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Keywords:

  • bone mineral density;
  • bone mineral content;
  • calcium absorption;
  • histomorphometry;
  • bone formation;
  • osteoclast

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The effects of the dietary Ca and P ratio, independent of any vitamin D effects, on bone mineralization and turnover was examined in 60 VDRKO mice fed different Ca/P ratio diets. High dietary Ca/P ratio promoted bone mineralization and turnover with adequate intestinal Ca and P transports in VDRKO mice.

Introduction: To clarify the effects of the dietary calcium (Ca) and phosphorus (P) ratio (Ca/P ratio) on bone mineralization and turnover in null-vitamin D signal condition, vitamin D receptor knockout (VDRKO) mice were given diets containing different Ca/P ratios.

Materials and Methods: Five groups of 4-week-old VDRKO mice, 10 animals each, were fed diets for 4 weeks. Group 1 was wild-type littermate mice, fed the diet containing 0.5% Ca and P (Ca/P = 1). Group 2 was the control and was fed a similar diet (Ca/P = 1). Groups 3, 4, 5, and 6 were fed the following diets:0.5% Ca and 1.0% P (Ca/P = 0.5), 1.0% Ca and 1.0% P (Ca/P = 1), 1.0% Ca and 0.5% P (Ca/P = 2), and 0.5% Ca and 0.25% P (Ca/P = 2).

Results and Conclusions: Compared with group 2, serum calcium and phosphorus levels in groups 4–6 significantly increased. Serum parathyroid hormone levels increased in group 3 and decreased in group 5. The amounts of intestinal calcium absorption decreased in groups 3 and 4. Phosphorus absorption increased in group 3 and decreased in groups 4–6. Bone mineral content (BMC) and bone mineral density (BMD) of the femur in group 3 significantly decreased and increased in group 5. In the primary spongiosa of the proximal tibia, the trabecular bone volume (BV/TV) and osteoid thickness (O.Th) in group 3 significantly increased, and decreased in group 6. In groups 5 and 6, the numbers of the trabecular osteoclasts increased. In groups 2 and 4, and the secondary spongiosa was identified in 5 of 10 mice. In group 3, there was no secondary spongiosa in either mouse. Osteoid maturation time (OMT) significantly decreased, and bone formation rate (BFR/BS) increased in groups 4–6. These data indicate that the dietary Ca/P ratio regulates bone mineralization and turnover by affecting the intestinal calcium and phosphorus transports in VDRKO mice. They may suggest the existence of Ca/P ratio-dependent, vitamin D-independent calcium and phosphorus transport system in the intestine.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Skeletal phenotype in a vitamin D receptor-ablated condition is characterized by impaired mineralization, reduced bone turnover, and abnormalities associated with low serum calcium and phosphorus levels and high serum parathyroid hormone (PTH) levels.(1) Normalization of serum calcium and phosphorus levels by the oral and intravenous treatment of calcium and phosphorus improves bone and mineral homeostasis in vitamin D receptor knockout (VDRKO) mice(1,2) and vitamin D-deficient rats.(3–6) These data indicate that bone mineralization and turnover are preserved if calcium and phosphorus treatments are properly maintained, even when the vitamin D receptor signal is lacking.

Intestinal absorption is the first gateway for calcium and phosphorus in the body. Net intestinal calcium absorption reflects the sum of the two different mechanisms: active, vitamin D-dependent absorption and passive absorption depending on the concentration gradient between intestinal lumen and blood.(7) The total amount of dietary calcium intake affects the level of absorption. On the other hand, excess in the available phosphate may reduce the passive shift of calcium by forming insoluble salts in the intestinal lumen. Thus, the relative ratio of calcium and phosphorus intakes seems to be another factor to regulate the intestinal calcium absorption. Epidemiologically, a reduced intake ratio of calcium to phosphorus is associated with a reduction in bone mineral density (BMD) in healthy perimenopausal women.(8,9) Recently, it was reported that excess intake of calcium without appropriate phosphorus supplementation reduced BMD in postemenopausal women.(10) In rats, chronic phosphorus supplementation decreased intestinal calcium absorption, leading to a decline in serum calcium concentration and resultant hyperparathyroidism.(11–13) However, these studies on the vitamin D-replete condition were not able to discriminate the role of the ratios of dietary calcium and phosphorus intakes on bone and mineral homeostasis.

