1,25-(OH)2D3 and calcium supplementation as well as their interaction in OVX rats were analyzed by two-way factorial ANOVA. Each bar represents the mean ± SEM of seven to eight animals. **, P < 0.01; ***, P < 0.001 (SHAM vs. OVX plus vehicle).

 
Two-way ANOVA revealed that 1,25-(OH)2D3 treatment of OVX rats resulted in a small, but significant (P = 0.028), rise in serum calcium (Fig. 2AGo). Serum and urinary phosphorus were not influenced by 1,25-(OH)2D3 treatment (data not shown). Serum alkaline phosphatase (Fig. 2BGo) and serum PTH (Fig. 2CGo) were suppressed by 1,25-(OH)2D3 administration to OVX rats (P < 0.001). Moreover, 1,25-(OH)2D3 treatment suppressed urinary DPD excretion (P = 0.013; Fig. 2DGo), and significantly increased the ratio of PYD/DPD in urine (P = 0.004; Fig. 2EGo). Calcium supplementation alone increased serum alkaline phosphatase activity (P = 0.001) and the ratio of PYD/DPD in urine (P = 0.016, Fig. 2EGo), but had no effect on serum PTH, serum calcium, or serum phosphorus (data not shown). Further, we found no significant interaction between 1,25-(OH)2D3 and calcium treatment for serum calcium, serum phosphorus (data not shown), serum alkaline phosphatase, serum PTH, or urinary excretion of phosphorus (data not shown) and collagen cross-links. Figure 3Go shows that urinary calcium excretion was significantly increased during treatment with 1,25-(OH)2D3 and calcium (P < 0.001 for 1,25-(OH)2D3 and P < 0.002 for calcium at 4, 5, and 6 months post-OVX). The two-way interaction between 1,25-(OH)2D3 and calcium was not significant at 4 (P = 0.154) and 5 (P = 0.065) months, but was highly significant at 6 months post-OVX (P < 0.001). Interestingly, we observed a steady increase in urinary calcium excretion over the treatment period in the groups receiving high dose 1,25-(OH)2D3, especially in combination with the calcium supplement. In both the OVX groups receiving high or low dose 1,25-(OH)2D3, addition of the dietary calcium supplement resulted in about a 2-fold augmentation of urinary calcium excretion, respectively (Fig. 3Go). The mean urinary calcium/creatinine ratio over the whole treatment period was similar in the group receiving high dose 1,25-(OH)2D3 alone (mean, 0.471 mmol/mmol) to that in the group receiving low dose 1,25-(OH)2D3 plus the calcium supplement (mean, 0.551 mmol/mmol).



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  		Figure 3. Urinary excretion of calcium (expressed as a ratio to creatinine excretion) plotted as a function of time post-OVX. Beginning 3 months post-OVX, OVX rats orally received either vehicle (Veh), a calcium supplement (Ca), low dose or high dose 1,25-(OH)2D3 (1,25D), or combinations of low dose and high dose 1,25-(OH)2D3 with the calcium supplement. Each data point is the mean ± SEM of five to eight animals.

 
Bone histomorphometry. By the end of the experiment, vehicle-treated OVX rats had lost 90% and 43% of tibial and vertebral cancellous bone, respectively, relative to SHAM controls (Figs. 4Go, 5AGo, and 6AGo). Vertebral cancellous bone area was almost identical to baseline levels in vehicle-treated SHAM animals at the end of the trial. Compared with pretreatment OVX rats (23.01 ± 1.0%), vertebral cancellous bone area in vehicle-treated OVX rats (21.08 ± 1.5%; Fig. 5AGo) showed little further decrease at the end of the experiment. In both vertebral and tibial bone, vehicle-treated OVX rats exhibited significant reductions in trabecular width (Figs. 5BGo and 6BGo) and trabecular number (Figs. 5CGo and 6CGo), and an increase in trabecular number per bone area (Figs. 5DGo and 6DGo) compared with SHAM controls.



