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Endocrinology Vol. 138, No. 6 2449-2457
Copyright © 1997 by The Endocrine Society

ARTICLES

Parathyroid Hormone Increases Bone Formation and Improves Mineral Balance in Vitamin D-Deficient Female Rats1

Anne Toromanoff, Patrick Ammann, Lis Mosekilde, Jesper Skovhus Thomsen and Jean-Luc Riond

University of Zurich, Institute of Animal Nutrition (A.T., J.-L.R.), Winterthurerstrasse 260, CH-8057 Zurich; and the Department of Internal Medicine, Division of Clinical Physiopathology, University Hospital of Geneva (P.A.), CH-1211 Geneva 14, Switzerland; and Institute of Anatomy, Department of Cell Biology, University of Aarhus (L.M., J.S.T.), DK-8000 Aarhus C, Denmark

Address all correspondence and requests for reprints to: Dr. Anne Toromanoff, University of Zurich, Institute of Animal Nutrition, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland. E-mail: toro{at}vetphys.unizh.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study was designed to investigate whether enhanced bone formation due to intermittent PTH administration is dependent on vitamin D metabolites.

Forty-eight female Sprague-Dawley rats were randomized into four groups: 1) vitamin D-sufficient, saline-injected (+D Sal); 2) vitamin D-sufficient, human (h) PTH-(1–38)-treated (+D PTH); 3) vitamin D-deficient, saline-injected (-D Sal); and 4) vitamin D-deficient, hPTH-(1–38)-treated (-D PTH) animals. The -D diet contained 2% calcium (Ca), 1.25% phosphorus (P), and 20% lactose to maintain normocalcemia and normophosphatemia despite vitamin D deficiency. The +D diet contained 0.8% Ca, 0.5% P, 20% lactose, and 1000 IU/kg vitamin D. After 45 days of either diet, the rats were injected with 50 µg/kg BW PTH or saline, sc, daily for 2 weeks.

Serum Ca, Mg, P, albumin, and creatinine were similar in all groups. PTH administration decreased endogenous PTH concentrations in the -D PTH compared with those in the -D Sal group. Serum alkaline phosphatase activity, bone mass measurements, dual energy x-ray absortiometric analysis of mineral density, and mechanical testing values in vertebrae and femora of the -D Sal animals did not significantly differ from those in +D Sal animals. Moreover, in both diet groups, PTH improved bone biochemical activity (as assessed by serum alkaline phosphatase), bone mass, mineral density, and biomechanical properties. These results indicate that mineral supply, more than vitamin D itself, may be important for normal bone mineralization and to enable PTH to enhance bone formation. A balance study performed during the last 3 days of the experiment revealed that PTH increased apparent intestinal magnesium absorption in the +D group only. Ca and P retention, however, were augmented in both diet groups after PTH treatment.

In conclusion, in normocalcemic and normophosphatemic -D rats, PTH treatment reduced the increased endogenous hormone concentration and improved Ca and P retention. Furthermore, PTH may have a vitamin D-dependent influence on intestinal magnesium absorption. Finally, short term PTH treatment is anabolic in bone of vitamin D-deficient rats when adequate mineral amounts are provided in the diet.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUMEROUS STUDIES have shown that intermittent administration of parathyroid extract, purified PTH, or synthetic PTH N-terminal fragments is anabolic in the bone of healthy (1, 2, 3, 4, 5) and osteopenic (6, 7, 8) animals (reviewed in Refs. 9 and 10). In the kidney, PTH enhances the 1{alpha}-hydroxylation of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D \[1,25-(OH)2D\]. This vitamin D metabolite, in turn, permits an optimal intestinal calcium (Ca) and phosphorus (P) absorption necessary to mineralize newly formed bone (11). 1,25-(OH)2D, like PTH, belongs to the most important group of bone regulatory hormones. It regulates osteoclastic differentiation from hematopoietic mononuclear cells, and osteoblastic functions and activity (12, 13, 14).

