Extrarenal metabolism of 25-hydroxycholecalciferol in the rat: Regulation by 1,25-dihydroxycholecalciferol

Summary To determine the role of the kidney in regulation of 25-hydroxycholecalciferol (25OHD₃, metabolism, the effects of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃] on³H-25OHD₃ were compared in intact and nephrectomized vitamin D-deficient rats. Sixteen hours after the intravenous administration of³H-25OHD₃, extracts of serum and pooled small intestinal mucosa were fractionated by Sephadex LH-20 column chromatography followed by high performance liquid chromatography. In intact rats, 1,25(OH)₂D₃ (50 ng/day i.p. for 7 days) increased mean serum³H-24,25-dihydroxycholecalciferol [³H-24,25(OH)₂D₃] from 2±2–210±80 fmol/ml (mean±1 SD), increased mean serum³H-25,26-dihydroxycholecalciferol [³H-25,26(OH)₂D₃] from 2±2–12±6 fmol/ml and lowered mean serum³H-1,25(OH)₂D₃ from 210±40–4±4 fmol/ml. Similarly, in nephrectomized animals, 1,25(OH)₂D₃ increased mean serum³H-24,25-(OH)₂D₃ from 6±11–115±30 fmol/ml and increased mean serum³H-25,26(OH)₂D₃ from 3±3–26 ± 10 fmol/ml. Nephrectomy increased serum³H-25(OH)D₃ in untreated (from 1450±225–2675±225 fmol/ml serum) and 1,25(OH)₂D₃ treated rats (from 1600±175–3075±100 fmol/ml).³H-1,25(OH)₂D₃ averaged 74±16% of total radioactivity in intestinal mucosa of untreated intact rats and was not detected in either the serum or intestinal mucosa of nephrectomized animals. The results suggest that in intact animals, extrarenal synthesis can account for substantial 24,25(OH)₂D₃ production and for most 25,26(OH)₂D₃ production. Further, the observed stimulation of production of 24,25(OH)₂D₃ and 25,26(OH)₂D₃ by 1,25(OH)₂D₃ in anephric — D rats providesin vivo evidence for regulation of extrarenal 25OHD₃: 24- and 26-hydroxylases.     

Effect of a long-term treatment with 1,25-dihydroxyvitamin D3 on osteocalcin in postmenopausal osteoporosis

Summary Serum bone Gla-protein (BGP or osteocalcin) was measured in 25 women with histologically confirmed postmenopausal osteoporosis before and during long-term treatment with 1 μg/day of 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃). Basal serum BGP was significantly lower in osteoporotic women (3.8±1.4 ng/ml) than in agematched controls (6.8±2.0 ng/ml). During 1,25(OH)₂D₃ therapy serum BGP increased so that the mean of the values observed on treatment (4.8±1.5) was significantly higher than the mean basal value. It is known that BGP synthesis is stimulated by 1,25 (OH)₂D₃ and that serum BGP is a specific marker of bone formation; therefore, it is possible that the low basal levels of osteocalcin we observed were related to the low serum 1,25(OH)₂D concentrations reported in osteoporotic women and that the increase in BGP levels observed under 1,25(OH)₂D₃ treatment was a consequence of osteoblast stimulation.     

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.     

Effects of 25‐hydroxyvitamin D3 on proliferation and osteoblast differentiation of human marrow stromal cells require CYP27B1/1α‐hydroxylase

