1,25 dihydroxyvitamin D3 alone or in combination with parathyroid hormone does not increase bone mass in young rats

Summary Parathyroid hormone (PTH) alone is known to increase bone mass, but clinical studies of osteoporotic men suggest that when 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) is given in combination with PTH, the effect on bone growth is enhanced. To determine if 1,25(OH)₂D₃ alone would stimulate bone growth, young male rats were given daily subcutaneous injections of either vehicle or 2.5, 5, 10, or 20 ng 1,25(OH)₂D₃ per 100 g body weight for 30 days. To determine if 1,25(OH)₂D₃ would augment the PTH anabolic response, rats were given daily subcutaneous injections of either vehicle for 12 days; or 4 μg/100 g hPTH alone or in combination with 5 ng/100 g 1,25(OH)₂D₃; or 8 μg/100 g hPTH alone or in combination with 5 ng/100 g 1,25(OH)₂D₃. Calcium (Ca), dry weight (DW), and hydroxyproline (Hyp) of the distal femur; the rate of mineralization in the metaphysis of the proximal tibia; and serum calcium and phosphate were measured. Low normocalcemic doses of 1,25(OH)₂D₃ did not significantly stimulate bone growth. 1,25(OH)₂D₃ did not augment the PTH-stimulated anabolic effect in young male rats. Low doses (2.5 and 5 ng) of 1,25(OH)₂D₃ were not hypercalcemic, and there was no increase in total bone calcium or dry weight although the 5 ng dose increased trabecular bone calcium. 1,25(OH)₂D₃ at 10 and 20 ng increased trabecular bone DW and Hyp, but mineralization was impaired and rats were hypercalcemic. 1,25(OH)₂D₃ in combination with PTH did not augment the PTH stimulation of bone growth as trabecular and cortical bone Ca, DW, and HYP were not increased in rats given both hPTH and 1,25(OH)₂D₃ compared with values for rats treated with hPTH alone.     

Induction of macrophagic and granulocytic differentiation of murine bone marrow progenitor cells by 1,25-dihydroxyvitamin D3

Summary 1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) was recently shown to promote maturation of 5-fluorouracil (5FU)-treated bone marrow cells by up-regulating macrophage-colony stimulating factor (M-CSF) receptors in the presence of interleukin la (IL-1). In order to reveal how 1,25(OH)₂D₃ interacts with colony-stimulating factors and regulates the differentiation of bone marrow progenitor cell populations, in the present study, natural bone marrow cells were isolated from untreated mice and used in a-minimum essential medium supplemented with 20% heat-inactivated horse serum without added appropriate cytokines. Under the conditions, cells spontaneously differentiated gradually with days of culture, as assessed by expression of macrophage differentiation antigens such as Mac-1 (CD11b) and F4/80. Both M-CSF and granulocyte macrophage-colony stimulating factor (GM-CSF) induced only Mac-1 antigen expression. Simultaneous treatment with M-CSF and 1,25(OH)₂D₃ enhanced the M-CSF's effect on expression of both antigens, although (1,25(OH)₂D₃) per se has no effect on the expression for up to 11 days. In addition, successive treatment with 1,25(OH)₂D₃ and M-CSF or GM-CSF dramatically enhanced expression of both antigens or Mac-1 antigen, respectively. Similarly, both simultaneous and successive treatment with 1,25(OH)₂D₃ and M-CSF significantly enhanced phagocytic activity and H₂O₂ production, whereas successive treatment with (1,25(OH)₂D₃) and GM-CSF significantly enhanced only phagocytic activity. Enzymehistochemical study demonstrated that cells treated simultaneously or successively with 1,25(OH)₂D₃ and M-CSF were strongly positive for nonspecific esterase (NSE), a macrophage-specific marker, and that simultaneous or successive treatment with 1,25(OH)₂D₃ and GM-CSF yielded cells strongly positive for NSE or for chloroacetate esterase (ChAE), a granulocyte-specific marker, respectively. These findings suggest that 1,25(OH)₂D₃ primes bone marrow progenitor cell populations not only to M-CSF but also to GM-CSF and thereby accelerates the CSFs-dependent differentiation of the cells to the macrophage or granulocyte.     