Although the regulatory effects of PTH and 1,25-(OH)2D in bone have been extensively studied, relatively few data concerning their mutual in vivo interactions are available (15, 16, 17, 18, 19). It remains to be clarified whether 1,25-(OH)2D (or any other vitamin D metabolite) is required for PTH to enhance bone formation. This question is of interest regarding a possible treatment of osteoporosis with PTH or anabolic PTH analogs (10). PTH-stimulated augmentation of the bone apposition rate has been shown to be dependent on the presence of vitamin D in rats fed normal or low Ca diets (16, 18). On the other hand, PTH-treated vitamin D-deficient rats were reported to have an increased bone mass compared with vitamin D-deficient untreated controls (20), pointing to a possible vitamin D-independent anabolic effect of PTH on bone. When vitamin D-deficient animals are made normocalcemic by experimental or dietary means, no osteomalacia occurs (21, 22, 23, 24, 25, 26). This suggests that the mineral supply, more than the direct action of vitamin D metabolites, may be critical for the mineralization of newly formed bone. To our knowledge, there is no report in the literature on the effect of PTH administration to vitamin D-deficient animals fed a high Ca and P diet.

The present study was designed to answer the question of whether the administration of PTH at a dose that is able to stimulate bone formation and increase the Ca and P balance in normal rats is also efficacious in vitamin D-deficient, normocalcemic and normophosphatemic animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, diets, and treatment
In a preliminary study performed to test the effects of experimental diets on serum vitamin D metabolites, 20 4-week-old female rats of the Sprague-Dawley strain (ZUR:SIV, Institute for Laboratory Animal Science, University of Zurich, Zurich, Switzerland), weighing 106 ± 4 g on arrival, were used. They were randomly assigned to 2 diet groups: 1 group received a vitamin D-deficient (-D) diet containing 2% Ca, 1.25% P, and 20% lactose (Kliba 343–08, Kaiseraugst, Switzerland). This diet has been reported to prevent hypocalcemia and hyperparathyroidism in vitamin D-deficient rats (25). The second group was fed a control (+D) diet with 0.8% Ca, 0.5% P, 1000 IU/kg vitamin D and 20% lactose (Kliba 343–03, Kaiseraugst, Switzerland). After 6 weeks, blood was collected from the tail vein under anesthesia with 100 mg/kg BW ketamine (Narketan, Chassot, Belp, Switzerland) and 5 mg/kg BW xylazine (Rompun, Bayer, Leverkusen, Germany), ip. Serum 25OHD was measured. After 8 weeks, blood was taken by cardiac puncture, and serum 25OHD and 1,25-(OH)2D were determined.

The main study involved 48 4-week-old female rats, weighing 98 ± 1 g, that were randomly assigned to 2 diet groups (+D and -D; n = 24/group). They were housed individually in hanging wire cages in a temperature-controlled room (21 C) with artificial lighting without a UV source. The light cycle was 14 h of light, 10 h of darkness. The rats were fed ad libitum and had free access to tap water, except during the balance study. Body weight and food and water intake were recorded daily throughout the experiment. Forty-five days after the beginning of the study, each diet group was randomly divided into 2 treatment groups (n = 12/group). The treated animals (+D PTH and -D PTH) were given 50 µg/kg BW PTH, sc, daily for 14 days and the controls (+D Sal and -D Sal) were injected with vehicle solution only. PTH [hPTH-(1–38); Sandoz, Basel, Switzerland; a gift from Dr. J. Gasser] was diluted in saline solution. On the day after the last PTH or control injection, blood was harvested from the heart in evacuated tubes (Vacutainer, Becton Dickinson, Basel, Switzerland) under sodium pentobarbital anesthesia (3 mg/kg BW, ip; Vetanarcol, Veterinaria, Zurich, Switzerland) and spun in a refrigerated centrifuge to separate the serum, which was frozen at -20 C until use. After death, the femora and the second, fifth, and sixth lumbar vertebrae were excised and frozen at -20 C for subsequent analysis.