1,25‐Dihydroxyvitamin D₃ [1,25(OH)₂D₃] has many noncalcemic actions that rest on inhibition of proliferation and promotion of differentiation in malignant and normal cell types. 1,25(OH)₂D₃ stimulates osteoblast differentiation of human marrow stromal cells (hMSCs), but little is known about the effects of 25‐hydroxyvitamin D₃ [25(OH)D₃] on these cells. Recent evidence shows that hMSCs participate in vitamin D metabolism and can activate 25(OH)D₃ by CYP27B1/1α‐hydroxylase. These studies test the hypothesis that antiproliferative and prodifferentiation effects of 25(OH)D₃ in hMSCs depend on CYP27B1. We studied hMSCs that constitutively express high (hMSCs^hi‐1α) or low (hMSCs^lo‐1α) levels of CYP27B1 with equivalent expression of CYP24A1 and vitamin D receptor. In hMSCs^hi‐1α, 25(OH)D₃ reduced proliferation, downregulated proliferating cell nuclear antigen (PCNA), upregulated p21^Waf1/Cip1, and decreased cyclin D1. Unlike 1,25(OH)₂D₃, the antiapoptotic effects of 25(OH)D₃ on Bax and Bcl‐2 were blocked by the P450 inhibitor ketoconazole. The antiproliferative effects of 25(OH)D₃ in hMSCs^hi‐1α and of 1,25(OH)₂D₃ in both samples of hMSCs were explained by cell cycle arrest, not by increased apoptosis. Stimulation of osteoblast differentiation in hMSCs^hi‐1α by 25(OH)D₃ was prevented by ketoconazole and upon transfection with CYP27B1 siRNA. These data indicate that CYP27B1 is required for 25(OH)D₃'s action in hMSCs. Three lines of evidence indicate that CYP27B1 is required for the antiproliferative and prodifferentiation effects of 25(OH)D₃ on hMSCs: Those effects were not seen (1) in hMSCs with low constitutive expression of CYP27B1, (2) in hMSCs treated with ketoconazole, and (3) in hMSCs in which CYP27B1 expression was silenced. Osteoblast differentiation and skeletal homeostasis may be regulated by autocrine/paracrine actions of 25(OH)D₃ in hMSCs. © 2011 American Society for Bone and Mineral Research.     

Different Effects of Insulin and Insulin-Like Growth Factors I and II on Osteoprogenitors and Adipocyte Progenitors in Fetal Rat Bone Cell Populations

We investigated the effects of insulin (1–1,000 nM), insulin-like growth factor (IGF)-I, and IGF-II (3–100 nM each) alone or together with 10 nM dexamethasone (DEX) or 10 nM 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) on proliferation and differentiation of adipocyte and osteoblast progenitors in bone cell populations derived from fetal rat calvaria. The effects on differentiation were evaluated by counting the number of bone or osteoid nodules and adipocyte colonies and the effects on proliferation, by measuring their size by image analysis. The types of cells studied were 1,25(OH)₂D₃- and DEX-responsive adipocyte progenitors and DEX-dependent and independent osteoprogenitors. Both IGF-I and IGF-II stimulated osteoprogenitor differentiation both alone and in the presence of DEX, while insulin stimulated osteoprogenitor differentiation only in the absence of DEX. Neither IGF-I/-II nor insulin affected proliferation of osteoprogenitors. Insulin had little effect on adipocyte differentiation by itself but strongly stimulated differentiation in the presence of either 1,25(OH)₂D₃ or DEX, while IGF-II stimulated adipocyte differentiation in both the absence and presence of 1,25(OH)₂D₃ or DEX. IGF-I by itself or in the presence of DEX strongly stimulated adipocyte cell differentiation but had little effect in the presence of 1,25(OH)₂D₃. Our results demonstrate that insulin, IGF-II, and IGF-I have specific and different effects on the differentiation and proliferation of different groups of progenitor cells.     

Vitamin D metabolism in pregnant and pseudopregnant rats: Identification of 25-hydroxycholecalciferol-1-hydroxylase in decidual tissue

Summary Elevated levels of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are found in late pregnancy but the factors responsible for this are not known. To determine if the maternal-fetal calcium flux or the presence of a previously described extrarenal 25-hydroxycholecalciferol-1-hydroxylase (25(OH)-D₃-1-hydroxylase) play a role, serum calcium and 1,25(OH)₂D₃ were measured in pregnant, nonpregnant, and decidua-bearing pseudopregnant rats. Serum calcium was 8.74± 0.26 mg/dl (mean±SEM) in nonpregnant rats. In pregnant rats, serum calcium was not significantly different from nonpregnant controls on day 12 and only slightly higher on day 15. Pseudopregnant rats were significantly hypercalcemic on days 12 (11.93±0.19 mg/dl) and 15 (11.45±0.23 mg/dl) compared with nonpregnant rats (P<0.001). In nonpregnant controls the serum level of 1,25(OH)₂D₃ was 44.6±6.3 pg/ml. Levels in pregnant rats were not significantly different on days 12 or 15 but tended to be higher by day 15 (75.2±19.7 pg/ml). Pseudopregnant rats had levels of 72.6±13.5 pg/ml on day 12 and 102.8±10.9 pg/ml on day 15, the latter of which was significantly higher than nonpregnant values (P<0.05). 25(OH)D₃-1-hydroxylase activity was determined in whole tissue homogenates of placenta and decidua. Placenta from pregnant rats and decidua from pregnant and pseudopregnant rats both formed putative 1,25(OH)₂D₃ in short-term incubation with 25(OH)D₃ as identified by comigration with authentic 1,25(OH)₂D₃ on high pressure liquid chromatography (HPLC). The results suggest that hypercalcemia during pseudopregnancy is due to unregulated production of 1,25(OH)₂D₃ by decidual tissue. Pregnant rats may not become hypercalcemic because the fetus acts as a calcium sink or the 25(OH)D₃-1-hydroxylase is under regulation in the pregnant state.     

