Intermittent administration of human parathyroid Hormone(1-34) prevents immobilization-related bone loss by regulating bone marrow capacity for bone cells in ddY mice.

ddY mice, 6 weeks of age, were neurectomized (Nx) in the right hindlimbs and sham-operated (Sham) in the left limbs for evaluation of the effects of intermittent injections of human parathyroid hormone (hPTH) on trabecular bone turnover and bone marrow cell development in unloaded and loaded limbs. Mice were given subcutaneous injections of hPTH(1-34) five times a week at a dose of 0 (vehicle), 4 (low dose), or 40 (high dose) microg/kg of body weight for 2, 4, or 6 weeks. Histomorphometric analyses of the trabecular bone of the proximal tibiae revealed that high-dose hPTH injections preserved the trabecular bone volume of the Nx limbs, which was reduced after neurectomy, at the same level as that of the contralateral Sham limbs. The mineral apposition rate in the Nx limbs was elevated to values above even that of the Sham limbs by high-dose hPTH injections. The bone formation rate reduced by neurectomy was maintained at the Sham level by low- and high-dose hPTH injections. The neurectomy-induced increase in osteoclast number was suppressed by high-dose hPTH injections. In the bone marrow cells, the numbers of nonadherent and adherent cells per tibia obtained from the Nx and Sham limbs did not change. The hPTH injections decreased the numbers of nonadherent cells and increased those of adherent cells in both the Nx and the Sham limbs, but the effects were less marked in the Nx than in the Sham limbs even at high-dose injections. The formation of osteogenic nodules in the marrow cultures obtained from the Nx limbs was decreased after surgery and was maintained at the level of the Sham limbs by high-dose hPTH injections. The number of osteoclast-like multinucleated cells was reduced in the Sham limbs by high-dose hPTH injections. The value was increased at 2 weeks after neurectomy, but it was maintained at the Sham level by high-dose hPTH injections through the experimental period. The numbers of colony forming units-fibroblastic, which were reduced by neurectomy, and those of colony forming units for granulocytes and macrophages were not altered by hPTH injections. These results demonstrate that intermittent high-dose hPTH administration in the Nx limbs as well as in the contralateral Sham limbs has similar anabolic effects, stimulating osteoblast cell lineage and suppressing osteoclast cell lineage. The anabolic effects at 4 microg were reduced, but the effects at 40 microg seemed to be less affected by unloading due to sciatic neurectomy.

Large-dose ascorbic acid administration suppresses the development of arthritis in adjuvant-infected rats.

We performed animal experiments to test the hypothesis that active oxygen species (AOS) play a major role in adjuvant-induced arthritis in rats and to determine whether large-dose ascorbic acid administration would suppress the development of arthritis, reducing the level of damaging AOS in the same animal model. Arthritis was induced in male Lewis rats by adjuvant injection into the base of the tail. Ascorbic acid at doses of 0.5, 1.0, and 2.0 g/kg body weight (BW) was injected intraperitoneally twice each week for 3 weeks (9 rats per group). The BW, hind paw edema, and arthritis score of the extremities were monitored during the period. On day 21, synovial tissues obtained from the ankle joints were examined histologically and for the activity of superoxide dismutase (SOD). The SOD activity in the red blood cells (RBC) was also measured. The arthritic control rats showed significant increases in paw volume and arthritis score from day 11. These changes were dose-dependently reduced by ascorbic acid administration. The infiltration of inflammatory cells into the synovial tissues was markedly decreased by ascorbic acid. The increases in SOD activities produced by the adjuvant injection were significantly reduced in both the synovium and the RBC at ascorbic acid doses of 1.0 and 2.0 g/kg BW. In conclusion, large-dose ascorbic acid administration reduced the increases in hind paw inflammatory edema, arthritis in the extremities, and infiltration of the inflammatory cells into the synovial tissue in the adjuvant-induced arthritis rats. Since these anti-arthritic effects were associated with a decrease in SOD activities in both the synovium and RBC, the decrease in SOD activity could be one of the mechanisms underlying the suppressive effects of large-dose ascorbic acid on the development of arthritis in this animal model, inhibiting the damaging AOS.

A novel activating mutation in calcium-sensing receptor gene associated with a family of autosomal dominant hypocalcemia.

Autosomal dominant hypocalcemia (ADH), caused by activating mutations of the calcium-sensing receptor (CaSR), is characterized by hypocalcemia with an inappropriately low concentration of PTH. Among 11 missense mutations of CaSR reported to date in patients with ADH or sporadic hypocalcemia, functional properties of 8 mutant CaSRs were characterized. Here, we describe a novel mutation of CaSR and its functional property in a family with ADH. The 41-yr-old male proband had asymptomatic hypocalcemia with a history of recurrent nephrolithiasis. His father had asymptomatic hypocalcemia, but his mother was normocalcemic. PCR-single strand conformation polymorphism and sequencing revealed that both the proband and the father had a novel heterozygous mutation in CaSR gene that causes lysine to asparagine substitution at codon 47 (K47N), which is in the extracellular domain of CaSR, like 6 of 11 known activating mutations. Using HEK293 cells transfected with wild-type or K47N CaSR complementary DNA, the intracellular Ca2+ concentration was assessed in response to changes in the extracellular Ca2+ concentration. The EC50 of the mutant CaSR for the extracellular Ca2+ concentration was 2.2 mmol/L and was significantly lower than that of wild-type (3.7 mmol/L). These results confirm that this mutation is responsible for ADH in this family. The fact that several inactivating mutations in familial hypocalciuric hypercalcemia occur in amino acid around K47 suggests the importance of the N-terminal portion of the receptor in extracellular Ca sensing.