Balance study
Fifty-five days after the beginning of the experiment, dietary Ca, magnesium (Mg), and P retention was determined during 3 days. The rats were given deionized water instead of tap water. Feces and urine were collected from each animal between two oral carmine red (Sigma Chemie, Buchs, Switzerland) administrations. Stool was separated from urine using a plastic grid placed under each cage. The feces samples were ashed in a muffle furnace at 600 C for 72 h; the ash was diluted in 25 ml 3 M HCl and analyzed for mineral content. Urine was collected on sheets of ashless filter paper (Schleicher and Schuell, Riehen, Switzerland) that were dried at 105 C for 24 h, weighed, and ashed at 600 C for 24 h. The ash was diluted in 7 ml 3 M HCl, and the mineral content was measured. The amounts of Ca, Mg, and P in the ashless paper itself were also determined. A standard curve was established, and the Ca, Mg, and P measurements were corrected for these blank values. The percent apparent intestinal absorption was calculated for each animal as ([(mean intake/24 h) - (mean fecal loss/24 h)]/[mean intake/24 h]) x 100. The balance was calculated as ([mean intake/24 h] - [(mean fecal loss/24 h) + (urinary loss/24 h)]).

Assessment of bone mass
The dry and ash weights of the bones were measured after 96 h at 105 C and 96 h at 600 C. The area and bone mineral content of the sixth lumbar vertebrae and the left femora were measured by a Hologic QDR-1000 dual energy x-ray absorptiometer as previously described (27), and the bone mineral density (BMD) was calculated from these measurements. The vertebrae and femora were analyzed as a whole, and four equal regions of the femora were also analyzed separately (28). The proximal region is described as R1, the proximal and distal midshafts as R2 and R3, and the distal femur as R4. In the R1, a frame including the femoral head with the external vertical limit being at one third the width of the trochanter major, and the inferior horizontal limit cutting the top of the trochanter minor, was separately analyzed (femoral neck region or R5).

Determination of biomechanical competence of lumbar vertebral body (L5)
The vertebrae were freed of soft tissue, and prepared and tested as previously described (29). Briefly, a central cylinder with plano-parallel ends and a height of approximately 3.6 mm was obtained from each vertebral body. The cylinder consisted of a central trabecular bone core and the cortical rim. Its volume was estimated by weighing before and after immersion in water, and its height was measured using a micrometer. The average cross-sectional area of each specimen was obtained by dividing the volume by the height. The vertebral body cylinders were tested along the proximal-distal axis in a materials-testing machine (Alwetron 250, Lorentzen and Wettre, Stockholm, Sweden) at a nominal deformation of 2 mm/min. Load and deformation were simultaneously recorded every 0.01 sec. The maximum load (expressed in Newtons) and the maximum deformation (in millimeters) were directly obtained from the load-deformation curves, and the following biomechanical parameters were calculated (30): the stress (load divided by area, in megapascals), the rigidity (or extrinsic stiffness, maximum slope of the load-deformation curve, in Newtons per mm), the energy absorbed by the bone tissue (i.e. the area under the load-deformation curve before the bone breaks (in millijoules), and the energy absorption divided by the volume of the vertebra (in millijoules per mm3).

Determination of biomechanical competence of the femoral neck
The femora were prepared and analyzed as previously descried (28). Briefly, the shaft was embedded in a methylmethacrylate cement (Technovit 4071, Haraeus Kulzer, Wehreim, Germany) up to the minor trochanter. A vertical load was exerted on the femoral head by a servo-controlled electromechanical system (Instron 1114, Instron Corp., High Wycombe, UK) at a speed of 2 mm/min. Load and deformation were simultaneously recorded every 0.01 sec. The rigidity and energy absorbed were calculated in the same way as for vertebrae.

Biochemical analysis
The serum concentrations of total Ca and Mg, inorganic P, albumin, alkaline phosphatase, and creatinine and the P content of urine and feces were analyzed with automatized colorimetric or photometric methods (Cobas-Mira, Hoffman-La Roche, Basel, Switzerland).

Serum N-terminal PTH, 25OHD, and 1,25-(OH)2D were measured with competitive protein-binding assays (Nichols Institute Diagnostics, Allschwil, Switzerland).