Human parathyroid hormone (1–34) and salmon calcitonin do not reverse impaired mineralization produced by high doses of 1,25 dihydroxyvitamin D3

Summary We have reported recently that pharmacologic doses of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) stimulated bone matrix formation but impaired mineralization. The objective of this study was to determine if parathyroid hormone (hPTH 1-34) or calcitonin (sCT) would mineralize the osteoid induced by 1,25(OH)₂D₃ in rat long bones. In one experiment, male Sprague-Dawley rats were given daily subcutaneous injections of vehicle: 8 μg hPTH(1-34); 125 ng 1,25(OH)₂D₃; or both 8 μg hPTH and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 12 days. In a second experiment, rats received daily injections of vehicle: 2 U sCT; 125 ng 1,25(OH)₂D₃; or both 2 U sCT and 125 ng 1,25(OH)₂D₃ per 100 g body weight for 18 days. Calcium (Ca), hydroxyproline (Hyp), and dry weight (DW) of the distal femur and serum calcium, phosphate, and serum bone Gla protein (BGP) were measured. In rats given both 1,25(OH)₂D₃ and hPTH, total bone DW and Hyp increased (P<.01) without a corresponding increase in bone Ca so that Ca/Hyp decreased 47% (P<.01) from control and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. In rats treated with both 1,25(OH)₂D₃ and sCT, total bone DW and Hyp increased while Ca decreased so that Ca/Hyp decreased 38% from control (P<.05), and remained comparable to values for rats treated with 1,25(OH)₂D₃ alone. These results indicate that hPTH or sCT, given by intermittent injection to rats for 12 or 18 days respectively, failed to mineralize the osteoid induced by high doses of 1,25(OH)₂D₃.     

Calcium regulating activity of 26,27-dialkyl analogs of 1α,25-dihydroxyvitamin D3

Summary A series of analogs of 1,25-dihydroxyvitamin D₃[1,25(OH)₂D₃] with alkyl substitutions in 26- and 27-positions were tested for calcium (Ca) regulating activity. The potencies of dialkyl analogs in stimulating bone resorption in neonatal mouse calvaria cultures were the highest in 1,25-dihydroxy-26,27-dimethylvitamin D₃[1,25(OH)₂-(Me)₂D₃], followed by 1,25(OH)₂D₃, 1,25-dihydroxy-26,27-diethylvitamin D₃[1,25(OH)₂(Et)₂D₃], and 1,25-dihydroxy-26,27-dipropylvitamin D₃[1,25(OH)₂(Pr)₂D₃] in that order. A similar order of potential regarding formation of osteoclast-like cells in mouse bone marrow cell cultures and on bone Ca mobilization with long-term vitamin D-deficient rats was observed in the same series. The relative potencies of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 1,25(OH)₂(Et)₂D₃, and 1,25(OH)₂(Pr)₂D₃ in competing with 1,25(OH)₂D₃ for binding to chick intestinal cytosol receptors were 1:1:0.16:0.036. A similar order of potential in case of intestinal Ca transport in situ was observed in the same series. The potencies of dialkyl analogs in competing with 25-hydroxy-vitamin D₃ for binding to rat serum vitamin D binding protein were much lower than that of 1,25(OH)₂D₃. Effect of 1,25(OH)₂(Me)₂D₃ on osteopenia in rats induced by ovariectomy and right sciatic neurotomy was higher than that of 1,25(OH)₂D₃. From these results, the lengthening by one carbon at 26- and 27-positions was shown to maintain the Ca regulatory activity of 1,25(OH)₂D₃.     

Calcium regulating activity of 24a-homo-24,24-difluoro-1α,25-dihydroxyvitamin D3 and 26,27-dimethyl-24,24-difluoro-1α,25-dihydroxyvitamin D3

Summary Two fluoro analogs of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24a-homo-24,24-difluoro-1,25-dihydroxyvitamin D₃ [24aF₂-homo-1,25(OH)₂D₃], and 26,27-dimethyl-24,24-difluoro-1,25-dihydroxyvitamin D₃ [24F₂-1,25(OH)₂(Me)₂D₃] were examined for calcium (Ca)-regulating activity. The objective of the present study was to determine whether or not fluoro substitution at 24-position would alter activities of the original compounds, that is, 26,27-dimethyl 1, 25-dihydroxyvitamin. D₃[1,25(OH)₂ (Me)₂D₃] and 24-homo-1,25-dihydroxyvitaminD₃[24homo-1,25(OH)₂D₃], respectively. The relative activities of 24aF₂-homo-1,25(OH)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 1,25(OH)₂D₃ in competing with 1,25(OH)₂D₃ for binding to chick intestinal cytosol receptor were 0.28:0.5:1.0. The relative potencies of the same series of compounds in competition for the vitamin D-deficient rat serum binding sites were 0.04:0.15:1. Bone-resorbing activities of two fluoro analogs in cultures of neonatal mouse parietal bones were more potent than that of 1,25(OH)₂D₃. Similar results were recognized in stimulating activities of osteoclast-like cell formation. Responses of two fluoro analogs to intestinal Ca absorption were similar to that of 1,25(OH)₂D₃. The potencies of 1,25(OH)₂D₃. and its fluoro analogs in bone Ca mobilization were the highest with 1,25(OH)₂D₃. followed by 24F₂ 1,25(OH)₂(Me)₂D₃ and 24aF₂-homo-1,25(OH)₂D₃, in that order. From these results and the data of Paulson et al. [24], fluoro substitution in 24-position of 1,25(OH)₂D₃. apparently does not alter their activities, hence, the fluoro substitution at 24-position of 1,25(OH)₂D₃. and the elongation of side chain of 1,25(OH)₂D₃. may not intensify Ca-regulating activity.     