Oral 1,25-dihydroxyvitamin D3 loading test in normal subjects and patients with chronic renal failure under haemodialysis

Summary: The present study was designed to determine the criterion for 1,25-dihydroxyvitamin D₃ (1,25 (OH)₂D₃) loading test in normal subjects and haemodialysis patients. Fourteen normal subjects were administered 1.0 μg of 1,25(OH)₂D₃ per os and serum 1,25(OH)₂D was monitored every hour up to 6 h afterwards under conditions of overnight fasting, and six haemodialysis patients were administered 2.0 μg of 1,25(OH)₂D₃ per os and serum 1,25(OH)₂D was monitored every 2 h up to 12 h afterwards. Peak time of serum 1,25 (OH)₂D varied between 2 and 5 h after administration in normal subjects. However, there was a good correlation between the maximum increment of 1,25(OH)₂D (maxΔ1,25(OH)₂D) and the increment at 4 h after administration (Δ1,25(OH)₂D(4 h)). the peak time of Δ1,25(OH)₂D in six haemodialysis patients was also at 4 h after administration. From these observations, Δ1,25(OH)₂D(4 h) was evaluated in subsequent studies. Twenty-six normal subjects and 24 haemodialysis patients were administered 0.5–2.0 μg of 1,25(OH)₂D₃ per os, according to their bodyweights, under conditions of overnight fasting. Blood samples were drawn for measuring 1,25(OH)₂D prior to and 4 h after administration. Δ1,25(OH)₂D(4 h) showed good correlation with the dose of 1, 25 (OH)2D3 adjusted by bodyweight (ng/kg bodyweight). the ratio of Δ1,25(OH)₂D(4 h) and adjusted dose of 1,25(OH)₂D₃ was more than 2.0 in all normal subjects (range: 1.97?2.89, mean ± SD: 2.38 ± 0.287). Moreover, the ratio of Δ1,25(OH)₂D(4 h) and adjusted dose of 1,25(OH)₂D₃ showed a good reproducibility (CV%= 5.7 Δ 0.32, n=5), and did not depend on the administered dose of 1,25(OH)₂D₃, suggesting that this ratio is a good parameter for the intestinal absorption of 1,25(OH)₂D₃. In haemodialysis patients, the mean ratio of Δ1,25 (OH)₂D(4 h) and adjusted dose of 1,25(OH)₂D₃ was 2.14 Δ 0.489, which was not significantly different from the ratio in normal subjects, suggesting that, fundamentally, there was no impairment of intestinal absorption of 1,25(OH)₂D₃ in these patients. However, low ratios of Δ(4 h) and the dose of 1,25(OH)₂D₃ with low basal levels of 1,25(OH)₂D were observed in some patients (less than 1.5 in four patients), suggesting that there exist haemodialysis patients with malabsorption of 1,25(OH)₂D₃. From these results, the criterion for normal response in 1,25(OH)₂D loading test was proposed, namely, that the ratio of Δ1,25(OH)₂D(4 h) and adjusted dose of 1,25(OH)₂D₃ be more than 2.0.     