The Ca and Mg contents of urine and feces were measured using atomic absorption spectrometry (Varian AA-1275, Varian International, Basel, Switzerland) after dilution with LaCl3.

Statistical analysis
Statistical analysis was performed using the Systat for Windows program (Systat version 5.0, Systat, Evanston, IL). All data are presented as the mean ± 1 SE. Comparisons between groups were made using Fisher’s least significant differences test after two-way ANOVA to examine the effects of diet, treatment, and interaction between diet and treatment. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth, and water and food intakes
There were no significant differences between groups for body weight at any time during the study. The final (day 59) body weights were 264 ± 11 g for the +D PTH animals, 260 ± 10 g for the +D Sal group, 263 ± 7 g for the -D PTH, and 266 ± 11 g for -D Sal. Daily food and water consumptions were similar in all groups (data not shown).

Serum biochemical analysis
In the -D group, serum 25OHD concentrations were under the detection limit of the assay (2.2 ng/ml) 6 (data not shown) and 8 weeks after starting the preliminary experiment (Table 1Go). After 8 weeks of the vitamin D-deficient regimen, 1,25-(OH)2D was still detectable in the serum of -D rats, but its concentration was significantly lower than that in the +D animals.


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  		Table 1. Serum 25OHD and 1,25-(OH)2D measured in female rats after 8 weeks of the experimental diets

 
The mean serum Ca, Ca corrected for albumin, Mg, P, and creatinine levels were not significantly different between groups (Table 2Go). PTH treatment significantly increased serum alkaline phosphatase when pooled -D PTH and +D PTH groups were compared with pooled +D Sal and -D Sal groups.


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  		Table 2. Serum biochemical parameters measured in the serum of rats after 59 days of the experimental diets and 14 days of daily saline (Sal) or hPTH-(1–38) injections

 
The serum rat PTH N-terminal level tended to be higher in the -D Sal animals than in the other groups (Fig. 1Go). In the -D group, PTH treatment significantly reduced immunoreactive PTH by 35% compared with that in the saline-treated animals.



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  		Figure 1. Rat PTH N-terminal was measured in the serum of the rats after 59 days of the experimental diets and 14 days of daily hPTH-(1–38) or saline injections. , +D Sal; {blacksquare}, +D PTH; {square}, -D Sal; {blacksquare}, -D PTH. **, P < 0.01 vs. respective Sal group.

 
Bone mass
In the -D Sal group, the wet, dry, and ash weights of the vertebrae and femora were not significantly different from those in the +D Sal animals (Table 3Go).


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  		Table 3. Mass measurements performed on the bones of the rats subjected to the experimental diets for 59 days and to 14 days of daily saline (Sal) or hPTH-(1–38) injections

 
hPTH-(1–38) treatment for 14 days increased bone mass in vertebrae and femora of the +D and -D animals (Table 3Go). The wet and dry weights of the lumbar vertebrae of the +D PTH rats increased slightly, but not significantly, compared with those in the control group. In the -D PTH rats, however, the wet, dry, and ash weights were significantly increased by 12%, 11%, and 11% compared with those in the -D Sal animals. The wet and dry weights in the -D PTH group reached significantly higher values than those in the +D PTH group. For the femora, the dry and ash weights of the +D PTH group were 2% and 3% higher than those in controls, but this increase was significant only for dry weight. In the -D PTH group, the dry and ash weights of the femora were 7% higher than those in the -D Sal animals.

BMD
PTH treatment augmented BMD in both vertebrae and femora of the +D and -D rats (Table 4Go). In the vertebrae of the +D PTH group, the increase reached 11% compared with that in the +D Sal rats, and in the -D animals the BMD of the PTH-treated group was 8% higher than that in the saline-injected group (Table 4Go, top). In the whole femora, the differences were also significant, but less pronounced (+3.5% in the +D PTH vs. +D Sal and +6% in the -D PTH group vs. -D Sal). When different regions of the femora were separately analyzed, the strongest effect of PTH occurred in the distal part (7% increase in the +D PTH vs. +D Sal, and 10% in the -D PTH vs. -D Sal group).