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  		Figure 4. Undecalcified, median sections of the first lumbar vertebrae of SHAM rats (A) and OVX rats treated for 3 months with vehicle (B), a calcium supplement (C), low dose (D) or high dose 1,25-(OH)2D3 (F), or combinations of low dose (E) and high dose (G) 1,25-(OH)2D3 with the calcium supplement. It is evident that calcium supplementation alone was without therapeutic effect, whereas treatment with calcitriol resulted in a dose-dependent increase in cancellous bone mass and an improvement of cancellous bone structure without obvious additional positive effects of calcium supplementation. Von Kossa/toluidine blue stain was used. Magnification, x13.

 


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  		Figure 5. Treatment effects of 1,25-(OH)2D3 and calcium supplementation on bone area (A), trabecular width (B), trabecular number (C), and trabecular number per bone area (D) in lumbar vertebral cancellous bone of osteopenic OVX rats 6 months post-OVX. Beginning 3 months post-OVX, OVX rats orally received vehicle (Veh), a calcium supplement (Ca), low dose or high dose 1,25-(OH)2D3 (1,25D), or combinations of low dose and high dose 1,25-(OH)2D3 with the calcium supplement. Statistical comparisons between SHAM and vehicle-treated OVX rats were performed using a two-sided t test. The treatment effects of 1,25-(OH)2D3 and calcium supplementation as well as their interaction in OVX rats were analyzed by two-way factorial ANOVA. Each bar represents the mean ± SEM of seven or eight animals. ***, P < 0.001 (SHAM vs. OVX plus vehicle).

 


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  		Figure 6. Treatment effects of 1,25-(OH)2D3 and calcium supplementation on bone area (A), trabecular width (B), trabecular number (C), and trabecular number per bone area (D) in proximal tibial cancellous bone of osteopenic OVX rats 6 months post-OVX. Beginning 3 months post-OVX, OVX rats orally received either vehicle (Veh), a calcium supplement (Ca), low dose or high dose 1,25-(OH)2D3 (1,25D), or combinations of low dose and high dose 1,25-(OH)2D3 with the calcium supplement. Statistical comparisons between SHAM and vehicle-treated OVX rats were performed using a two-sided t test. The treatment effects of 1,25-(OH)2D3 and calcium supplementation as well as their interaction in OVX rats were analyzed by two-way factorial ANOVA. Each bar represents the mean ± SEM of seven or eight animals. **, P < 0.01; ***, P < 0.001 (SHAM vs. OVX plus vehicle).

 
Factorial ANOVA of the histomorphometric data in the OVX groups showed that 1,25-(OH)2D3 treatment of osteopenic OVX rats significantly increased cancellous bone mass (P < 0.001), trabecular width (P < 0.001), and trabecular number (P < 0.001) and decreased trabecular number per bone area (P < 0.001) in lumbar vertebral bone (Figs. 4Go and 5Go). The effects of 1,25-(OH)2D3 treatment on vertebral cancellous bone area and trabecular width appeared to be dose dependent. Although we found a significant treatment effect of 1,25-(OH)2D3 on trabecular number in the vertebrae, a closer scrutiny of the data revealed that trabecular number in 1,25-(OH)2D3-treated rats did not increase relative to OVX pretreatment control levels (Table 2Go). We observed no woven bone formation in 1,25-(OH)2D3-treated animals. Calcium supplementation had no treatment effect. Further, there was no interaction between the treatment effects of 1,25-(OH)2D3 and calcium. In the proximal tibial metaphysis (Fig. 6Go), there was only a weak treatment effect of 1,25-(OH)2D3 on cancellous bone mass (P = 0.021). However, there were pronounced effects of 1,25-(OH)2D3 therapy on trabecular width (P < 0.001) and on trabecular number per bone area (P = 0.003) in tibial cancellous bone. Again, calcium supplementation had no effect. In contrast to the vertebrae, there was a weak interaction between 1,25-(OH)2D3 and calcium on trabecular width (P = 0.015) and trabecular number per bone area (P = 0.048) in the tibiae.