Elongation of the side chain of analogs of 1α,25-dihydroxyvitamin D3 prevents osteopenia in a rat model

Five analogs of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] [1], 26,27-dimethyl-1,25-dihydroxyvitamin D₃ [1,25(OH)₂(Me)₂D₃] [2], 26,27-dimethyl-1,25-dihydroxyvitamin D₃ [1,25(OH)₂(Et)₂D₃] [3], 26,27-dipropyl-1, 25-dihydroxyvitamin D₃ [1,25(OH)₂(Pr)₂D₃] [4], 26,27-dimethyl-24, 24-difluoro-1,25-dihydroxyvitamin D₃ [24F₂-1,25(OH)₂(Me)₂D₃, and [5] 24a-homo-24,24-difluoro-1,25-dihydroxyvitamin D₃ [24aF₂-homo-1,25(OH)₂D₃] were investigated to clarify the possibility that prevents osteopenia induced in rats by ovariectomy and sciatic neurotomy. The objective of our studies was to determine whether these analogs may be effective for treatment of subjects with osteoporosis. 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃ prevented decreases in bone mineral density (BMD) of the femur, as measured by dual energy X-ray absorptiometry (DXA). The potency of 1,25(OH)₂(Me)₂D₃ in this test was higher than that of 1,25(OH)₂D₃. The potencies of 24F₂-1,25(OH)₂(Me)₂D₃ and 24aF₂-homo-1,25(OH)₂D₃ were similar to that of 1,25(OH)₂D₃. On the other hand, though 1,25(OH)₂(Et)₂D₃ and 1,25(OH)₂(Pr)₂D₃ had a preventive effect on the decease in BMD, the potency of two analogs was lower than that of 1,25(OH)₂D₃. Decreases in cortical and trabecular bone areas of the femur were prevented by three analogs of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃. Serum calcium (Ca) concentration was elevated at the last administration of three analogs of 1,25(OH)₂D₃, 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃ and 24aF₂-homo-1,25(OH)₂D₃. Decreases in the Ca concentration in untreated rats were noted a few days after the last administration. In light of these positive effects, we are continuing research on 1,25(OH)₂(Me)₂D₃, 24F₂-1,25(OH)₂(Me)₂D₃, and 24aF₂-homo-1,25(OH)₂D₃ as putative treatment for osteoporosis.     

Stimulation of undermineralized matrix formation by 1,25 dihydroxyvitamin D3 in long bones of rats

Summary We previously reported that pharmacologic doses of 1,25 dihydroxyvitamin D₃ (1,25-(OH)₂D₃) given for 2–3 days, inhibited osteoblastic collagen synthesis in young rats. In this study, we tested the effects of 5, 25, and 125 ng of 1,25(OH)₂D₃ injected subcutaneously into 6-week-old rats for 12 or 18 days. In rats given 125 ng, cortical bone of distal half femurs exhibited decreased calcium (Ca) content but dry weight and hydroxyproline (Hyp) content were no different from control. Trabecular bone Ca was not different from control but dry weight and Hyp were increased. When cortical and trabecular bone were combined, there was a decrease in Ca, an increase in Hyp, and a 50% decrease in Ca:Hyp. Fluorescent labels given after 8 days of treatment were either diffuse or absent in calcified sections from rats given 125 ng, indicating impaired mineralization. The 25 and 125 ng doses produced hypercalcemia with normal serum phosphate. There was a dose-related increase in serum immunoreactive bone gla protein (BGP) and serum 1,25(OH)₂D₃ and a decrease in serum 25 (OH)D₃. At the 5 ng dose, no adverse effects were seen on body growth. With 25 ng and 125 ng, growth was inhibited. Increased serum urea nitrogen and histologic evidence of nephrocalcinosis occurred at the 125 ng dose. When 125 ng was given for 12 days and then withdrawn for 6 days, systemic toxicity decreased and bone Hyp and Ca increased so that Ca:Hyp remained low and comparable to that of rats treated with 1,25(OH)₂D₃ continuously We conclude that pharmacologic doses of 1,25(OH)₂D₃ stimulate trabecular bone matrix formation but produce impairment of mineralization, despite a high Ca×Pi product.     

