1,25(OH)2D and 24,25(OH)2D production in the developing kidney

To clarify the state of vitamin D production by the developing kidney, firstly, we measured serum levels of 1,25(OH)₂D and 24,25(OH)₂D in humans of different ages (pregnant and nonpregnant women, adult males, children and newborn infants) and secondly, we measured 1- and 24-hydroxylase activity in the kidney mitochrondria of rats at different ages. The mean serum levels of 1,25(OH)₂D in pregnant women, cord blood and newborns were significantly higher than those in children and non-pregnant women and adult males. In newborns, the level increased with gestational age. Synthesis of 1,25(OH)₂D was, at least in part, under the control of the fetus and newborn, rather then being solely a reflection of the conditions prevailing in the mother. The 1-hydroxylase activity in mitochondria was highest in the 1- to 2-month-old rats, and it decreased gradually thereafter. The change in 1-hydroxylase activity with age was due to a change in the V_max of the system.     

Oral 1,25(OH)2D3 pulse therapy

Conclusion In our experience, after a few months of therapy, every patient showed a marked improvement in both X-ray abnormalities derived from osteitis fibrosa and symptoms of renal osteodystrophy, especially bone pain, unless the serum phosphorus level was very high. The effectiveness of this therapy on the suppression of PTH secretion apparently depends on the initial PTH level, and also on the size of the gland itself. One of the major current difficulties in this therapy is the prevention of hypercalcemia when calcium carbonate is used. The calcium concentration of the dialysate must be reduced to 2.5 mEq/l not only for pulse therapy, but also for conventional therapy by vitamin D with calcium carbonate. Parathyroidectomy should be indicated only for the patient who does not respond to pulse therapy.     

Biochemical characterization of 1,25(OH)2D3 receptors in chick embryonal duodenal cytosol

Summary This study presents measurements of serum vitamin D metabolites, calcium and phosphorus as well as measurements of the equilibrium dissociation constant for duodenal 1,25(OH)₂D₃ receptor in 15-, 18-, 19-, and 20-day chick embryos in comparison to that in 1- and 118-day-old chicks and to vitamin D-deficient chicks. The present results showed that: (a) serum 1,25(OH)₂D and 24,25(OH)₂D levels rise from 15 and 18 to days 19 and 20 of embryonic development while serum phosphate levels are stable; (b) serum calcium levels rise at hatching to adult levels; (c) the duodenal 1,25(OH)₂D₃ receptor is detectable in 15-day-old embryo and has a Kd similar to that of 118-day-old vitamin D-replete chicks; and (d) the activity of 1,25(OH)₂D₃ receptor in chick duodenal cytosol is maximal at hatching.     

Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

Summary We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)₂D₃ and for glucocorticoids, and have examined the modulation of the 1,25(OH)₂D₃ receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)₂D₃ receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)₂D₃; this phenomenon occurred in a dosedependent fashion for triamcinolone (10⁻⁹ to 10⁻⁷ M) with a maximum increase of 1,25(OH)₂D₃ receptor concentration of ⋍twofold. During the culture period, the 1,25(OH)₂D₃ receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)₂D₃ receptor concentration was higher in the triamcinolone-treated cells.     

Modulation by epidermal growth factor of the basal 1,25(OH)2D3 receptor level and the heterologous up-regulation of the 1,25(OH)2D3 receptor in clonal osteoblast-like cells