Neither treatment of OVX rats with 1,25-(OH)2D3 nor treatment with calcium had any significant effect on osteoid perimeter, MS/OS ratio, bone formation rate (Fig. 7Go, A–C), osteoid width (data not shown), or osteoid maturation time (data not shown). However, 1,25-(OH)2D3 treatment reduced osteoclast numbers in vertebral cancellous bone (P = 0.001; Fig. 7DGo). Further, we found a significant interaction between the treatment effects of calcium and 1,25-(OH)2D3 for the MS/OS ratio (P = 0.005) and osteoclast number (P = 0.002). An unexpected finding in this study was that dietary calcium supplementation alone tended to further increase osteoclast numbers relative to those in OVX vehicle controls (Fig. 7DGo). In contrast, biochemical markers of bone resorption in urine tended to be lower in this group than in vehicle-treated OVX animals (Fig. 2Go, D and E). The reason for this discrepancy is not known.



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  		Figure 7. Treatment effects of 1,25-(OH)2D3 and calcium supplementation on osteoid perimeter (A), MS/OS ratio (B), bone formation rate (C), and osteoclast number (D) in lumbar vertebral cancellous bone of osteopenic OVX rats 6 months post-OVX. Beginning 3 months post-OVX, OVX rats orally received vehicle (Veh), a calcium supplement (Ca), low dose or high dose 1,25-(OH)2D3 (1,25D), or combinations of low dose and high dose 1,25-(OH)2D3 with the calcium supplement. Statistical comparisons between SHAM and vehicle-treated OVX rats were performed using a two-sided t test. The treatment effects of 1,25-(OH)2D3 and calcium supplementation as well as their interaction in OVX rats were analyzed by two-way factorial ANOVA. Each bar represents the mean ± SEM of six to eight animals. *, P < 0.05 (SHAM vs. OVX plus vehicle).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the prerequisites to perform this study was a basal diet with a reduced calcium content that would allow the administration of 1,25-(OH)2D3 at largely different doses and that, with the help of a calcium supplement, would make it possible to achieve similar urinary excretions of calcium in the OVX rats receiving high dose 1,25-(OH)2D3 alone and the group receiving low dose 1,25-(OH)2D3 plus the calcium supplement. Although the basal diet containing 0.25% calcium and 0.6% phosphorus used in our investigation did induce a small and transient reduction in vertebral cancellous bone mass and also generated changes in bone mineralization, the 6-month-old female Fischer rats used in our study were able to adapt to this diet without permanent cancellous bone loss caused by a persistent negative calcium balance. However, although the rats were allowed to adapt for 1 month to the changed diet before the start of the experiment, the adaptation process to the reduced calcium diet took longer than 4 months before the rats seemed to be in a steady state of bone and calcium metabolism.

The major untoward side-effects of chronic treatment with active vitamin D metabolites in humans and animals are hypercalcemia and hypercalciuria. The current study has further corroborated the idea that the toxicity of 1,25-(OH)2D3 is mainly determined by dietary calcium intake. The rats in our experiment tolerated high doses of 1,25-(OH)2D3 without any severe side-effects when fed a diet with a reduced calcium content. The normal range for the urinary calcium/creatinine ratio is about 0.3–0.7 mmol/mmol in rats of the same strain and age when fed a standard rat chow containing 0.9% calcium and 0.7% phosphorus (15). Therefore, only the group receiving high dose 1,25-(OH)2D3 in combination with the calcium supplement can be considered hypercalciuric in the present experiment compared with rats receiving a standard rat diet. The rats fed the diet containing 0.25% calcium ingested about 25 mg calcium/day in our study. Using the metabolic body weight (BW0.75) for calculation, this corresponds to a daily calcium intake of about 1.7 g in a 60-kg human. In the groups of rats receiving the diet supplemented with calcium, the mean ingested amount of calcium was approximately 60 mg/day, corresponding to a daily intake of 4.0 g calcium in a 60-kg human. Therefore, the oral calcium supplementation administered in our experiment corresponds to a very large calcium supplement when extrapolated to the clinical situation in humans. When the metabolic body weight is used as a basis for calculation, the low dose (0.025 µg/kg·day) of 1,25-(OH)2D3 used in our experiment corresponds to a dose of about 0.4 µg/day, and the high dose (0.1 µg/kg·day) compares to a dose of about 1.5 µg/day in a 60-kg human.