Calcium regulating activity of 26,27-dimethyl analog of 24R,25-dihydroxyvitamin D3

To determine the possibility that methyl substitution in 26- and 27-positions of 24R,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] alters activities of the original compound, the effects of 24,25(OH)₂D₃ on calcium (Ca) regulating activity were compared with those of its methyl analog [24,25(OH)₂(CH₃)₂D₃] in addition to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. 24,25(OH)₂D₃ at 10^-6 M and 24,25(OH)₂(CH₃)₂D₃ at 10^-7 M and above significantly stimulated both bone resorption in neonatal mouse calvaria cultures and formation of osteoclast-like multinucleated cells (MNC) in mouse bone marrow cultures. A stimulative effect of 1,25(OH)₂D₃ on bone resorption and MNC formation was recognized in very low concentrations (10^-11 M and above). Although a potency of 24,25(OH)₂(CH₃)₂D₃ in stimulating bone calcium (Ca) mobilization and intestinal Ca transport was higher than that of 24,25(OH)₂D₃, the potencies of both compounds were similar to that of 1,25(OH)₂D₃ unlike in vitro experiments. As 1,24R,25-trihydroxy-26,27-dimethylvitamin D₃ showed almost the same effect as 24,25(OH)₂(CH₃)₂D₃, the dihydroxy form is suggested to be hydroxylated at 1 position and converted to trihydroxy form in vitamin D-deficient rats. From these results, methyl substitution in 26- and 27-position of 24,25(OH)₂D₃ was found to elevate Ca regulating activity of the original compound. In addition, it is suggested that the basis for a similarity in potency between 1,25(OH)₂D₃ and 24,25(OH)₂D₃ or its dimethyl analog in vitamin D-deficient rats is likely the result of 1 -hydroxylation.     

Inhibition by aminohydroxypropylidene bisphosphonate (AHPrBP) of 1,25(OH)2 vitamin D3-induced stimulated bone turnover in the mouse

Summary The purpose of this study was to evaluate whether the 1,25(OH)₂D₃-induced increased bone mineralization in the mouse occurs in response to stimulation of bone resorption. In order to inhibit bone resorption, 35-day-old mice were given 16 μmol/kg/day of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) for 10 days, the first injection occurring 3 days prior to the continuous infusion of 0.06, 0.13, or 0.20 μg/kg/day of 1,25(OH)₂D₃ for 7 days. Two groups of mice were treated with AHPrBP or 1,25(OH)₂D₃ alone. The skeletal changes were assessed by histomorphometric study of caudal vertebrae after double³H-proline and double tetracycline labelings for evaluation of the matrix apposition rate (MaAR) and mineral apposition rate (MiAR), respectively. Treatment with AHPrBP alone or combined to 1,25(OH)₂D₃ decreased the number of acid phosphatase-stained osteoclasts and reduced the endosteal MaAR and MiAR and the amount of osteoid. When given alone, 1,25(OH)₂D₃ increased serum calcium above normal, enhanced the number of histochemically active osteoclasts, and stimulated the endosteal MiAR. Pretreatment with AHPrBP blocked both the increase in serum calcium and the stimulation of the MiAR induced by 1,25(OH)₂D₃ infusion though serum 1,25(OH)₂D₃ levels rose according to the dose given. The results show that 1) the serum calcium and the bone resorbing responses to 1,25(OH)₂D₃ infusion are prevented by pretreatment with AHPrBP, and 2) the stimulatory effect of 1,25(OH)₂D₃ on the mineralization rate is blocked when bone resorption is inhibited. The data indicate that 1,25(OH)₂D₃ promotes bone mineralization in the mouse mainly in response to stimulation of bone resorption.     

