Uptake of fluorine in cortical and trabecular bone.

Nuclear magnetic resonance techniques can measure the fluoride levels in bone of the finger after a patient has ingested F in the treatment of osteoporosis, but does uptake in cortical bone reflect uptake in the critical trabecular bone? Investigation has been made of the relative uptake of fluoride from drinking water into trabecular and cortical bone of the rat. For fine detail of the uptake of F into the femur and vertebra, microprobe techniques were used with a spatial resolution of 10 microns; for broader studies, treating the femur as representative of cortical bone and the vertebra as typical of trabecular bone, chemical techniques using spectroscopy and ion-selective electrodes were employed. The conclusion is that in the rat uptake of F by cortical bone is indicative of uptake by trabecular bone, and that therefore as a working hypothesis NMR measurement of F in the finger may be taken as reflecting uptake of F by trabecular bone.

The effects of fluoride on ectopic bone formation.

The effects of fluoride (F) on ectopic bone formation induced in rats by implants of demineralized cortical bone tissue were studied. Test rats received sodium fluoride (NaF), 6 mmol/liter in drinking water, and controls received fluoride-free water. Implant accumulations of tracer hydroxyproline ([3H](OH)P), 45Ca, and stable Ca were determined 24 h after injections of tritiated proline ([3H]P) and 45Ca, to estimate rates of collagen synthesis, mineralization, and net mineral mass, respectively. Conventional histology on undemineralized implant sections was done. Mineralized bone was first observed by implant histology, 2 weeks after implantation and continued to increase up to 8 weeks. A few chondrocytes were observed. Prior to bone formation, dense fibrous tissue was observed within the marrow space of the original implant. The rate of collagen synthesis peaked at 1 week, again at 3 weeks, and then continued at a slower rate up to 8 weeks. The rates of mineralization paralleled the rates of collagen synthesis between 2 and 8 weeks, indicating bone mineralization over this period. During the first 2 weeks after implantation no mineral deposition was observed. The initial peak of collagen synthesis without mineralization (0-2 weeks) indicates fibrous tissue formation and is in agreement with the histological analysis. Fluoride treatment increased rates of collagen synthesis during both the initial period of fibrous tissue formation and later bone formation. The ratio of mineralization rate to collagen synthesis rate (45Ca/[3H](OH)P) was decreased by fluoride throughout the 2-8 week period, but net mineral mass was comparable to control rats by 8 weeks, indicating that fluoride delays, but does not prevent, bone mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoride inhibition of bone mineralization may depend on physical/chemical properties.

The properties of fluoride (F) incorporation into bone powder in vitro were studied in an attempt to clarify possible mechanism for F inhibition of bone mineralization and soft tissue mineral deposition in vivo. Rat bone powder was prepared with minimal physical and chemical stress, and exposed to F (less than 1 mmol) in aqueous solutions buffered to pH 7.4. Initial experiments confirmed reported data that the rates of F incorporation into bone mineral in vitro resemble F clearance from blood in vivo. The rate of F incorporation was inversely proportional to the amount of F added to supernatants and decreased exponentially with time. Following F incorporation, the bone mineral became less soluble, with solubility inversely proportional to bone F concentration. However, a fraction (about 20%) of F freshly incorporated in bone powder dissolved readily in pH 7.4 buffer and appears to be adsorbed F. The fraction of incorporated F that was readily dissolved decreased with time over the initial 24 h after incorporation, was proportional to the amount of F freshly incorporated, and was associated with an inhibition of further F incorporation.

The relationship between bone apposition rate and vitamin D activity in phosphate-deficient rats.