Summary The effects of epidermal growth factor (EGF) on basal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) receptor level and on parathyroid hormone (PTH)-induced 1,25-(OH)₂D₃ (OH)₂D₃ receptor up-regulation were studied in the phenotypically osteoblastic cell line UMR 106. EGF in concentrations exceeding 0.1 ng/ml reduced the number of 1,25(OH)₂D₃ binding sites without changing the binding affinity. Maximal reduction was 30% at about 1 ng/ml. This reduction was independent of a change in cAMP content. EGF dose-dependently attenuated both PTH-induced 1,25(OH)₂D₃ receptor up-regulation and PTH-stimulated cAMP production without and effect on the ED₅₀ of the PTH effects. For both PTH responses the IC₅₀ and the maximal effective dose were similar, 0.1 ng/ml an 1 ng/ml EGF, respectively. Reduction was first seen at 0.01 ng/ml EGF. At this concentration. EGF reduced PTH-stimulated 1,25-(OH)₂D₃ receptor binding without an inhibition of the cAMP response. Time-course studies with 1 ng/ml EGF revealed that at 2 h preincubation EGF reduced the heterologous up regulation by PTH, and maximal inhibition was seen after 4 h. In contrast, PTH-stimulated cAMP production was just significantly inhibited only after 6 h, with 60% inhibition after 24 h preincubation. The effects of prostaglandin E₂ and forskolin on both 1,25(OH)₂D₃ binding and cAMP production were inhibited in a similar fashion. On the other hand, dibutyryl cAMP- and 3-isobutyl-1-methylxanthinestimulated 1,25(OH)₂D₃ binding were not affected by EGF. Taken together, our results demonstrate that EGF reduces both the basal number of 1,25(OH)₂D₃ binding sites and the heterologous up-regulation of the 1,25(OH)₂D₃ receptor. The current data suggest that EGF reduces heterologous upregulation of the 1,25(OH)₂D₃ receptor independent of as well as dependent on the cAMP messenger system. The EGF effect is not primarily located at the PTH receptor, at cAMP phosphodiesterase, or at protein kinase A level.     

24,25(OH)2D3, bone formation,and bone resorption in vitamin D-deficient,azotemic rats

Summary Bone formation, mineralization, and resorption were measured in vitamin D-deficient, azotemic rats given two different dosages of 24,25(OH)₂D₃ daily and in vehicle-treated controls (C). The intraperitoneal administration of 65 pmol over a 10 day period corrected the hypocalcemia observed in C, whereas 130 pmol produced mild hypercalcemia. Both dosages reduced osteoid width, osteoid area, and mineralization front width form control values. The rates of bone and matrix formation were unaffected by treatment. In C, matrix formation exceeded bone formation and resulted in osteoid accumulation; both dosages of 24,25(OH)₂D₃ reversed this relationship such that bone formation exceeded matrix formation in each treatment group. The rates of osteoid maturation and initial mineralization increased during repletion with 24,25(OH)₂D₃ at both dosage levels. However, the serum calcium concentration was correlated with both osteoid maturation rate (r=0.68,P<0.01) and initial mineralization rate (r=0.63,P<0.01) when all three experimental groups were considered. Bone resorption was unchanged from control values during treatment with 24,25(OH)₂D₃. The results suggest that 24,25(OH)₂D₃ promotes the maturation and mineralization of osteoid, and that this metabolite differs in its effects on bone formation and resorption. It is not clear, however, that the changes in bone dynamics observed are independent of the calcemic response induced by metabolite repletion under the conditions of this experiment.     

Effects of phosphate and 1,25(OH)2D3 on in vitro bone collagen synthesis in the hypophosphatemic mouse

Summary Calvarial bones from hypophosphatemic (Hyp) mice and normal littermates were cultured in a chemically defined medium to determine: (a) the effect of medium phosphate (Pi) concentration (1, 2, and 3 mM) on collagen synthesis; (b) the effect of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] (10⁻¹²M–10⁻⁷M) on collagen synthesis; and (c) whether bone responsiveness to 1,25(OH)₂D₃ was affected by changes in medium Pi concentration. Bone collagen synthesis was evaluated by measuring [³H]hydroxyproline formation. The distribution of labeled hydroxyproline between bone explant and culture medium (total and dialyzable fraction) was studied. These experiments confirm that 1,25(OH)₂D₃ inhibits specifically bone collagen synthesis in vitro. We did not detect any effect of medium Pi concentration on basal collagen synthesis but were able to demonstrate that lowering medium Pi concentration increased the 1,25(OH)₂D₃-induced inhibition of collagen synthesis. Bones from both genotypes responded to 1,25(OH)₂D₃, but modulation of this response by changes in Pi concentration was altered in Hyp bone as, in contrast to normal bone, its response to 1,25(OH)₂D₃ was unaffected when medium Pi concentration was decreased from 3 to 2 mM. These findings support the hypothesis of an altered response of bone to 1,25(OH)₂D₃ in the Hyp mouse.     