The effect of 1,25 dihydroxycholecalciferol on parathyroid hormone secretion by monolayer cultures of bovine parathyroid cells

Summary Controversy exists over a direct effect of 1,25(OH)₂D₃ on PTH secretion. To investigate the possibility that the suppressive effect of 1,25(OH)₂D₃ on PTH secretion may be demonstrable in 1,25(OH)₂D₃-depleted tissue and/or after prolonged periods of exposure to 1,25(OH)₂D₃, primary monolayer cultures of bovine parathyroid cells were established in 1∶1 DMEM/Ham's F-12 media supplemented with 2% calf serum but not 1,25(OH)₂D₃. Ionized calcium was maintained at 1.0 mM. Experiments were performed on 4-day-old culture cells. PTH concentration was measured using both a mid-region/carboxyl and an amino-terminal PTH antisera. 1,25(OH)₂D₃ at a concentration of 0.1 ng/ml suppressed PTH secretion by 32±7% after 48 hours. High calcium concentration (2.0 mM) suppressed PTH secretion by 37±10% and this effect was not additive over that of 1,25(OH)₂D₃. PTH secretion rate recovered fully 48 hours after normalization of the external calcium concentration but not after the removal of 1,25(OH)₂D₃. It is concluded that 1,25(OH)₂D₃ directly suppresses PTH secretion by monolayer culture of bovine parathyroid cells.     

Comparison of metabolism of vitamins D2 and D3 in children with nutritional rickets

Children with calcium‐deficiency rickets may have increased vitamin D requirements and respond differently to vitamin D₂ and vitamin D₃. Our objective was to compare the metabolism of vitamins D₂ and D₃ in rachitic and control children. We administered an oral single dose of vitamin D₂ or D₃ of 1.25 mg to 49 Nigerian children—28 with active rickets and 21 healthy controls. The primary outcome measure was the incremental change in vitamin D metabolites. Baseline serum 25‐hydroxyvitamin D [25(OH)D] concentrations ranged from 7 to 24 and 15 to 34 ng/mL in rachitic and control children, respectively (p < .001), whereas baseline 1,25‐dihydroxyvitamin D [1,25(OH)₂D] values (mean ± SD) were 224 ± 72 and 121 ± 34 pg/mL, respectively (p < .001), and baseline 24,25‐dihydroxyvitamin D [24,25(OH)₂D] values were 1.13 ± 0.59 and 4.03 ± 1.33 ng/mL, respectively (p < .001). The peak increment in 25(OH)D was on day 3 and was similar with vitamins D₂ and D₃ in children with rickets (29 ± 17 and 25 ± 11 ng/mL, respectively) and in control children (33 ± 13 and 31 ± 16 ng/mL, respectively). 1,25(OH)₂D rose significantly (p < .001) and similarly (p = .18) on day 3 by 166 ± 80 and 209 ± 83 pg/mL after vitamin D₂ and D₃ administration, respectively, in children with rickets. By contrast, control children had no significant increase in 1,25(OH)₂D (19 ± 28 and 16 ± 38 pg/mL after vitamin D₂ and D₃ administration, respectively). We conclude that in the short term, vitamins D₂ and D₃ similarly increase serum 25(OH)D concentrations in rachitic and healthy children. A marked increase in 1,25(OH)₂D in response to vitamin D distinguishes children with putative dietary calcium‐deficiency rickets from healthy children, consistent with increased vitamin D requirements in children with calcium‐deficiency rickets. © 2010 American Society for Bone and Mineral Research     

Suggestion of a deficient osteoblastic function in diabetes mellitus: The possible cause of osteopenia in diabetics

Summary The mechanism underlying diabetic osteopenia is still unclear and may involve osteoblastic activity and/or the deficit of insulin's anabolic action. Bone gla protein (BGP) is synthesized by the osteoblast and its synthesis increases with 1,25(OH)₂D₃ and fluoride. Because 1,25(OH)₂D₃ also stimulates insulin secretion, sodium fluoride administration can be used to investigate deficient osteoblastic activity in diabetics, as reflected by BGP levels. BGP was determined before and after administering sodium fluoride at a dosage of 50 mg/day/15 days to three groups: 14 patients with insulin-dependent diabetes, 16 diabetics on oral antidiabetic treatment, and 25 controls, all of similar age, sex, and characteristics. Basal BGP values (mean±SD) were low in diabetics on insulin treatment (4.3±1.1 ng/ml) and in diabetics on oral antidiabetics (5.8±1.2 ng/ml) as compared with controls (6.5±0.7 ng/ml) (P<0.001 and <0.05, respectively). After giving fluoride, BGP values did not change in the two diabetic groups but did vary in controls (8.1±0.6 ng/ml,P<0.001). These results suggest that deficient osteoblast function could be responsible for osteopenia in diabetics.     