1,25-(OH)2D3 therapy reduced bone resorption in OVX rats in the current study, as measured by osteoclast numbers in cancellous bone and urinary collagen cross-links excretion. An antiresorptive effect of chronic 1,25-(OH)2D3 administration has also been reported in previous studies in rats (16, 17, 18) and humans (19, 20). In contrast to the effects on cancellous bone mass, however, we found a significant interaction between the effects of 1,25-(OH)2D3 and calcium on osteoclast numbers, i.e. the suppressive effects of 1,25-(OH)2D3 therapy on osteoclast numbers were enhanced by supplemental dietary calcium. Moreover, compared with this study, the antiresorptive effects of chronic administration of 1,25-(OH)2D3 appear to be much more pronounced in rats receiving a normal rat diet containing 0.9% Ca and 0.7% phosphorus (15). Therefore, the effects of 1,25-(OH)2D3 therapy on osteoclast numbers may at least in part be mediated indirectly through effects on calcium homeostasis. Mature osteoclasts do not contain 1,25-(OH)2D3 receptors (6). 1,25-(OH)2D3 treatment had a significant inhibitory effect on PTH secretion in the current experiment. Because PTH is one of the most important regulators of osteoclast number and activity in vivo (21), it is likely that the suppressive effects of 1,25-(OH)2D3 on bone resorption are mediated through down-regulation of PTH secretion. However, we observed no interaction between calcium supplementation and 1,25-(OH)2D3 on serum PTH. Thus, the treatment effects of 1,25-(OH)2D3 and calcium on osteoclast number and serum PTH levels showed some discrepancies, and it is unclear at present whether there are additional factors other than PTH involved in the suppression of osteoclastic bone resorption by 1,25-(OH)2D3. Nevertheless, the current experiment indicates that high doses of chronically administered 1,25-(OH)2D3 have antiresorptive effects even in combination with a diet reduced in calcium content.

The results of the present study have clearly shown that a 3-month therapeutic administration of 1,25-(OH)2D3 to osteopenic OVX rats increases cancellous bone mass and improves histomorphometric indexes of cancellous bone structure in the lumbar vertebrae and also partially in the tibiae. Relative to that in vehicle-treated OVX controls, vertebral cancellous bone mass was increased by 22% and 23% in OVX rats receiving low dose as well as by 34% and 39% in OVX rats receiving high dose 1,25-(OH)2D3, alone or in combination with the calcium supplement, respectively. The reduced therapeutic efficacy of 1,25-(OH)2D3 in the proximal tibia is probably related to the severe osteopenia that was present in this bone site at the start of therapy. Similar observations have been made with therapeutic administration of PTH to osteopenic OVX rats (22). In agreement with earlier studies conducted in our laboratory (15, 23), the present experiment has shown that the main microanatomical mechanism that increases cancellous bone mass in rats treated with active vitamin D metabolites is an increase in the trabecular width of existing bone spicules, not an increase in trabecular number. As longitudinal bone growth in the lumbar vertebrae of 9-month-old Fischer rats is already undetectable (24), the increase in vertebral cancellous bone mass and trabecular width in response to 1,25-(OH)2D3 therapy observed in this study cannot solely be explained by the antiresorptive activity of this compound. Rather, this effect can only be caused by a situation in which bone formation exceeds bone resorption. At the end of the trial, i.e. after 3 months of 1,25-(OH)2D3 therapy, there were no significant effects of 1,25-(OH)2D3 on cancellous bone formation. However, although 1,25-(OH)2D3 treatment diminished bone resorption in OVX rats in our study, it had no suppressive effect on the bone formation rate, thus probably shifting the balance of bone turnover in favor of bone formation. A previous investigation (8) in normal rats has shown that bone formation was temporarily increased with a maximum 10–15 days after short term administration of high dose 1,25-(OH)2D3. Therefore, it may alternatively be possible that 1,25-(OH)2D3 transiently stimulated bone formation during the initial phase of treatment, and that bone formation had returned to OVX vehicle control levels by the end of the current study.