Lack of influence of 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion from normal or hyperplastic glands

Summary The role of 24,25(OH)₂D₃ on parathyroid gland function remains controversial. The present studies were performedin vitro using (a) dispersed normal bovine parathyroid cells (bPTC) and (b) dispersed canine PTC (cPTC) prepared from glands of normal dogs, dogs with chronic renal failure (CRF), and dogs with CRF treated with 24,25(OH)₂D₃, 2.5 μg orally every day for more than 6 months. Bovine parathyroid cells were incubated for up to 180 min at 0.5, 1.0, and 3.0 mM external calcium in the presence or absence of 24,25(OH)₂D₃ (100 or 1000 nM). Similar experiments were conducted with cells incubated for 24 h in the presence of either the ethanol vehicle or 24,25(OH)₂D₃ (1000 nM). Parathyroid hormone secretion, measured in the supernatant by both C-terminal and N-terminal assays, did not show any differences between control and experimental groups at any time interval. Canine parathyroid cells obtained from uremic animals showed an average threefold increase in the total amount of PTH secreted, on a per cell basis over 180 min at 0.5 mM Ca²⁺, when compared with normal controls. However, there was no significant difference in PTH secretion at any level of calcium concentration between the cells obtained from parathyroid glands of CRF dogs and 24,25(OH)₂D₃-treated CRF dogs. Acute exposure to 24,25(OH)₂D₃ (1000 nM)in vitro of the cells obtained from the glands of CRF dogs also had no effect on PTH secretion. We conclude that 24,25(OH)₂D₃ has no direct effect on PTH secretion from dispersed parathyroid cells of either normal or uremic animals.     

Control of plasma 1,25-(OH)2-vitamin D concentrations by calcium and phosphorus in the rat: Effects of hypophysectomy

Summary The mechanism by which dietary phosphate deprivation elevates plasma 1,25-(OH)₂-D levels is not known. To evaluate the role of the pituitary in regulating plasma 1,25-(OH)₂-D concentrations, the responses of plasma 1,25-(OH)₂-D to dietary phosphate deprivation and, separately, to dietary calcium deprivation were evaluated in intact and hypophysectomized male rats. Among intact and hypophysectomized rats eating normal diets, plasma 1,25-(OH)₂-D levels averaged 228±76 and 148±62 pmol/1, respectively (P<0.01). During dietary phosphate deprivation, plasma 1,25-(OH)₂-D levels rose to 1160±260 in intact rats and fell to 90±26 pmol/l in hypophysectomized rats (P<0.001). By contrast, during dietary calcium deprivation, plasma 1,25-(OH)₂-D levels rose in both intact and hypophysectomized animals to 856±107 and 742±279 pmol/l, respectively (NS). In response to dietary phosphate deprivation, serum calcium concentrations rose as 1,25-(OH)₂-D concentrations rose in intact rats but remained at control levels in hypophysectomized rats. These results support the hypothesis that a pituitary hormone acting either directly or indirectly on the kidney mediates the increase in plasma 1,25-(OH)₂-D during dietary phosphate deprivation. The hypercalcemia that occurs in rats during dietary phosphate deprivation appears to depend on the elevation of plasma 1,25-(OH)₂-D.     

1,25-dihydroxyvitamin D3 causes an increase in the number of osteoclastlike cells in cat bone marrow cultures

Summary The direct effect of 1,25(OH)₂D₃ upon osteoclast formation from precursor cells is still unknown. In the present experiments we have tested the effects of 1,25(OH)₂D₃ on the generation of osteoclastlike cells in cat bone marrow cultures. These cultures contain proliferating nonattached mononuclear cells and precursor cells that subsequently attach to the culture flask surface and then fuse to form multinucleated osteoclastlike cells. After 7 days of culture we separated the nonattached precursor cells from the attached cells and studied the effects of 1,25(OH)₂D₃ (10⁻¹⁰ M–10⁻⁸ M) on multinucleated cell formation in these two cell populations. In cultures derived from the non-attached precursor cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) resulted in a 180% increase in the number of attached mononuclear cells and a 90% increase in the number of nuclei contained within multinucleated cells. These effects were dose-dependent. 1,25(OH)₂D₃ did not have a consistent effect on the number of nonattached precursor cells. In cultures derived from attached cells, 7 days of treatment with 1,25(OH)₂D₃ (10⁻⁸ M) induced a 50% increase in the number of mononuclear attached cells and a 40% increase in the number of nuclei within polykaryons. The most likely explanation for these results is that 1,25(OH)₂D₃ promotes the differentiation and subsequent adhesion of nonattached precursor cells, stimulates proliferation of attached mononuclear precursor cells, and possibly stimulates fusion of these attached precursor cells.     