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  		Table 4. Bone mineral density of the rats subjected to the experimental diets for 59 days and to 14 days of daily saline or hPTH-(1–38) injections

 
Mechanical properties of the bones
The diet regimen itself did not significantly affect any of the tested biomechanical parameters in the vertebrae or femoral neck (Table 5Go). In the vertebrae, PTH treatment improved the maximal load in both diet groups slightly, but not significantly, but when this parameter was normalized for cross-sectional area (stress), the increase was significant in both -D and +D groups (+D PTH and -D PTH were 18% and 21% higher compared with +D and -D Sal groups). The rigidity tended to increase in the -D PTH animals compared with that in the -D Sal group. In both +D and -D groups, PTH augmented the energy absorbed by the bone (respectively, 24% and 20%), but this increase was significant only in the +D animals. However, when the energy absorbed was normalized by the volume of the vertebrae, the values were significantly higher in both +D and -D PTH groups (38% increase for the +D PTH group compared with the +D Sal and 24% increase for the -D PTH group compared with the -D Sal animals).


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  		Table 5. Biomechanical properties tested on the bones of the rats after 59 days of the experimental diets and 14 days of daily saline or hPTH-(1–38) injections

 
In the femoral neck, the maximum load increased by 14% in the -D PTH rats compared with that in the -D Sal group, but this increase was not significant. In contrast to the vertebra, the energy absorbed by the femoral neck was not significantly affected by PTH in either -D or +D animals. The rigidity increased after PTH treatment in both +D and -D groups (34 and 18%), but the increase was significant only in the +D animals.

Balance study
PTH treatment significantly increased the percent apparent Ca absorption (Fig. 2AGo) by 63% in the +D group and by 40% in the -D. It did not significantly affect urinary Ca excretion in the +D group (Fig. 2BGo). In the -D PTH group, however, urinary Ca was 30% lower than the level in the -D Sal animals. PTH significantly increased Ca balance in both dietary groups (Fig. 2CGo). The effect of the treatment was greater in +D (+68%) than in -D (+44%) animals.



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  		Figure 2. Dietary Ca retention was measured during the last 3 days of the experiment, starting after 55 days of the experimental diets and 11 days of daily saline or hPTH 1–38 injections. , +D Sal; {blacksquare}, +D PTH; {square}, -D Sal; {blacksquare}, -D PTH. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. respective Sal group).

 
The diet significantly altered the effect of PTH treatment on the apparent intestinal Mg absorption. PTH slightly, but significantly, increased (by 6%) the apparent intestinal absorption of Mg in the +D group (Fig. 3AGo) and was without effect on this parameter in the -D animals. The diet affected urinary Mg excretion, which was 28% lower in the -D than in the +D group (Fig. 3BGo). Mg balance was not influenced by the treatment, but the -D rats retained significantly more Mg than the +D animals (Fig. 3CGo).



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  		Figure 3. Dietary Mg retention was measured during the last 3 days of the experiment, starting after 55 days of the experimental diets and 11 days of daily saline or hPTH-(1–38) injections. , +D Sal; {blacksquare}, +D PTH; {square}, -D Sal; {blacksquare}, -D PTH. **, P < 0.01 (vs. Sal group). §, P < 0.05; §§, P < 0.01 (vs. respective +D group).

 
The diet affected the apparent P absorption, which was reduced by 32% in the -D animals compared with that in the +D group (Fig. 4AGo). PTH treatment significantly increased the apparent intestinal P absorption by 15% when both +D PTH and -D PTH were compared with both +D and -D saline groups. Urinary P excretion was significantly reduced by 22% in the -D group compared with that in the +D animals (Fig. 4BGo). The treatment had no significant effect on this parameter. The diet had no significant influence on P balance, but PTH treatment did. Both PTH-treated groups (+D and -D) conserved 28% more P than either saline group (Fig. 4CGo).