It is well known that 1,25-(OH)2D3 enhances the intestinal absorption of calcium (19, 25, 26, 27). This effect was documented in our study by a significant stimulation of urinary calcium excretion in 1,25-(OH)2D3-treated OVX rats and a significant interaction between the effects of 1,25-(OH)2D3 and calcium supplementation on urinary calcium excretion at the end of the study. Therefore, if the bone anabolic actions of 1,25-(OH)2D3 were mediated indirectly through the improvement of calcium balance, one would expect a significant interaction between the effects of 1,25-(OH)2D3 and calcium supplementation. This was not the case however. Factorial ANOVA showed that the only determinant of the bone anabolic response in vertebral and tibial cancellous bone of osteopenic OVX rats was the dose of 1,25-(OH)2D3 administered. The calcium supplement showed no treatment effect, and there was no interaction between the effects of 1,25-(OH)2D3 and calcium on vertebral and tibial cancellous bone mass, i.e. supplemental dietary calcium did not augment the bone anabolic action of 1,25-(OH)2D3. Taken together, these data strongly indicate that a direct action of 1,25-(OH)2D3 on bone is the major component of the bone anabolic effects of 1,25-(OH)2D3 in osteopenic OVX rats. A direct stimulating effect of 1,25-(OH)2D3 on bone turnover and remodeling activity in vivo has also been suggested by earlier studies (5, 28, 29), and a recent investigation showing that short term administration of high dose 1,25-(OH)2D3 augments bone formation and increases the number of osteoblast precursor cells in rat bone marrow (8) further corroborated the idea that the anabolic effects of 1,25-(OH)2D3 are mediated through a direct pharmacological influence on cells of the osteoblastic lineage in bone. Therefore, although evidence from vitamin D-deficient rats, vitamin D receptor knockout mice, and patients with hereditary vitamin D-resistant rickets has suggested that 1,25-(OH)2D3 is not essential for the normal function of bone cells (30, 31, 32), pharmacological administration of 1,25-(OH)2D3 appears to be able to directly elicit an anabolic response in bone that can be used to increase cancellous bone mass in an osteopenic skeleton. Hopefully, further elucidation of the mechanisms underlying this bone anabolic effect of 1,25-(OH)2D3 may lead to improved treatment strategies for osteoporosis in the future. Although one should generally be cautious when extrapolating animal experimental findings to humans, this study would suggest that clinical treatment of osteoporotic patients with higher doses of vitamin D metabolites with a maintenance level of dietary calcium intake may be superior to treatment with lower doses of vitamin D metabolites in combination with a calcium supplement.


   Acknowledgments
 
The authors acknowledge the excellent technical assistance of Stefanie Engert, Barbara Amann, Claudia Baumeister, and Solveig Wiesmayr. We thank Prof. Klaus Osterkorn for his help with the statistical analyses.