1,25 dihydroxyvitamin D3 modifies cyclosporine-induced bone loss

Summary We have previously shown that cyclosporin A (CsA) produces high bone remodeling with resorption exceeding formation and loss of bone volume in the rat. This may have important clinical implications where CsA is widely used in organ transplantation. 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a bone mineralizing hormone which also has immune modifying properties. Consequently, we studied the effect of combined CsA and 1,25(OH)₂D₃ administration over 28 days in four groups of rats. Group A received vehicle (n=10), group B CsA (15 mg/kg) (n=10) alone, group C 1,25(OH)₂D₃ plus CsA (n=15), and group D 1,25(OH)₂D₃ alone (20 ng/100 g) (n=15). Rats were bled periodically at day 0, 7, 14, and 28 and Ca, parathyroid hormone (PTH), 1,25(OH)₂D, osteocalcin (bone Gla-protein, BGP), BUN, and creatinine were measured. Rats were sacrificed on day 28 and bones were examined histomorphometrically. Compared to controls, CsA resulted in significant elevation of BGP and a transient increase in 1,25(OH)₂D with excess bone remodeling and loss of bone volume. 1,25(OH)₂D₃ administration produced hypercalcemia, a significant rise in BGP, with suppression of PTH and increased osteoid volume. Combined therapy prevented the loss of bone volume probably due to increased osteoid tissue and enhanced osteoblast activity. Renal dysfunction, a side-affect of CsA, was not a factor. In conclusion, 1,25(OH)₂D₃ combined with CsA restores bone volume which is accompanied by increases in serum calcium and BGP.     

Vitamin D metabolites prevent vertebral osteopenia in ovariectomized rats

Summary The present study investigated the prophylactic effects of vitamin D metabolites and vitamin D metabolite combinations on static and dynamic, tetracycline-based, histomorphometric parameters in the axial skeleton of ovariectomized rats. Forty-three Fischer-344 rats (10 weeks old, 130 g each body weight, BW) were either bilaterally ovariectomized (OVX) or sham-operated (SHAM). The rats were allocated into the following groups: SHAM; OVX; OVX+7.5 ng 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]/rat/day; OVX +15 ng 1,24R,25-trihydroxyvitamin D₃ [1,24,25-(OH)₃D₃]/rat/day; OVX+75 ng 24R,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃]/rat/day; OVX+7.5 ng 1,25(OH)₂D₃/rat/ day+15 ng 1,24,25(OH)₃D₃/rat/day; OVX+7.5 ng 1,25(OH)₂D₃/rat/day+75 ng 24,25(OH)₂D₃/rat/day. The vitamin D metabolites were fed orally starting 4 weeks after surgery. Urine and blood samples were collected 12 and 16 weeks postovariectomy, respectively. Sixteen weeks after surgery, all rats were sacrificed, and the first lumbar vertebrae were processed undecalcified for histomorphometric analysis. Ovariectomy induced a highly significant reduction (P<0.001) of cancellous bone mass in the secondary spongiosa of the lumbar vertebral body. The bone loss in OVX rats was accompanied by a distinct elevation of all histomorphometric parameters of bone formation and resorption. 1,25(OH)₂D₃ and both vitamin D metabolite combinations significantly raised serum calcium levels and prevented the bone loss by inhibiting the increased bone resorption in OVX rats. In the applied dosage, 1,24,25(OH)₃D₃ and 24,25(OH)₂D₃ alone were ineffective in preserving the cancellous bone of the lumbar vertebra in OVX rats. We conclude that the oral prophylactic application of low doses of active vitamin D metabolites can effectively prevent the osteopenia induced by ovariectomy in the axial skeleton of the rat.     

Vitamin K2 promotes 1α,25(OH)2 vitamin D3-induced mineralization in human periosteal osteoblasts

The effect of vitamin K on mineralization by human periosteal osteoblasts was investigated in the absence and presence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃). Vitamin K₁ and K₂, but not vitamin K₃, at 2.5 M enhanced in vitro mineralization when cells were cultured with vitamin K for 20 days after reaching confluence in vitro. Vitamin K₂ (2-methyl-3-all-trans-tetraphenyl-1,4-naphthoquinone: menatetrenone) was the most potent of these vitamin K analogs; it slightly inhibited alkaline phosphatase (ALP) activity. Human osteoblasts were mineralized and showed the enhanced ALP activity on treatment with 10^-9 M of 1,25(OH)₂D₃ for 20 or 25 days after confluence. Vitamin K₂ promoted the 1,25(OH)₂D₃-induced mineralization, but slightly inhibited the 1,25(OH)₂D₃-induced ALP activity. Moreover, vitamin K₂ enhanced the 1,25(OH)₂D₃-induced osteocalcin accumulation in the cells and the extracellular matrix (cell layer), but inhibited the osteocalcin content in the medium produced by the 1,25(OH)₂D₃ treatment. However, vitamin K₂ alone did not induce osteocalcin production in the human osteoblasts. On Northern blot analysis, osteocalcin mRNA expression on 1,25(OH)₂D₃-treated cells was enhanced by vitamin K₂ treatment, but vitamin K₂ alone did not induce osteocalcin mRNA expression. Warfarin blocked both the 1,25(OH)₂D₃-induced osteocalcin production and the accumulation in the cell layer, and also blocked the 1,25(OH)₂D₃ plus vitamin K₂-induced osteocalcin production and the accumulation in the cell layer. The 1,25(OH)₂D₃-induced mineralization promoted by vitamin K₂ was probably due to the enhanced accumulation of osteocalcin induced by vitamin K₂ in the cell layer. However, we concluded that the mineralization induced by vitamin K₂ alone was due to the accumulation of osteocalcin in bovine serum on the cell layer, since osteocalcin extracted from the cell layer was not identified by specific antiserum against human osteocalcin, which does not cross-react with bovine osteocalcin. These results suggest that the mechanism underlying the mineralization induced by vitamin K₂ in the presence of 1,25(OH)₂D₃ was different from that of vitamin K₂ alone, and that osteocalcin plays an important role in mineralization by osteoblasts in vitro.     