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  		Figure 4. Dietary P retention was measured during the last 3 days of the experiment, starting after 55 days of the experimental diets and 11 days of daily saline or hPTH-(1–38) injections. , +D Sal; {blacksquare}, +D PTH; {square}, -D Sal; {blacksquare}, -D PTH. §§, P < 0.01 (vs. respective +D group). #, P < 0.05 (both -D and +D PTH vs. both -D and +D Sal).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present experiment was undertaken to examine whether enhanced bone formation due to intermittent PTH administration occurs in vitamin D-deficient normocalcemic and normophosphatemic animals. The results showed that bone mass, BMD, and biomechanical properties of the -D rats were comparable to those of the +D animals. Moreover, compared with respective controls, both -D and +D rats had increased bone formation after PTH treatment.

As previously shown (31, 32, 33), the diet regimen devised by Kollenkirchen et al. (25) proved to be a good tool, as normocalcemia and normophosphatemia could be achieved in all vitamin D-deficient rats. Another crucial point in a study on vitamin D-deficient animals is to assess the degree of vitamin D deficiency. In the preliminary experiment, the serum 25OHD level of each rat in the vitamin D-deficient group was below the limit of detection of the assay. These rats still had detectable serum 1,25-(OH)2D, which is a relatively common feature in studies of vitamin D-deficient animals, as complete depletion is difficult to achieve (34). Interestingly, the rats fed the +D diet also had low serum 1,25-(OH)2D concentrations. Plasma 1,25-(OH)2D levels have been reported to be about 25–50 pg/ml in normal adult rats (34). We used the same RIA to measure serum 1,25-(OH)2D concentrations in 12-week-old female rats fed a standard laboratory chow without lactose. In these rats, the mean serum 1,25-(OH)2D concentration was about 75 pg/ml (our unpublished observations), much higher than that in the +D animals of the present study. The low serum 1,25-(OH)2D level measured in the present experiment (9 ± 2 pg/ml) is, therefore, not likely to be due to an underestimation caused by any technical problem, but may be related to the presence of lactose in our control diet. Lactose increases Ca bioavailability (35, 36), and serum 1,25-(OH)2D concentrations and intestinal actions have been shown to be decreased by high Ca intake in vitamin D-deficient and -sufficient rats (31, 36, 37). Bruns et al. (37) reported that an increase in the Ca content of the diet, from 0.1% to 1.6%, led to a 5-fold decrease in the circulating 1,25-(OH)2D concentrations in normal nonlactating female rats. In vitamin D- and lactose-supplemented animals, the Ca and P requirements may be very low. As the regulation of renal 1{alpha}-hydroxylase is determined by the Ca and P needs of the organism (13), the 1{alpha}-hydroxylase activity and/or amount may be strongly down-regulated in the +D rats. In earlier work using the same -D diet, no +D controls with lactose were used (25, 31, 32, 33). There is, to our knowledge, no data in the literature about 1,25-(OH)2D concentrations in vitamin D-replete, lactose-supplemented animals.

Although the measured total serum Ca and phosphate concentrations were in the normal range in all groups, endogenous PTH levels were slightly increased in the -D, saline-injected rats, compared with those in the vitamin D-sufficient control animals. This slight secondary hyperparathyroidism may be necessary for the animals of the -D Sal group to maintain normocalcemia. It may be allowed and/or strengthened by the lack of the negative feedback control of 1,25-(OH)2D on the synthesis and secretion of PTH (38, 39). This finding, nevertheless, contrasts with previous reports of some experiments using the same diet (25, 31, 32). This discrepancy may be caused by the sex (all of the cited studies were performed in male rats, and only females were used in the present experiment) or the strain of the animals. Brown et al. (33), however, who also used the same diet, reported significantly higher PTH concentrations in vitamin D-deficient control rats than in vitamin D-deficient 1,25-(OH)2D-replete animals, without enlargement of the parathyroid gland in the first group.

As previously observed in humans (40), PTH administration was paradoxically able to reduce the circulating immunoreactive PTH N-terminal concentration. We interpreted this result as a reduction of the synthesis and/or the secretion and/or an increase in the peripheral metabolism of the endogenous hormone in the -D group, because the blood was taken 24 h after the last injection, and the half-life of PTH in vivo is on the order of a few minutes (39). The mechanism by which exogenous PTH may exert this negative regulation of endogenous circulating PTH concentrations remains to be determined.