   Footnotes
 
1 Presented in part at the 10th Workshop on Vitamin D, Strasbourg, France, May 1997, and at the 19th Annual Meeting of the American Society of Bone and Mineral Research, Cincinnati, OH, September 1997. This work was supported by Grant Er 223/1-1 from Deutsche Forschungsgemeinschaft (to R.G.E.). Back

Received February 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gallagher JC, Goldgar D 1990 Treatment of postmenopausal osteoporosis with high doses of synthetic calcitriol. A randomized controlled study. Ann Intern Med 113:649–655
  2. Tilyard MW, Spears GF, Thomson J, Dovey S 1992 Treatment of postmenopausal osteoporosis with calcitriol or calcium. N Engl J Med 326:357–362[Abstract]
  3. Shiraki M, Ito H, Orimo H 1993 The ultra long-term treatment of senile osteoporosis with 1{alpha}-hydroxyvitamin D3. Bone Miner 20:223–234[Medline]
  4. Fujita T 1992 Vitamin D in the treatment of osteoporosis. Proc Soc Exp Biol Med 199:394–399[Medline]
  5. Gallagher JC, Recker RR 1985 A comparison of the effects of calcitriol or calcium supplements on bone in postmenopausal osteoporosis. In: Norman AW, Schaefer K, Grigoleit H-G, von Herrath D (eds) Vitamin D. Chemical, Biochemical and Clinical Update. de Gruyter, Berlin, pp 971–975
  6. Narbaitz R, Stumpf WE, Sar M, Huang S, DeLuca HF 1983 Autoradiographic localization of target cells for 1{alpha},25-dihydroxyvitamin D3 in bones from fetal rats. Calcif Tissue Int 35:177–182[CrossRef][Medline]
  7. Ikeda T, Kohno H, Yamamuro T, Kasai R, Ohta S, Okumura H, Konishi J, Kikuchi H, Shigeno C 1992 The effect of active vitamin D3 analogs and dexamethasone on the expression of osteocalcin gene in rat tibiae in vivo. Biochem Biophys Res Commun 189:1231–1235[CrossRef][Medline]
  8. Erben RG, Scutt AM, Miao D, Kollenkirchen U, Haberey M 1997 Short-term treatment of rats with high dose 1,25-dihydroxyvitamin D3 stimulates bone formation and increases the number of osteoblast precursor cells in bone marrow. Endocrinology 138:4629–4635[Abstract/Free Full Text]
  9. Reichel H, Koeffler HP, Norman AW 1989 The role of the vitamin D endocrine system in health and disease. N Engl J Med 320:980–991[Medline]
  10. Colwell A, Russell RGG, Eastell R 1993 Factors affecting the assay of urinary 3-hydroxy pyridinium crosslinks of collagen as markers of bone resorption. Eur J Clin Invest 23:341–349[Medline]
  11. Schenk RK, Olah AJ, Herrmann W 1984 Preparation of calcified tissues for light microscopy. In: Dickson GR (ed) Methods of Calcified Tissue Preparation. Elsevier, Amsterdam, pp 1–56
  12. Erben RG, Kohn B, Rambeck WA, Zucker H 1990 Histomorphometric analysis of the rat proximal tibial metaphysis by "linear scanning." Scanning Microsc 4:625–638[Medline]
  13. Baron R, Vignery A, Neff L, Silverglate A, Santa Maria A 1983 Processing of undecalcified bone specimens for bone histomorphometry. In: Recker RR (ed) Bone Histomorphometry: Techniques and Interpretation. CRC Press, Boca Raton, pp 13–35
  14. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2:595–610[Medline]
  15. Erben RG, Bromm S, Stangassinger M 1998 Short-term prophylaxis against estrogen depletion-induced bone loss with calcitriol does not provide long-term beneficial effects on cancellous bone mass or structure in ovariectomized rats. Osteoporos Int 8:82–91[CrossRef][Medline]