Effect of 24R,25-dihydroxyvitamin D3 on the formation and function of osteoclastic cells.

Previous reports demonstrated that the administration of large doses of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] to animals with normal vitamin D supply causes an increase in bone volume with reduced bone resorption and decreased osteoclast number. The present study was undertaken to clarify if 24R,25(OH)2D3 has any inhibitor effect on the formation and function of osteoclasts. The effect of 24R,25(OH)2D3 on the formation of osteoclastic cells was examined by measuring the number of tartrate-resistant acid phosphatase-positive multinucleated cells (MNCs) formed from hemopoietic progenitor cells obtained from spleens of 5-fluorouracil-treated mice. Treatment with 1,25(OH)2D3 or parathyroid hormone fragment 1-34 [PTH(1-34)] stimulated osteoclast-like MNC formation in a dose-dependent manner. Addition of 24R,25(OH)2D3 alone showed a weak stimulatory effect on MNC formation at 10(-6) M, which appeared to be due to its binding to 1,25(OH)2D3 receptors. In contrast, when 24R,25(OH)2D3 was added together with 1,25(OH)2D3 or PTH(1-34), it inhibited osteoclast-like MNC formation stimulated by these hormones. A significant inhibition of MNC formation was observed with 10(-7) M 24R,25(OH)2D3, and the stimulatory effect of 1,25(OH)2D3 or PTH(1-34) was almost completely eliminated with 10(-6) M 24R,25(OH)2D3. Neither 24S,25(OH)2D3 nor 25(OH)D3 exhibited a similar inhibitory effect. The effect of 24R,25(OH)2D3 on the resorptive function of osteoclasts was examined by measuring the formation of resorption pits by mouse bone cells on dentine slices. Treatment with 24R,25(OH)2D3 also inhibited the resorption pit formation stimulated by 1,25(OH)2D3 or PTH(1-34) with similar dose response.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypomagnesemia with increased metabolism of parathyroid hormone and reduced responsiveness to calcitropic hormones.

A patient with severe hypomagnesemia due to chronic alcoholism is presented who repeatedly exhibited marked hypocalcemia with a dissociation between radioimmunoassay findings for mid region of parathyroid hormone (PTH-M) and immunoradiometric assay findings of serum intact PTH (PTH-intact). Serum PTH-M was moderately elevated whereas serum PTH-intact was in a low normal range every time when her serum magnesium (Mg) concentration was markedly reduced. There was also a marked reduction in serum osteocalcin concentration. Supplementation of Mg resulted in a sharp increase in serum PTH level with a rapid disappearance of the dissociation between the two immunoassays of PTH. Shortly after serum PTH and 1,25(OH)2D levels reached their peak, serum osteocalcin started to increase, and was elevated into a supranormal level with normalization of serum Ca concentration. Mg is thought to act as a mimic/antagonist of calcium (Ca), and high extracellular Ca is shown to cause an inhibition of secretion with a stimulation of degradation of PTH. Thus, these observations are consistent with the hypothesis that Mg deficiency causes an increase in the metabolism of PTH and a reduction in the secretion of bioactive intact PTH by increasing the sensitivity of parathyroid cells to Ca. In addition, the fact that hypocalcemia disappeared concomitant with a marked increase of serum osteocalcin from undetectable levels suggest that refractoriness of bone to calcitropic hormones is present which plays a significant role in the development of hypocalcemia under hypomagnesemia, and that serum osteocalcin can be a good marker for the assessment of the responsiveness of bone to calcitropic hormones in these patients.

Erythrocytosis in hypophosphatemic rickets: irreversible complication due to nephrocalcinosis after vitamin D and phosphate therapy.

A patient with hypophosphatemic vitamin D-resistant rickets developed secondary erythrocytosis during treatment with large doses of vitamin D2 and phosphate. Erythrocytosis was accompanied by a fall in circulating plasma volume and appeared to have developed as a consequence of nephrocalcinosis because it occurred after the appearance of nephrocalcinosis following several episodes of hypercalcemia and hyperphosphatemia. Nephrocalcinosis and erythrocytosis did not disappear even after recovery of renal function. Thus, the present observations point to the importance of preventing these irreversible complications that could cause renal failure, erythrocytosis, and thrombotic events during the management of hypophosphatemic vitamin D-resistant rickets.