In rats, phosphorus deficiency (P-) has been shown previously to stimulate the linear bone apposition rate (BAR) and this P- effect is dependent on adequate intake of vitamin D. To investigate further the relative importance of the vitamin D3 metabolites, 1,25(OH)2D3, 24,25(OH)2D3, and 25(OH)D3, in BAR stimulation, we studied, in P- rats, the relationships between BAR and plasma levels of these three vitamin D3 metabolites following vitamin D3 deprivation. Three groups of rats were placed on diets differing only in phosphorus (P) and vitamin D3(D3) content, with one group diet deficient in both P and D3, one diet, P-, D3 replete, and one diet both P and D3 replete. Plasma levels of the three vitamin D3 metabolites, plasma Ca and P, isotopic Ca absorption and BAR measurements were carried out at 1, 3, and 5 weeks after onset of the test diets. In P-, D3 replete rats, both plasma levels of 1,25(OH)2D3 and BAR were increased throughout the 1 to 5 week study period, while 25(OH)D3 and 24,25(OH)2D3 levels were not significantly different from P and D3 replete controls. In P-, D3 restricted rats, BAR was decreased by one week, prior to any reduction in plasma levels of 25(OH)D3 and 24,25(OH)2D3 and while plasma 1,25(OH)2D3 levels were still well above control values. In this P- rat model, the vitamin D dependent BAR stimulation does not appear to be directly related to alterations in the plasma levels of 1,25(OH)2D3, 24,25(OH)2D3, or 25(OH)D3.

The effect of fluoride on nephrocalcinosis in rats.

To investigate the effects of fluoride on soft tissue calcification, female weanling rats were fed a nephrocalcinogenic diet and NaF in drinking water over a 4 week period. The diet contained adequate Ca (0.5%) and high phosphorus (1.0%, P). The nephrocalcinosis is attributed to the relatively low dietary Ca/P ratio since addition of Ca to provide a Ca/P ratio of 2.0 prevents kidney calcification. With NaF in drinking water at levels of 1.19 to 4.76 mmol/L kidney calcification was decreased from 127 +/- 24 to 17.3 +/- 1.7 mumol/g wet weight, with no significant differences over this dose range. With the increasing NaF doses, serum F, at 4 weeks, increased from 4.4 +/- 0.8 to 36.5 +/- 6.0 mumol/L compared to untreated F levels of 1.2 +/- 0.1 mumol/L. Bone histology showed no evidence of F stimulation with any of these NaF doses. Previously reported work has shown that, for weanling rats on this diet, greater than 4.8 mumol/L NaF in drinking water is required to produce histological fluorosis within 5 weeks. To inhibit kidney calcification, NaF treatment must be maintained throughout the 4-week study period since calcification occurred if NaF was withheld over either the initial or final 2-week period. These findings indicate a possible therapeutic value of NaF, clinically, in the prevention of soft tissue calcification.

The effect of diet calcium on fluoride toxicity in growing rats.

The effect of dietary Ca in response to fluoride (F) treatment was investigated in rats. Rats were maintained on either adequate (0.5%) or high (2.0%) dietary Ca and given for 5 weeks, NaF in drinking water. The minimum NaF levels that inhibited body growth and reduced survival were 300 mg/L with 0.5% diet Ca and 550 mg/L with 2.0% diet Ca. With these toxic F doses, bone histology showed increased formation surfaces and thickened osteoid seams (osteoid index 6-7%). Fluoride doses 30% below toxic levels (200 and 350 mg/L for 0.5 and 2.0% diet Ca, respectively) had no demonstrable effect on bone. Additional diet Ca reduced F absorption from 76 +/- 3 to 47 +/- 3% for 0.5 and 2.0% diet Ca, respectively. Comparable absorbed doses of F produced comparable effects on bone and body growth but, with additional dietary Ca, these effects were observed with 50% lower serum and bone F levels. Variable response to NaF therapy can be produced in rats by alterations in dietary Ca alone. Results indicate that for clinical treatment the NaF dose needs to be adjusted on an individual basis but neither serum nor bone F levels can be used reliably to establish optimal doses.

Plasma vitamin D metabolite levels in phosphorus deficient rats during the development of vitamin D deficient rickets.