An increase in dietary calcium improved bone mineralization and turnover,(1,2) and a restriction of phosphorus intake improved hypocalcemia, bone mineral content (BMC), and strength of femur in VDRKO mice.(14) Thus, we hypothesized that the dietary calcium and phosphorus ratio (Ca/P ratio) plays an important role in bone and mineral homeostasis the in null vitamin D signal condition. In this experiment, we compared serum levels and intestinal absorptions of calcium and phosphorus, BMC and BMD, and bone mineralization and turnover in VDRKO mice fed diets containing different ratios of calcium and phosphorus.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Experimental design

Wild-type and VDRKO mice established by Yoshizawa et al.(15) were bred and maintained on laboratory chow containing 1.0% calcium and phosphorus (CE-2; Clea, Tokyo, Japan). Null mutant mice were obtained by intercrossing a heterozygous a VDRKO female and a heterozygous male, and the wild-type littermate mice were used for the analyses. After weaning at 3 weeks of age, all mice were fed the control diet containing 0.5% calcium (Ca) and 0.5% phosphorus (P). Then, VDRKO mice were divided into five dietary groups of 10 mice each (5 males and 5 females) and fed one of five diets: the control diet (Ca/P = 1.0; group 2), a diet containing 0.5% calcium and 1.0% phosphorus (Ca/P = 0.5; group 3), a diet containing 1.0% calcium and phosphorus (Ca/P = 1.0; group 4), a diet containing 1.0% calcium and 0.5% phosphorus (Ca/P = 2.0; group 5), or a diet containing 0.5% calcium and 0.25% phosphorus (Ca/P = 2.0; group 6). All test diets contained 20% lactose and were composed of calcium carbonate (CaCO3) and potassium dihydrogenphosphate (KH2PO4) as the dietary calcium and phosphorus source. Amounts of calcium and phosphorus in the test diets were confirmed in advance. Wild-type littermates (group 1) were continually fed the control diet during the experimental period. Mice were allowed free access to the assigned diet and water for 4 weeks. The animals were housed individually in stainless steel cages in a room maintained at 22°C with a 12-h light-dark cycle. At the end of the period, blood was collected from the abdominal aorta while the animal was anesthetized; the animal was killed, and both tibias and femurs were obtained for further analyses. The Tokyo University of Agriculture Animal Use Committee approved the study, and the animals were maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals of the University.

Balance study

The amount of food ingested was measured weekly. All feces and urine were collected from each mouse. There was a fine-meshed steel net under the wire cage; all urine passed through the net and was collected separately from feces. The absorption of calcium and phosphorus during the 4-week period was calculated as the difference between the amounts of oral intake and fecal excretion. The retention of calcium and phosphorus in the body was calculated as the difference between the amount of absorption and excretion in urine. Thus, their absolute amount (mg) and percent (%) values were calculated from the following equations: intestinal absorption (mg) = oral intake (mg) − fecal out put (mg), intestinal absorption (%) = intestinal absorption (mg)/oral intake (mg) × 100, retention (mg) = intestinal absorption (mg) − urine excretion (mg), and retention rate (%) = retention (mg)/oral intake (mg) × 100.

Measurements of calcium and phosphorus contents in feces and urine

Feces and urine were collected throughout the experimental period. They were dried, ashed, and solubilized with 1N HCl solution. Calcium content was analyzed by atomic absorption spectrometry (Shimadzu AA640–13; Shimadzu, Kyoto, Japan) according to the method of Gimblet et al.(16) Phosphorus content was measured colorimetrically by the method of Gomori.(17) The absorption of calcium and that of phosphorus during the 4-week period was calculated as a balance between the amounts of those intakes and of fecal excretion. The absorption rate of calcium and that of phosphorus were each calculated as a percentage of the absorption against to the amount of intake.

Blood chemistry

The blood samples were centrifuged at 3000g for 15 minutes, and the supernatants were used as serum samples. Serum calcium and phosphorus were analyzed using the same methods with those for feces and urine samples. Serum PTH levels were assayed by means of an enzyme-immunoassay kit (Immutopics, Inc., San Clemente, CA, USA), which is a two-site immuno-radiometric assay (IRMA) that uses two different goat antibodies to measure both intact rat PTH and its N-terminal fragment.(18)

Bone length and minerals

Length of femur was measured using a micrometer. BMC (mg) and BMD (mg/cm2) values were measured by DXA (DCS-600A; Aloka, Tokyo, USA) adapted for use in the case of small animals.