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cellsIn vitro

Summary Binding of [³H] 1,25 (OH)₂D₃ and effects of 1,25 (OH)₂D₃ on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [³H] 1,25 (OH)₂D₃ by a 3.5 S macromolecule with DNA binding, K_D 6.2×10⁻¹⁰M and N_max 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10⁻⁸ M 1,25 (OH)₂D₃, but not 25 (OH)D₃, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.     

Histological observations on the failure of rachitic rat bones to respond to 1,25/OH)2D3

Summary Rachitic rats, maintained on diets with low or normal P contents, were given daily intraperitoneal doses of 1,25(OH)₂D₃ or 25OHD₃ at levels of 100 or 200 ng. Plasma chemistry was measured and the ash content and histological appearance of the bones investigated. Using labeled material it was shown that the dosing levels of 1,25(OH)₂D₃ employed ensured a higher than normal plasma concentration of that metabolite over the period between doses. 1,25(OH)₂D₃ was not as effective as 25OHD₃ in raising bone ash or reducing the amount of osteoid. The difference between the effects of the metabolites was evident at both dietary P levels, but more marked at the higher P level. In contrast, the metabolites reduced the width of the epiphyseal plate to an approximately similar degree, and this is possibly the reason why there are discrepancies between previous reports of the effectiveness of 1,25(OH)₂D₃ compared with 25OHD₃ or vitamin D₃. Dosing with 1,25(OH)₂D₃ failed to maintain a constant plasma P_i value over the period between doses in animals fed the low P diet.     

Effects of 1,25(OH)2D3 on bone tissue in the rabbit: Studies on fracture healing,disuse osteoporosis,and prednisone osteoporosis

Summary A closed tibial fracture, which was controlled by an intramedullary stainless steel pin, was created in 16 rabbits. Eight rabbits were treated with 75 ng of 1,25(OH)₂D₃ daily as subcutaneous (s.c.) injections. After three weeks, the fractured tibia resisted a force of 101,7±21.0 Newtons in the control group and 57.3±8.0 Newtons in animals given 1,25(OH)₂D₃ (m±SE,P<0.05). In another group of eight rabbits, the left hindleg was immobilized in a plastic splint. Four rabbits were given 75 ng of 1,25(OH)₂D₃/day s.c. and the effect of immobilization was studied on the calcaneus. Bone ash/cm³ of the calcaneus on the immobilized side was decreased by 11±2% in control rabbits and by 20±2% in the group treated with 1,25(OH)₂D₃ indicating a more advanced immobilization osteoporosis (m±SE,P<0.05), which was also demonstrated by studies of bone density. Eighteen rabbits were used in a study of the effects of 1,25(OH)₂D₃ on the development of prednisolone osteoporosis. The dose of prednisolone was 2.5 mg per day, given by the oral route. After four months, the density of the femur was 1.53±0.02 g/cm² in control rabbits and 1.42±0.01 in prednisolonetreated animals (P<0.01). In rabbits additionally given 1,25(OH)₂D₃, the mean value for bone density was further lowered (n.s.). It appears that 1,25(OH)₂D₃ exaggerates disuse osteoporosis and prednisolone osteoporosis and impairs fracture healing in rabbits. These results differ from what has been shown earlier with 1,25(OH)₂D₃ treatment in the rat.     

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.     

Inhibitory effects of 9-cis and all-trans retinoic acid on 1,25(OH)2 vitamin D3-induced bone resorption

The effects of retinoic acid (RA), and calcitriol are mediated by specific nuclear receptors (RARs and VDR, respectively). Induction of RAR and VDR responsive elements in target genes requires a cofactor, the retinoid-X-receptor (RXR), with its ligand 9-cis RA. We have previously demonstrated the expression of RARs and RXRs in osteoblasts, and herein investigated the effects of the retinoids all-trans RA and 9-cis RA alone and combined with calcitriol on bone resorption in vitro, measured by ⁴⁵Ca-release from prelabeled neonatal mouse calvarial bones. All-trans RA and 9-cis RA were powerful stimulators of bone resorption and essentially equipotent. At threshold concentrations (1 nM) both 9-cis RA and at-RA markedly inhibited the resorption induced by calcitriol (1 pM). The findings are compatible with a physiological role for retinoids in bone metabolism.     