Idiopathic juvenile osteoporosis: Evidence of normal osteoblast function by 1,25-dihydroxyvitamin D3 stimulation test

Summary Idiopathic juvenile osteoporosis (IJO) is a rare form of bone demineralization that occurs during childhood. The mechanism of bone loss is unclear. Some bone hystomorphometric studies have found osteoblast failure and decreased bone formation in the affected patients whereas others have reported increased bone resorption. To elucidate this issue, we studied osteoblast function in six patients with IJO (five males, one female; aged 2.3–14.6 years) and five healthy sex- and age-matched subjects (four males, one female; aged 2.0–15.1 years) measuring serum values of osteocalcin under basal condition and during an osteoblast stimulation test performed by oral 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] administration (1.8 g/1.73 m²/daily). After a baseline day (day 0), all the subjects (patients and controls) received 1,25(OH)₂D₃ in four divided doses for 6 days (days 1–6). Fasting blood samples were obtained every morning (0800 h) for the determination of serum osteocalcin. Baseline osteocalcin levels were not significantly different between IJO and controls (13.58±6.05 ng/ml versus 16.04±5.09 ng/ml, respectively) even if two patients had low osteocalcin values. During 1,25(OH)₂D₃ administration, serum osteocalcin values significantly increased (P<0.001) from baseline in both children with IJO and controls, reaching peak values not significantly different in the two groups. Our results do not support the hypothesis that defective osteoblast function is the primary factor of bone demineralization in IJO.     

The dichotomy in the effects of 1,25 dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on bone γ-carboxyglutamic acid-containing protein in serum and bone in vitamin D-deficient rats

Summary Vitamin D-deficient, second generation, rachitic rats showed significant decrease in bone Gla protein (BGP) levels in circulation and in the skeleton. 1,25 dehydroxyvitamin D₃ (1,25 (OH)₂D₃) exhibited the most potent influence on serum BGP levels in a dose-dependent manner. At a dose 25 ng/100 g body weight 1,25 (OH)₂D₃ showed a cumulative effect, i.e., the longer the treatment, the more circulating BGP was detected 24,25 dehydroxyvitamin D₃ (24,25(OH)₂D₃) at the same doses did not show similar effect on the serum BGP levels, regardless of the serum calcium levels. Bone BGP levels assayed at various sites representing endochondral and intramenbranous ossification demonstrated an opposite pattern. 1,25(OH)₂D₃ administration was not sufficient to restore bone BGP levels to normalcy, whereas in animals treated with 24,25(OH)₂D₃ bone BGP and calcium levels were significantly higher than control (Vitamin D₃-repleted) levels. The present results can be explained by the dual action of 1,25 (OH)₂D₃ on both synthesis and release of BGP by bone turnover, whereas 24,25 (OH)₂D₃ stimulates synthesis and accumulation of BGP in bone. These observations imply that caution is required in the interpretation of clinical data based solely on serum BGP determination.     

Effects of vitamin D-binding protein on bone resorption stimulated by 1,25 dihydroxyvitamin D3

Summary Vitamin D and its metabolites are tightly bound to the serum vitamin D-binding protein (DBP) and only the free hormone is considered to be physiologically active. On the other hand, DBP could interact with cell membranes and even favor its intracellular entry. The present study was undertaken to examine the effects of DBP on bone resorption stimulated by 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Forelimb bones from 19-day-old fetal rats were cultured for 5 days in the presence of purified human or rat serum albumin (hSAP or rSAP) and 1,25(OH)₂D₃, with or without human or rat DBP (hDBP or rDBP). Bone resorption was assessed by measuring the release of previously incorporated⁴⁵Ca. We found that the resorptive response to 1,25(OH)₂D₃ was minimally altered by hDBP (5 μM). The minimal effects of hDBP on 1,25(OH)₂D₃ activity on rat bones might be explained by a 6-fold lower affinity of hDBP (1.1×10⁷ M⁻¹) than rDBP (5.9×10⁷ M⁻¹) for 1,25(OH)₂D₃ or by species differences in cellular recognition of DBP. In a homologous rat system, however, rDBP at low (0.5 μM) or physiological (5 μM) concentration significantly decreased 1,25(OH)₂D₃-induced bone resorption. These data therefore support the hypothesis that free rather than DBP-bound 1,25(OH)₂D₃ is physiologically important.     