Our work corroborates earlier studies showing that bone in vitamin D-deficient rats may be normal or slightly subnormal when the mineral supply is adequate (21, 23, 24, 25, 26, 31). Bone mass (as assessed by wet, dry, and ash weights), BMD, mechanical properties, and biochemical activity (assessed by serum alkaline phosphatase levels) were all comparable to the control +D values. This confirms and further supports previous findings (23, 24) indicating that the direct role of 1,25-(OH)2D in bone is less important than its action on the intestine. Moreover, the results presented here demonstrate that in vitamin D-deficient rats, when the mineral supply is maintained high enough by an adequate diet, PTH increases bone mass, BMD, and biomechanical properties, suggesting an augmentation of osteoid production and mineralization in addition to an improvement of the quality of the bones, even in vitamin D-deficient rats. This might appear to be in contradiction to results reported previously (16, 17). However, the experimental conditions in these studies were different from ours. Tam et al. (16) used vitamin D-deficient, parathyroid extract-treated rats fed a low Ca diet. Marcus et al. (17) used a normal Ca diet that probably did not allow sufficient intestinal Ca mobilization, and the vitamin D-deficient thyroparathyroidectomized rats were continuously infused with PTH. Continuous PTH infusions have been shown to have greater catabolic than anabolic effects on bone (2, 41). We provided evidence that the anabolic effect of PTH on bone is not dependent on vitamin D, at least after a short term PTH treatment.

For some parameters (e.g. bone mass of the vertebrae and BMD of the femora), the effect of PTH seemed to be more pronounced in -D than in +D animals. There may be several reasons for this. First, as the endogenous PTH was reduced by the PTH administration in the -D group, a possible negative action of the endogenous hormone on bone may have been suppressed in the -D PTH rats. This reduction of a catabolic effect may be additive to the anabolic effect of the exogenous PTH. Second, the bone resorption may have been impaired by the vitamin D deficiency, and the PTH-induced bone formation may have occurred in partial absence of resorption and, for this reason, be more rapidly obvious than that in normal animals. This hypothetical impaired bone resorption could occur without inhibiting the formation, as the anabolic action of PTH on bone has been shown to be independent of bone resorption in the rat model (42). Finally, it has been shown that 1,25-(OH)2D infusion may retard bone mineralization (43); its very low level in the -D group may have allowed a faster mineralization of the PTH-stimulated, newly formed bone than in normal (+D) conditions.

As previously reported, PTH treatment was able to stimulate the fractional apparent intestinal Ca, Mg, and P absorption in the +D group (44, 45, 46). For Ca and P, this enhancement is thought to be due to the PTH-stimulated increase in renal hydroxylation of 25OHD to 1,25-(OH)2D, which directly acts on the active absorption in the intestine (47). The stimulation of intestinal Mg absorption may also occur through vitamin D-dependent mechanisms and/or be inhibited by the high dietary Ca and P contents of the vitamin D-deficient diet, because it was completely abolished in the -D group. 1,25-(OH)2D administration to vitamin D-deficient rats has been shown to stimulate intestinal Mg absorption (48), and increased dietary Ca and P levels have been shown to decrease it (49, 50).

In the vitamin D-deficient rats, we cannot completely rule out the hypothesis that the remaining 1,25-(OH)2D amounts together with an intestinal up-regulation of the vitamin D receptor (VDR) may be responsible for the normal apparent intestinal Ca absorption observed. This, however, seems unlikely; first, because the apparent intestinal Ca and P absorptions were unequally affected by vitamin D-deficient diet, and second, because the vitamin D levels and effects are impaired when vitamin D-deficient animals are fed a high Ca diet (36, 37), and an up-regulation of the intestinal VDR in vitamin D deficiency has never been reported. More probably, the vitamin D-deficient animals of this study lacked the homologous up-regulation of the VDR by 1,25-(OH)2D (13). This might be strengthened by the negative effect of PTH on the intestinal VDR homologous up-regulation (51). However, as PTH treatment in vitamin D-deficient rats decreased the endogenous PTH levels, the homologous up-regulation of the VDR might have been less blocked in the -D PTH than in the -D Sal group. In this case, small circulating amounts of 1,25-(OH)2D might have more effect in the -D PTH than in the -D Sal animals, as the effect of vitamin D is dependent on receptor number and occupancy (13).