Plasma levels of the vitamin D metabolites were related to changes in bone morphology during the development of rickets in rats deprived of phosphorus and vitamin D. Weanling rats were studied at 1, 3, and 5 wk after onset of diets deficient in phosphorus or in both phosphorus and vitamin D. Bone histology and morphometry were carried out and measurements were made of 45Ca and 32P absorption, serum Ca and P, and plasma 25(OH)D3, 24,25(OH)2D3 and 1,25(OH)2D3. After 1 wk of vitamin D restriction, the plasma levels of 25(OH)D3 and 24,25(OH)2D3 were non-detectable (less than 0.5 and less than 0.8 ng/ml). The plasma 1,25(OH)2D3 level was elevated at 1 wk (105.5 pg/ml) and fell to 19 pg/ml by 5 wk. At 1 wk mild rachitic lesions in epiphyseal cartilage were observed despite the elevated 1,25(Oh)D3 level. Serum Ca and P levels and values for 45CA and 32P absorption decreased and the severity of the rickets increased with the fall in plasma levels of 1,25(OH)2D3. In Vitamin D replete, phosphate deficient rats the epiphyseal cartilage was normal throughout the 5 wk study period. Our results provide further evidence that physiological levels of 1,25 (OH)2 D3 will not prevent rickets without adequate plasma concentrations of either 25(OH)D3 or 24,25(OH)2D3.

Protective effect on vitamin D2 on bone apposition from the inhibitory action of hydrocortisone in rats.

Using the technique of short interval sequential tetracycline labeling, it was documented that the apposition of mineralized bone matrix in adult male Sprague-Dawley rats was inhibited by hydrocortisone. The inhibition occurred as early as six days after the onset of the treatment and was dose dependent over a dose range of 0.62 to 20 mg per kg body weight per day. Vitamin D2 supplements by injection protected bone from this hydrocortisone action. 64 I. U. of vitamin D2 injected daily was able to prevent the inhibition of bone apposition by 20 mg per kg body weight per day of hydrocortisone. The results imply that vitamin D or its metabolites may compete with hydrocortisone in some cellular mechanisms and support the usefulness of vitamin D supplements in the treatment and the prevention of steroid-induced osteoporosis.

Differences between the effects of phosphate deficiency and vitamin D deficiency on bone metabolism.

It has been widely believed that phosphate deficiency causes osteomalacia. Based on this belief, the rickets of familial hypophosphatemia has been attributed to phosphate deficiency associated with the hypophosphatemia. The present studies on rats have, however, demonstrated significant differences between the effects of phosphate deficiency on bone metabolism and the characteristic features of rickets. Weanling rats, maintained on a mildly phosphate deficient diet, had hypercalcemia and hypophosphatemia, and impairment of body growth, bone growth, and bone mineralization. The maximum effect was observed at 5 wk; between 5 and 20 wk the rats improved despite persistent hypophosphatemia. Histologically, at 5 wk the bone showed thick unmineralized osteoid seams covering most bone surfaces, but the epiphyseal cartilage was normal. In addition, the excess osteoid readily incorporated tetracycline indicating normal mineralization and, based on a new sequential pulse labeling technique, the linear bone apposition rate (LBA) was significantly (p < 0.001) increased above control values. This increase was observed within the initial 4 days of phosphate (P) deficiency and persisted up to 15 wk. This effect of P deficiency on LBA was dependent on vitamin D activity. At 4 wk, the mean LBA was 0.106 +/- 0.003 (1 SE) in control rats, 0.149 +/- 0.008 microns/hr in P deficient rats, 0.083 +/- 0.004 microns/hr in vitamin D deficient rats and 0.086 +/- 0.006 microns/hr in rats deficient in both P and vitamin D. We have reported a similar increase in LBA with parathyroid hormone activity. With vitamin D deficiency, phosphate deficient rats showed all the characteristic features of rickets; disorganization of epiphyseal cartilage, excessive unmineralized osteoid, and reduced mineralization based on the incorporation of tetracycline. We conclude that the effects of phosphate deficiency on bone metabolism more closely resembles the effects of PTH activity than the characteristic effects of osteomalacia and rickets.