Bone-resorbing activities of 24-epi-1α-hydroxyvitamin D2 and 24-epi-1α,25-dihydroxyvitamin D2

Bone-resorbing activities of 24-epi-1-hydroxyvitamin D₂ [24-epi-1(OH)D₂], 24-epi-1,25-dihydroxyvitamin D₂ [24-epi-1,25(OH)₂D₂], and 1,24S,25-trihydroxyvitamin D₂ [1,24S,25(OH)₃D₂], which might be a metabolite of 24-epi-1,25(OH)₂D₂, were investigated. In an in vitro bone resorption test, the activity of 24-epi-1(OH)D₂ was similar to that of 1-hydroxyvitamin D₃ [1(OH)D₃] at 10^-9 M-10^-6 M. The activity of 24-epi-1,25(OH)₂D₂ was weaker than that of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] at 10^-11 M-10^-8 M. On the other hand, the activity of 1,24S,25(OH)₃D₂ was similar to that of 24-epi-1,25(OH)₂D₂ at 10^-11 M-10^-9 M. In the formation assay of osteoclast-like cells, the activity of 24-epi-1(OH)D₂ was weaker than that of 1(OH)D₃ at 10^-7 M. The activity of 24-epi-1,25(OH)₂D₂ was almost similar to that of 1,25(OH)₂D₃ at 10^-11 M-10^-7 M. The activity of 1,24S,25(OH)₃D₂ was significantly weaker than that of 24-epi-1,25(OH)₂D₂ at 10^-11 M-10^-9 M. In the two experiments, the potencies of 24-epi-1,25(OH)₂D₂ were about 100 times higher than those of 24-epi-1(OH)D₂. In an in vivo/in vitro bone resorption test, the activity of 24-epi-1(OH)D₂ was almost similar to those of 1(OH)D₃ and 1,25(OH)₂D₃ and higher than those of 24-epi-1,25(OH)₂D₂ and 1,24S,25(OH)₃D₂. 24-epi-1-(OH)D₂ and 1(OH)D₃ were longer lasting than 24-epi-1,25(OH)₂D₂ and 1,25(OH)₂D₃ in this experiment. These results suggested that 24-epi-1(OH)D₂ as well as 1(OH)D₃ was converted into dihydroxy form in vivo.     

The influence of dihydroxylated vitamin D metabolites on bone formation in the chick

Summary The ability of 1,25(OH)₂D₃ and of 24,25(OH)₂D₃ to prevent or to heal rickets in chicks was evaluated by studies of plasma biochemistry, growth plate histology, bone morphometry and microradiography, and bone mineralization. 1,25(OH)₂D₃ at a dose of 100 ng/day produced fewest abnormalities compared with vitamin D₃-treated control chicks. Bone growth was slightly greater than vitamin D₃-treated controls in chicks given a lower dose of this metabolite; the reverse was observed in chicks given a higher dose. 24,25(OH)₂D₃ was less effective than 1,25(OH)₂D₃ in preventing rickets even at doses as high as 400 ng/day. Treatment of rachitic chicks with doses of 24,25(OH)₂D₃ up to 300 ng/day produced no healing effect on the bone lesions, in marked contrast to the beneficial effects observed with 1,25(OH)₂D₃.     

Effect of 1,25-dihydroxycholecalciferol on adenosine 3′,5′-cyclic monophosphate production in calvaria of mice

Summary 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃), added at doses which stimulate bone resorption in the system used, did not increase adenosine 3,5-cyclic monophosphate (cAMP) production in vitro in mouse calvaria incubated for up to 48 h. Thus 1,25-(OH)₂D₃ does not appear to stimulate bone resorption through involvement of an increased cAMP production.     