The vitamin D-independent mechanisms of Ca and P absorption are thought to be mainly nonsaturable and to occur by a paracellular pathway (52, 53). Although these mechanisms may be considered physiologically more important than the vitamin D-dependent active processes, because the bulk of Ca, Mg, and P is absorbed in this way (54), their regulations are not really understood. They are not thought to be subject to any rapid hormonal regulation, but a long term adaptive one has never been excluded. A possible regulation of the paracellular pathway has been postulated (55). Lactose is thought to increase intestinal Ca and P absorption by passive and perhaps also active mechanisms (56, 57, 58). For Ca, the effect of lactose has been shown to occur independently of vitamin D (35). In the present experiment, PTH might have been able to further increase the lactose-enhanced intestinal Ca and P absorption in both +D and -D animals. A vitamin D-independent increase in Ca absorption was also demonstrated during pregnancy and lactation in the rat, which may not only be due to the intestinal hypertrophy observed in these conditions (59, 60). A similar adaptation mechanism(s) may have occurred in the -D, PTH-treated animals. Moreover, as PTH receptor messenger RNA was identified in the rat intestine (61), the hormone may act directly in this organ. In normal chickens, PTH is able to rapidly stimulate intestinal Ca and P absorption in vivo (62, 63). This direct effect of PTH also occurs in isolated rat intestinal cells in vitro, independently of any new 1{alpha}-hydroxylation of 25OHD (64, 65).

PTH treatment did not affect Ca, Mg, or P excretion in the +D group. Our method of urine collection does not allow us to correct for the glomerular filtration rate, so the present data on mineral excretion have to be interpreted with caution. The diet affected mineral excretion more than the treatment. In the -D animals, PTH significantly decreased the excretion of Ca, probably by enhancing the tubular Ca reabsorption (66). The urinary Mg excretion was markedly decreased in the -D animals. This effect may be due to the suppression of the 1,25-(OH)2D-stimulated increase in Mg excretion that has been previously described (67). The elevated PTH in the -D Sal group or the PTH injections in the -D PTH group may also be partly responsible for this effect, as PTH has been demonstrated to increase renal Mg reabsorption (68). However, this hypothesis is not very likely, as P excretion was also decreased in the -D animals. It is difficult to impute this effect to PTH, because PTH decreases renal P reabsorption (66), at least in normal animals.

In conclusion, a short term intermittent PTH administration may have anabolic effects on bone of vitamin D-deficient rats when adequate mineral amounts are provided in the diet. Furthermore, under conditions of adaptation to a long term vitamin D deficiency, PTH treatment is able to reduce the increased endogenous hormone concentration and to improve dietary Ca and P retention. On the other hand, PTH may have a vitamin D-dependent influence on intestinal Mg absorption.


   Acknowledgments
 
The authors gratefully acknowledge the expert technical assistance of Isabelle Badoud, Rhea Forrer, Birthe Gylling-Jørgensen, Brigitte Küffer, and Barbara Schneider, and the help and critical advice of Dr. Elvira Del Prete, Dr. Jürg A. Gasser, Dr. Nese Kocabagli, Dr. Deborah Langford, Dr. Thomas Lutz, and Prof. Marcel Wanner.


   Footnotes
 
1 This work was supported by the Swiss National Research Foundation (Grant 32–33853-92). Presented in part at the 50th Meeting of the German Society for Nutrition Physiology, Gottingen, Germany, February 1996 (Proc Soc Nutr Physiol 5, Abstract 58, 1996), and at the Second Meeting of the Swiss Bone and Mineral Society, Bern, Switzerland May 2, 1996. Back

Received December 16, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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