Effects of weight loss on serum 1,25-(OH)2-vitamin D concentrations in adults: A preliminary report

Summary During a review of 42 metabolic studies in healthy women and men we observed that serum 1,25-(OH)₂-D concentrations were directly correlated to the observed daily changes in body weight (r=0.68;P<0.001) and to caloric intake/kg/day (r=0.39;P=0.01). These relationships could not be accounted for by related and physiologically expected changes in serum Ca or iPTH concentrations. However, serum 1,25-(OH)₂-D concentrations were observed to be inversely correlated to serum PO₄ levels (r=−0.44;P=0.004). In addition, serum PO₄ levels were inversely correlated to the daily changes in body weight (r=−0.40;P=0.009). Since dietary sodium intake averaged 142 mmol/day, it is unlikely that the observed changes in weight were the result of changes in salt and water balance. Thus it seems reasonable to speculate that serum 1,25-(OH)₂-D concentrations may vary directly with energy balance, as reflected by changes in body weight. This effect may be mediated by alterations in PO₄ metabolism. The accurate assessment of serum 1,25-(OH)₂-D levels thus appears to require several measurements over time periods during which body weight is stable.     

Enhanced suppression of 1,25(OH)2D3 and intact parathyroid hormone in Graves' disease as compared to toxic nodular goiter

Summary 1,25(OH)₂D₃, 25OHD₃, and intact parathyroid hormone, as well as various parameters of calcium-phosphorus metabolism were measured in 38 patients with Graves' disease (GD) and in 24 patients with toxic nodular goiter (TNG). Plasma 1,25(OH)₂D₃ levels were lower in GD patients (82 ±29 pmol/liter) than in those with TNG (155±32 pmol/liter) (P<0.0005). The mean value of 1,25(OH)₂D₃ in 45 controls was intermediate between the two groups of patients (140±41) and the difference was statistically significant. GD patients before and after treatment had higher alkaline phosphatase (P<0.05), lower intact parathyroid hormone (PTH) (P<0.05), and lower 1,25(OH)₂D₃ levels (P<0.0005 in the hyperthyroid andP<0.01 in the euthyroid state) than TNG patients. We conclude that increased skeletal calcium resorption is due to elevated levels of T₃ causing suppression of 1,25(OH)₂D₃ production and of PTH levels in both groups of patients albeit of different degrees. Furthermore, we postulate that the profound suppression of 1,25(OH)₂D₃ in GD is secondary to an immune-mediated phenomenon.     

Effect of vitamin D3, 25-hydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion

Summary The active vitamin D metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] causes marked suppression of both pre-proparathyroid hormone messenger RNA (pre-proPTH mRNA) and parathyroid hormone (PTH) secretion. These effects are dose dependent and reversible when tested in anin vitro primary tissue culture cell system using normal bovine parathyroid cells. In the current studies, the precursors of 1,25(OH)₂D₃ and the related metabolite 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], were used in the same culture system to test for possible regulatory effects. The results were compared with identically prepared cells exposed to 1,25(OH)₂D₃. In short-term studies (30–120 minutes), none of the vitamin D-related compounds produced any effect on PTH secretion. In long-term studies (24–48 hours, using primary tissue culture in the presence of test agents), neither vitamin D₃ nor 25(OH)D₃ affected PTH secretion or pre-proPTH mRNA over the concentration range 10⁻¹¹–10⁻⁷M. On the other hand, 24,25(OH)₂D₃ produced significant suppression of both pre-proPTH mRNA (77% of control,P<.01) and PTH secretion (75% of control,P<.005) at 10⁻⁷ M. By comparison, 10⁻¹¹ M 1,25(OH)₂D₃ produced levels of suppression (25–30%) of both pre-proPTH mRNA and PTH secretion comparable to 10⁻⁷ M 24,25(OH)₂D₃, while even greater suppression (40–50%) occurred at 10⁻⁹-10⁻⁷ M 1,25(OH)₂D₃. From these studies, we conclude that vitamin D₃ and 25(OH)D₃ do not have significant effects on PTH synthesis and secretion over the range of doses tested. Compared with 1,25(OH)₂D₃, 24,25(OH)₂D₃ exhibits mild suppression at pharmacologic concentrations. The effect of 24,25(OH)₂D₃ prabably occurs through weak interaction of 24,25(OH)₂D₃ with the 1,25(OH)₂D₃ receptor.     

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.