The amino acid sequence of porcine intestinal calcium-binding protein.

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.

Phophate-induced renal calcification in the rat.

Studies were done to investigate nephrocalcinosis produced in weanling female Wistar rats fed pelleted, semisynthetic diets. The rats were fed diets varying in concentrations of Ca and P supplied as inorganic salts for periods of 4--6 weeks and results compared with control rats fed laboratory rodent chow for the same period of time. Measurement of renal Ca and P concentrations showed that nephrocalcinosis was produced by semisynthetic diets with inorganic phosphate concentrations as low as 0.5% on a weight basis; in contrast, rats fed regular laboratory chow (P = 0.72%) showed no evidence of nephrocalcinosis. The severity of the lesion was proportional to dietary phosphate concentrations from 0.5 to 1.0% but other dietary factors modified the severity of the lesion. With the lower dietary phosphate of 0.5%, increasing dietary Ca from 0.5 to 1.0% decreased the severity of the renal calcification. Decreasing protein concentrations from 25 to 15% casein increased the severity of the renal lesions. Other dietary factors also appear to modify the phosphate-induced nephrocalcinosis since no lesions occurred in rats on laboratory chow. It is suggested that the availability of dietary phosphate may be a factor. The phosphate in the semisynthetic diets was totally inorganic while the natural foods of laboratory chow contain, at least in part, organic phosphate.

Bone mineral measurements of the central skeleton by in vivo neutron activation analysis for routine investigation os osteopenia.

In vivo neutron activation analysis has been used to measure bone mineral content in the central skeleton where osteoporotic fractures occur. To be of diagnostic value, the results must be normalized for body size. From data obtained from 74 healthy children and adults up to 55 years of age, we have found that the calcium in the central skeleton is approximately proportional to the cube of the subject's height. The correlation for the adults alone has an r value of 0.81. When data from both adults and children are used, r = 0.95. The validity of this cubic height relationship to the Ca concentration measurements has been further substantiated by studies on rats. The total femur calcium content of 110 rats from weanling to 25 weeks of age was proportional to the overall femur (length) 3.6 (or (length)2.6 per unit length) with r = 0.99. When the level of Ca content is related to data on normal subjects of the same body size (giving the Calcium Bone Index or CaBI) a good separation is obtained between normal volunteers and osteoporotic subjects. Volunteers who were 20 to 55 years of age had CABI 1.0 "/- .12 (SD) while osteoporotics had CaBI 0.69 +/- .10 (SD). When the calcium content as determined by in vivo activation analysis is expressed as a CaBI, it provides a powerful tool for the diagnosis of osteopenia. We suggest that all bone measurements, including peripheral ones, be normalized for body size in order to increase their diagnostic value.

Rat intestinal and renal calcium-binding proteins.

The effect of vitamin D activity on the major renal Ca-binding protein has been compared with that on the intestinal Ca-binding protein. Using a method based on Ca-binding capacity, these proteins were measured in vitamin D deficient rats after vitamin D treatment for varying periods up to 5 days. Since P deficiency has been shown to stimulate synthesis of the active metabolite 1,25-dihydroxycholecalciferol, a similar experiment was done on rats fed a P-deficient diet for periods up to 21 days. The renal Ca-binding protein was unchanged by vitamin D treatment to vitamin D deficient rats and was only slightly increased (50%) by phosphate deficiency. By comparison, the intestinal protein was increased twofold by vitamin D treatment and fivefold by phosphate deficiency. Results indicate that vitamin D activity has no direct effect on the major renal Ca-binding protein.

Radioimmunoassay studies of intestinal calcium-binding protein in the pig. I. Identification of intestinal calcium-binding protein in blood and response to a low calcium diet.