  16. Wronski TJ, Halloran BP, Bikle DD, Globus RK, Morey Holton ER 1986 Chronic administration of 1,25-dihydroxyvitamin D3: increased bone but impaired mineralization. Endocrinology 119:2580–2585[Abstract]
  17. Erben RG, Weiser H, Sinowatz F, Rambeck WA, Zucker H 1992 Vitamin D metabolites prevent vertebral osteopenia in ovariectomized rats. Calcif Tissue Int 50:228–236[CrossRef][Medline]
  18. Erben RG, Bante U, Birner H, Stangassinger M 1997 1{alpha}-hydroxyvitamin D2 partially dissociates between preservation of cancellous bone mass and effects on calcium homeostasis in ovariectomized rats. Calcif Tissue Int 60:449–456[CrossRef][Medline]
  19. Gallagher JC, Jerpbak CM, Jee WS, Johnson KA, DeLuca HF, Riggs BL 1982 1,25-Dihydroxyvitamin D3: short- and long-term effects on bone and calcium metabolism in patients with postmenopausal osteoporosis. Proc Natl Acad Sci USA 79:3325–3329[Abstract/Free Full Text]
  20. Need AG, Horowitz M, Philcox JC, Nordin BE 1985 1,25-Dihydroxycalciferol and calcium therapy in osteoporosis with calcium malabsorption. Dose response relationship of calcium absorption and indices of bone turnover. Miner Electrolyte Metab 11:35–40[Medline]
  21. Martin TJ, Raisz LG, Rodan G 1987 Calcium regulation and bone metabolism. In: Martin TJ, Raisz LG (eds) Clinical Endocrinology of Calcium Metabolism. Marcel Dekker, New York, pp 1–52
  22. Qi H, Li M, Wronski TJ 1995 A comparison of the anabolic effects of parathyroid hormone at skeletal sites with moderate and severe osteopenia in aged ovariectomized rats. J Bone Miner Res 10:948–955[Medline]
  23. Erben RG, Bante U, Birner H, Stangassinger M 1997 Prophylactic effects of 1,24,25-trihydroxyvitamin D3 on ovariectomy-induced cancellous bone loss in the rat. Calcif Tissue Int 60:434–440[CrossRef][Medline]
  24. Erben RG 1996 Trabecular and endocortical bone surfaces in the rat: modeling or remodeling? Anat Rec 246:39–46[CrossRef][Medline]
  25. Pansu D, Bellaton C, Roche C, Bronner F 1983 Duodenal and ileal calcium absorption in the rat and effects of vitamin D. Am J Physiol 244:G695–G700
  26. Brommage R, Binacua C, Carrié A-L 1995 The cecum does not participate in the stimulation of intestinal calcium absorption by calcitriol. J Steroid Biochem Mol Biol 54:71–73[CrossRef][Medline]
  27. Chabanis S, Hanrotel C, Duchambon P, Banide H, Kubrusly M, Aymard P, Lacour B, Drueke T 1994 Duodenal calcium binding protein and active calcium transport in rats: are they functionally related? Nephrol Dial Transplant 9:1402–1407[Abstract/Free Full Text]
  28. Marie PJ, Travers R 1983 Continuous infusion of 1,25-dihydroxyvitamin D3 stimulates bone turnover in the normal young mouse. Calcif Tissue Int 35:418–425[CrossRef][Medline]
  29. Malluche HH, Matthews C, Faugere MC, Fanti P, Endres DB, Friedler RM 1986 1,25-Dihydroxyvitamin D maintains bone cell activity, and parathyroid hormone modulates bone cell number in dogs. Endocrinology 119:1298–1304[Abstract]
  30. Weinstein RS, Underwood JL, Hutson MS, DeLuca HF 1984 Bone histomorphometry in vitamin D-deficient rats infused with calcium and phosphorus. Am J Physiol 246:E499–E505
  31. Walters MR, Kollenkirchen U, Fox J 1992 What is vitamin D deficiency? Proc Soc Exp Biol Med 199:385–393[Medline]
  32. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, Jurutka PW 1998 The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 13:325–349[CrossRef][Medline]



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