Bone histomorphometry

Bone labeling with an intramuscular injection of calcein (6 mg/kg body weight) was performed 7 and 3 days before death. The samples of left proximal tibias were fixed in 100% ethanol after the adherent soft tissues were trimmed, after which the samples were embedded in methyl methacrylate (MMA) and Villanueva's bone staining. Serial undecalcified 10-μm-thick frontal sections were obtained with a Reichert-Jung microtome (Model 2050 Supercut; Reichert-Jung, Heidelberg, Germany). The sections were subjected to osteoid staining using Goldner's method. The right proximal tibia specimens were embedded in paraffin after decalcification with 10% buffered EDTA. Decalcified 5-μm-thick specimens were stained for TRACP. Histomorphometry was performed with a semiautomatic image-analyzing system linked to a light microscope (Cosmozone 1S; Nikon, Tokyo, Japan).(19) In the metaphyseal region, the cancellous bone area 0.5 mm distal from the growth plate was observed under a fluorescence microscope. The primary spongiosa was distinguished by the presence of intratrabecular cartilage cores and by its diffuse calcein uptake, with little or no delineation of mineralizing front. The secondary spongiosa contained few cartilaginous cores, and there was a distinct mineralizing front at the trabecular surface.

Measured parameters in histomorphometry

MMA sections of Villanueva's staining were measured to determine the percentage of trabecular bone volume to tissue volume (BV/TV, %) and fluorescence labeling. Double-labeled surfaces to total labeled surface (dLS/BS, %) were obtained from measurement of the trabecular perimeter at 100-fold magnification, and interlabel thicknesses (Ir.L.Th, μm) were obtained at 200-fold magnification. The mineral apposition rate (MAR, μm/day) was calculated as π/4 × Ir.L.Th/4. The ratio of bone formation rate to bone surface (BFR/BS, μm3/μm2/day) was calculated by the formula MAR × dLS/BS. Osteoid surface (OS/BS, %), osteoid volume (OV/BV, %), and osteoid thickness (O.Th, μm) were measured in sections stained for osteoid by Goldner's staining. Osteoid maturation time (OMT, day) was calculated as O.Th/MAR. Trabecular osteoclast number (Oc.N/BS, n/mm) was measured in sections subjected to TRACP staining. TRACP+ cells that formed resorption lacunae at the surface of the trabeculae and contained one nucleus or more were identified as osteoclasts.

The growth plate width and length of the primary spongiosa were measured along the longitudinal axis of the specimen. In the area of primary spongiosa, the parameters of BV/TV, OV/BV, OS/BS, O.Th, and Oc.N/BS were obtained. In the secondary spongiosa, the values of BV/TV, OV/BV, OS/BS, O.Th, BFR/BS, OMT, and Oc.N/BS were obtained.

Statistical analysis

Results were expressed as mean ± SD. To assess the effect of lacking VDR gene, the data from group 2 were initially compared with those of group 1 by Student's t-test after the F test and Mann-Whitney's U-test. To determine the effects of diets in VDRKO mice, data in groups 2–6 were evaluated by one-way ANOVA followed by Fisher's protected least significant difference (PLSD) test after Bartlett test. Data in groups 3–6 were compared with those in group 2. In case significant differences were found between SD values using the F test and Bartlett test, Mann-Whitney's U-test was performed after the Kruskall-Wallis test. A p value of less than 0.05 was considered significant. The analyses were performed using StatView 5.0 software (Macintosh; Apple Computer Inc., Cupertiono, CA, USA).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

All data did not significantly differ between male and female mice. Thus, both males and females were included in the results.

Body weight gain and intakes of food

The amounts of food intake and weight gain in group 3 were significantly smaller than those in group 2 (Table 1). The weight gain in group 5 was significantly larger than that in group 2. Compared with group 2, the amounts of calcium intake significantly decreased and phosphorus intake increased in group 3. In group 4, both of these intakes increased. Calcium intake in group 5 increased, and phosphorus intake in group 6 decreased.

Table Table 1. Food Intake and Growth Performance During Experimental Period
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Serum chemistry and PTH levels

In group 2, serum calcium levels were significantly lower and PTH levels were significantly higher than the corresponding values in group 1 (Table 2). Compared with group 2, PTH values in group 3 significantly increased. In groups 4–6, serum calcium and phosphorus levels increased. Serum PTH levels in group 5 decreased.

Table Table 2. Serum Chemistry at the End of the Experiment
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Intestinal calcium and phosphorus absorptions, excretions, and retentions

Compared with group 2, the amounts (mg) of calcium absorption and excretion in group 3 significantly decreased (Table 3). The excretion rates (%) of calcium also decreased. In group 4, the amount and rate values of calcium absorption and retention significantly decreased. The rate values of calcium absorption and retention decreased in group 5 and increased in group 6.