We have developed a radioimmunoassay for porcine intestinal calcium-binding protein (CaBP) and have used it to detect CaBP in pig plasma. Plasma CaBP is identical to intestinal CaBP on the basis of immunological activity, molecular size, and molecular charge properties. The plasma CaBP concentration was greater in the portal blood than in mixed venous blood, suggesting that blood CaBP originates in the gut. Two of four 15-week-old littermate pigs were placed on a low calcium diet (0.15% calcium, 0.65% phosphorus) and two on a control diet (0.65% calcium, 0.65% phosphorus). After 2 weeks, the entire small intestine was removed and divided into nine 1.8-m segments. CaBP was assayed in both plasma and intestinal mucosa. When the two pigs on a low calcium diet were compared with two control pigs, there was a general increase in immunoreactive CaBP in both plasma and intestinal mucosa. However, there was no increment in immunoreactive CaBP in the first 1.8-m segment of small intestine. Seventy-one percent of the increment in CaBP occurred distal to the first two segments. The largest fractional low calcium diet effect occurred in the ileum. The mean CaBP concentration for the total small intestine increased by a factor of 1.9. The plasma CaBP concentration increased by a factor of 2.6. In these pigs, plasma CaBP was a more reliable indicator of change in CaBP status than was the measurement in the proximal gut segment which contained the duodenum. The assay of CaBP in blood is convenient and may obviate the sampling errors inherent in intestinal biopsy.

Radioimmunoassay studies of intestinal calcium-binding protein in the pig. II. The distribution of intestinal CaBP in pig tissues.

Using a specific radioimmunoassay for porcine intestinal calcium-binding protein (CaBP), we have measured the concentration of CaBP in the various tissues and organs of normal pigs. Intestinal CaBP was present in highest concentration in the upper small intestine, with lower concentrations in the distal small intestine. Intestinal CaBP was also found, in lower concentrations, in kidney, liver, thyroid, pancreas, and blood. In all other tissues, including parathyroid, bone, skeletal muscle, and brain, CaBP immunoreactivity was undetectable or less than in blood. The elution profile of calcium-binding activity and immunoreactivity from gel filtration analysis of kidney and parathyroid extracts suggest that the calcium-binding protein in the parathyroid gland, and the major calcium-binding protein(s) in the kidney, are chemically and immunochemically different from intestinal CaBP.

Intestinal calcium-binding protein in animals fed normal and rachitogenic diets: I. Rat studies.

Measurements were made of duodenal calcium-binding protein (CaBP) on rats during development of rickets and, subsequently, following vitamin-D2 treatment. Results showed a poor inverse correlation between duodenal CaBP and rickets. In rats fed a phosphate-deficient rachitogenic diet, duodenal CaBP concentration finally fell below detectable limits, but CaBP was still readily measurable 2 weeks after rickets was clearly established. Following a massive dose of vitamin D2 (50 000 I.U.) to rachitic animals, CaBP was formed. However, a small dose of vitamin D2 (500 I.U. daily for 4 days) was insufficient to demonstrate CaBP synthesis than vitamin-D treatment alone. The rachitogenic diet supplemented with phosphate, which caused osteoporosis but not rickets, inhibited CaBP synthesis. The results suggest that nutritional deficiencies from the rachitogenic diet, in addition to vitamin-D deficiency, inhibited CaBP synthesis.

Intestinal calcium-binding protein in animals fed normal and rachitogenic diets: II. Pig studies.

Experiments were carried out in pigs to investigate the relationship between concentration of intestinal calcium-binding protein (CaBP) and the presence of rickets. Pigs made rachitic by a diet deficient in calcium and in vitamin D had concentrations of intestinal CaBP no less than values obtained on control pigs. These experiments, together with earlier work on rats (Can. J. Physiol. Pharmacol. 1975. 53, 137--143), demonstrate a poor inverse correlation between levels of intestinal CaBP and rickets.