Alkaline phosphatase inhibition by levamisole prevents 1,25-dihydroxyvitamin D3-stimulated bone mineralization in the mouse

Summary To determine the relationship between alkaline phosphatase (AP), 1,25(OD)₂D₃ and bone formationin vivo, we have examined the effects of levamisole, a stereospecific inhibitor of AP on bone formation and on 1,25(OH)₂D₃-stimulated bone mineralization in the mouse. Normal mice were injected daily with levamisole at doses of 40 and 80 mg/kg/b.w. The compound was given alone or in combination with 1,25(OH)₂D₃ infusion (0.05 μg/kg/d) for 7 days. Treatment with levamisole alone inhibited the serum AP activity (mainly of skeletal origin in mice) by 18.4 and 61.3% for the low and high dose respectively. No deleterious effect on body growth, tibia length, and bone cells population was detected. The moderate inhibition of AP activity produced by the lower dose of levamisole alone (18.4%) or in combination with 1,25(OH)₂D₃ (37.9%) was associated with a reduced endosteal matrix apposition rate (MaAR) determined by double³H-proline labeling method. This effect was related to a levamisole-induced fall in serum phosphate. Despite the moderate inhibition of AP activity, the mineral apposition rate (MiAR) determined by the double tetracycline labeling method remained normal. Moreover, 1,25(OH)₂D₃ infusion still resulted in increased MiAR which was stimulated to the same extent as in the absence of levamisole. By contrast, the more severe inhibition of AP activity induced by 80 mg/kg of levamisole alone (61.3%) or in combination with 1,25(OH)₂D₃ (45.8%) inhibited both the MaAR and the MiAR and prevented the stimulatory effect of 1,25(OH)₂D₃ on bone mineralization. The data show that AP activity affects the bone matrix and mineral apposition ratesin vivo and that severe inhibition of AP activity inhibits the 1,25(OH)₂D₃-induced stimulation of bone mineralization in the mouse.     

1,25-Dihydroxyvitamin D3 promotes prostaglandin E1-induced differentiation of HL-60 cells

Human promyelocytic HL-60 cells can be induced by biochemical agents to differentiate in vitro towards divergent types of myelomonocytic cells. It has been reported that prostaglandin E₁ (PGE₁) can induce granulocytic differentiation and that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) can induce monocytic differentiation. We have now examined the effects of these compounds, both alone and in combination, on HL-60 cell differentiation. PGE₁ (1 g/ml) or 1,25(OH)₂D₃ (10 nM) each inhibited cell proliferation over 48–96 hours of treatment, but combined treatment with both agents was necessary to produce a strong inhibition. The percentage of HL-60 cells that can reduce nitroblue tetrazolium (NBT) (a characteristic index of early monocytic or granulocytic differentiation) increased 13-fold within 72 hours of PGE₁ treatment, and 1,25(OH)₂D₃ produced a fivefold stimulation. However, combined treatment (PGE₁ plus 1,25(OH)₂D₃) produced a dramatic 35-fold increase. HL-60 cells did not produce significant levels of nitric oxide (NO) before 48 hours in culture, and treatment with PGE₁ or 1,25(OH)₂D₃ did not significantly increase cellular NO elaboration over control levels. However, combined treatment produced a striking 12-fold increase over control levels. Similarly, combined treatment was necessary to obtain the maximal time-dependent stimulation of cellular lactate dehydrogenase (LDH) activity (a marker of granulocytic differentiation) as well as acid phosphatase (ACP) activity. During this same period of time, PGE₁, but not 1,25(OH)₂D₃, markedly stimulated cellular claboration of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-, and 1,25(OH)₂D₃ cotreatment strongly augmented these effects. Thus, combined treatment with 1,25(OH)₂D₃ plus PGE₁ generally augmented the apparent conversion of these cells, producing synergistic (multiplicative) or additive effects. Furthermore, PGE₁ induced within 48 hours the more general phenotypic changes classically associated with the differentiation of these cells: increased expression of chloroacetate esterase (ChAE) (a granulocytic marker), decreases in the nuclear/cytoplasmic ratio (characteristic of development beyond the promyelocyte/myelocyte stage), and major alterations in morphology from floating spherical cells to loosely adherent, elliptical polygons. 1,25(OH)₂D₃ had little effect itself on most of these parameters, but augmented the morphological changes induced by PGE₁ treatment. Within 48 hours, the ability of these cells to reduce the tetrazolium salt WST-1, a general measure of cellular metabolic activity, was increased by PGE₁, but not by 1,25(OH)₂D₃; however, the combination of 1,25(OH)₂D₃ and PGE₁ again produced the strongest stimulation. Similarly, only PGE₁ significantly reduced intracellular ATP levels, but combined treatments produced a more pronounced decrease. In summary, our findings suggest that PGE₁, not 1,25(OH)₂D₃, is sufficient to promote rapid in vitro differentiation of HL-60 cells along the granulocytic pathway; however, the PGE₁-induced conversion of these cells is markedly augmented by cotreatment with 1,25(OH)₂D₃. In addition, these converted HL-60 cells preferentially utilize the glycolytic pathway, rather than the citric acid cycle, for production of ATP, a metabolic characteristic that resembles that described for mature granulocytes.