Table Table 3. Balance Study During Experimental Period*
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The amounts and rates of phosphorus absorption in group 2 significantly increased compared with group 1. Compared with group 2, these values increased in group 3 and decreased in groups 4–6. The amounts of phosphorus excretion increased and that of retention decreased in group 3. In group 4, the parameters of phosphorus excretion and retention, except the amount of excretion, significantly decreased. In groups 5 and 6, the amount and rate values of phosphorus excretion significantly decreased. The amounts of phosphorus retention decreased in group 6.

Bone length and minerals

Bone length, BMC, and BMD in group 2 were significantly smaller than the corresponding values in group 1 (Table 4). Compared with group 2, these parameters in group 3 were significantly reduced. In group 4, the length value increased. In group 5, all these parameters significantly increased.

Table Table 4. Bone Length, BMC, and BMD in Femur
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Growth plate thickness, the longitudinal length of the primary spongiosa area, and histomorphometry of the primary spongiosa

In group 2, the values of growth plate thickness and the longitudinal length of the primary spongiosa area were significantly larger than the values in group 1 (Table 5). Compared with group 2, the values of these parameters in group 3 significantly increased. Length of the primary spongiosa increased in group 4. These parameters in group 6 were decreased. In group 2, BV/TV, OV/BV, OS/BS, and O.Th were significantly larger and the value of Oc.N/BS was significantly smaller than the respective values in group 1. Compared with group 2, the values of BV/TV and O.Th in group 3 significantly increased. In groups 5 and 6, the values of Oc.N/BS increased. The values of BV/TV and O.Th in group 6 decreased.

Table Table 5. Growth Plate Thickness and Longitudinal Length of the Primary Spongiosa Area and Histomorphometry of the Primary Spongiosa in Tibia*
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Histomorphometry of the secondary spongiosa

The region of the secondary spongiosa with distinct mineralization front was identified in all mice in groups 1, 5, and 6 (Table 6). In groups 2 and 4, the region was identified in 5 of 10 mice. In group 3, there was no secondary spongiosa in either mouse (Fig. 1). In group 2, the values of BV/TV, OV/BV, and O.Th were significantly larger than the values in group 1. Compared with group 2, the values of BV/TV and OMT significantly decreased, and the values of OS/BS and BFR/BS increased in group 4. In group 5, the values of BV/TV, O.Th, and OMT decreased, and BFR/BS increased. In group 6, the values of BFR/BS increased, and those of O.Th and OMT decreased.

Table Table 6. Histomorphometry of the Secondary Spongiosa in Tibia
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Figure FIG. 1.. The trabecular bone of the proximal metaphysis in the tibia. A-F are samples from groups 1–6, respectively. Undecalcified sections observed under fluorescent microscope, ×10. (A; group 1) The trabecular bone with distinct mineralization front. (B; group 2, and D; group 4) The trabecular bone with diffuse calcein labeling (the primary spongiosa) and a few trabecular bones with distinct mineralization front (secondary spongiosa). (C; group 3) The trabecular bone with diffuse labeling. Mineralization front was not seen in the trabeculae. (E; group 5, and F; group 6) The trabecular bone with distinct mineralization front.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

This study demonstrated that bone mineralization and turnover were regulated by the dietary Ca/P ratios in VDRKO mice. Increases in the dietary Ca/P ratio from 0.5 to 2 increased the amounts of intestinal calcium absorptions and serum calcium levels. Intestinal phosphorus absorptions inversely related to the dietary Ca/P ratios. Serum PTH levels decreased and the values of femoral BMC and BMD increased. BFR/BS, O.Th, and OMT in the secondary spongiosa of the proximal tibia improved, and Oc.N/BS in the primary spongiosa increased.

In VDRKO mice fed the diet containing 0.5% calcium and phosphorus with a Ca/P ratio of 1, the parameters of bone mineral homeostasis such as serum calcium and phosphorus levels, BMC and BMD of the femur, and the bone tissue turnover of the tibia were all reduced compared with the wild-type mice fed a similar diet.(14,15) The non-vitamin D-dependent calcium transport was apparently preserved by the large concentration gradients between the intestinal lumen and blood.(20,21) Thus, the retention of calcium in VDRKO mice was maintained at similar levels with those in the normal littermates (NLMs). However, lowered serum calcium levels resulted in the smaller bone length, BMC, and BMD. Parameters of bone turnover and mineralization such as BFR/BS and OMT in the secondary spongiosa of the tibia were also reduced. Thus, when fed the diet containing 0.5% calcium and phosphorus with Ca/P ratio of 1, in the null vitamin D signal condition, bone and mineral homeostasis seem to be maintained by lowering serum calcium level and reducing the functional reservoir of bone mineral.

In VDRKO mice fed the diet with Ca/P ratio of 1, but with higher concentrations of calcium and phosphorus (1%), serum calcium level improved compared with that of NLMs. However, the BMC and BMD of the femur remained similar with those in the VDRKO mice fed the diet containing 0.5% calcium and phosphorus. While the amount of calcium available in the intestine increased, the calcium absorption was reduced. Whereas the bone length in the mice fed 1.0% calcium and phosphorus was larger than the length in mice fed 0.5% calcium and phosphorus, the mineralization and turnover of the proximal spongiosa in the tibia remained similar. The secondary spongiosa was identified in only 5 of 10 mice in both groups. In the secondary spongiosa, BFR/BS and OMT values were improved, but trabecular osteoclasts did not increase. These data indicate that the improvements of mineralization and turnover of trabecular bone were limited by the increases in the amounts of dietary calcium and phosphorus at Ca/P ratio of 1.

When the dietary Ca/P ratio increased from 1 to 2 in VDRKO mice, BMC and BMD values of the femur recovered to the level of the NLMs. The effects of increasing dietary calcium and those of reducing phosphorus were apparently similar in increasing serum calcium levels. The rate values of calcium absorption reduced in the mice given the higher calcium diet, and the values increased when given the lower phosphorus diet. The amounts of intestinal calcium absorption thus maintained similar in both groups of mice. These data suggest that the dietary Ca/P ratios affect serum calcium levels and bone mineral by adjusting intestinal calcium absorption. Bone length increased and the remodeling of the primary spongiosa to the secondary spongiosa occurred in both groups of mice. The values of OV/BV, O.Th, BFR/BS, and OMT in the secondary spongiosa were also similar. Thus, it is suggested that the effects of the dietary amounts of calcium and phosphorus on bone mineralization and turnover were minimum at the dietary Ca/P ratio of 2, as they were at the Ca/P ratio of 1. The reason for increases in osteoclast number with the increase in the dietary Ca/P ratio from 1 to 2 in these mice is not certain. Signaling other than the VDR pathway could facilitate osteoclastogenesis(22) when mineralization is recovered in the null vitamin D signal condition.

When the dietary Ca/P ratio decreased from 1 to 0.5 by increasing phosphorus content, an increase in phosphorus absorption was consistent with the available amount of phosphorus in intestine. However, reductions in the amounts and ratios of intestinal calcium absorption were larger than expected from the amounts of oral calcium intake. Reductions in calcium and phosphorus retentions were consistent with the reductions in BMC and BMD of the femur. Thus, it is obvious that a reduction in dietary Ca/P ratio decreased the intestinal calcium transport, reducing serum calcium levels, and led to the reduction in the bone mineral reservoirs. Reduction of phosphorus retention seemed to be caused by a marked increase in serum PTH level, resulting in an increase in the urinary excretion of phosphorus. Bone histomorphometry indicated further loss of mineralization and bone turnover including the loss of tissue remodeling from the primary spongiosa to the secondary spongiosa. No increase in osteoclast number in face of PTH increase could be related to the increase in the unmineralized osteoid tissue, which osteoclasts were not able to resorb. Thus, a reduction of Ca/P ratio in the diet seemed to reduce the total bone mineral storage and turnover by affecting intestinal calcium and phosphorus absorption. The apparent dissociation in the marked loss of BMC values and the relative maintenance of calcium retention could indicate the increase in soft tissue calcification such as nephrocalcinosis and vascular wall calcification,(11,13) which we did not examine in this experiment.

In this experiment, we found that the values of dietary Ca/P ratios critically regulate bone mineralization and turnover in VDRKO mice. Intestinal calcium and phosphorus absorption seemed to depend not only on the amounts of intakes, but also on the dietary Ca/P ratio. Molecular mechanism for the regulation of intestinal calcium and phosphorus absorption by the dietary Ca/P ratio is not certain. It may be possible to attribute the effect for the solubility of calcium and phosphorus in intestinal lumen when concomitantly present. However, because serum phosphorus levels increased before serum calcium levels decreased in normal rats when phosphorus intake was increased,(23) the existence of Ca/P ratio-dependent calcium and phosphorus transport system in VDRKO mice could be assumed.

In conclusion, the present data indicate that the dietary Ca/P ratios could regulate bone mineralization and turnover by affecting intestinal calcium and phosphorus absorption in VDRKO mice. The data suggest the existence of Ca/P ratio-dependent, vitamin D-independent calcium and phosphorus transport system in intestine. Because we did not examine the normal mice, the role of the dietary Ca/P ratios in the presence of VDR signal system is still uncertain, but it may affect bone and mineral homeostasis in the normal or subnormal vitamin D signal condition.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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