Serum 1,25-dihydroxyvitamin D concentrations in premature infants: Preliminary results

Summary Serum 1,25(OH)₂D concentrations were measured in serial serum samples from 19 premature infants of 29.6±1.3 weeks gestation and 1,129±159 g birthweight. 1,25(OH)₂D was always normal or elevated and mean concentrations increased with age (adult, 55.2±13; infants, 1–2 weeks, 81.5±37.7 pg/mg; 3 weeks, 65±21; 6 weeks, 90.0±17.3; 9 weeks, 99.0±25.1; 12 weeks, 103.3±26.6 pg/ml). No correlation was seen with 25-OHD. Infants given 800 IU D₂ supplements had lower 1,25(OH)₂D levels than infants given 400 IU D₂. Breast fed infants had initially higher 1,25(OH)₂D levels; however, this was not sustained. These preliminary data suggest that premature infants regulate 1,25(OH)₂D production similar to more mature infants and children. Whether the premature infant has a normal gastrointestinal and/or bone responsiveness to 1,25(OH)₂D and whether these elevated 1,25(OH)₂D concentrations are “adequately elevated” requires further study. NIH Grant 2R01HD-09998-06.     

Local injection of 1,25-dihydroxyvitamin D<Subscript>3</Subscript> enhanced bone formation for tooth stabilization after experimental tooth movement in rats

The present investigation evaluated the effect of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on alveolar bone formation during tooth movement in rats. Orthodontic elastics were inserted between the maxillary first and second molars on bilateral sides in male rats. 1,25(OH)₂D₃ was injected locally, at the concentration of 10^–10M, once every 3 days in the submucosal palatal area of the root bifurcation of the molar on the right side. Histomorphometric analysis revealed that tooth movement without application of 1,25(OH)₂D₃ decreased the mineral appositional rate (MAR) on the compression area at 7 days. Repeated injections of 1,25(OH)₂D₃ in the orthodontically treated animals distinctly stimulated alveolar bone formation on the mesial side at 14 days. There was a significant increase in MAR associated with elevated osteoblast surface (Ob.S/BS) value on the tension surface. These findings suggest that local application of 1,25(OH)₂D₃ enhances the reestablishment of supporting tissue, especially alveolar bone of teeth, after orthodontic treatment.     

1,25 Dihydroxyvitamin-D3 Attenuates the Confluence-Dependent Differences in the Osteoblast Characteristic Proteins Alkaline Phosphatase, Procollagen I Peptide, and Osteocalcin

In the present study a cell culture model of primary human osteoblasts based on degrees of confluence was investigated by measuring basal and 1,25(OH)₂D₃stimulated levels of the osteoblast characteristic proteins alkaline phosphatase (AP), procollagen I-peptide (PICP), and osteocalcin (OC), as well as the corresponding gene expression. Primary osteoblast-like cell cultures from seven donors were treated in the second passage with 1,25(OH)₂D₃ (5 × 10⁻⁸ M for 48 hours) and investigated at four stages of confluence (stage I 50%, stage II 75%, stage III 100%, and stage IV 7 days postconfluence). In untreated cultures passing through the different stages of confluence, we saw a 1.8-fold increase of AP activity, a 2.3-fold increase of OC secretion, but a decrease of PICP levels to 0.36-fold. Gene expression showed only minor variation between the different confluence stages. 1,25(OH)₂D₃ did not significantly affect PICP production. Alkaline phosphatase protein was stimulated during proliferation until confluence, with no effect thereafter. Surprisingly, OC secretion and mRNA expression were stimulated in all four stages to the same absolute level independent of basal values. We conclude that our results correspond to other studies showing differentiation-stage dependent changes of basal levels of osteoblast-specific proteins. However, 1,25(OH)₂D₃ stimulation decreased the confluence-dependent difference for AP and abolished it for osteocalcin, thus leading to a more differentiated phenotype of the osteoblast. Therefore, 1,25(OH)₂D₃ stimulation might improve the reproducibility of results obtained at different confluence stages from cultures of clinical samples. Received: 25 November 1997 / Accepted: 2 September 1998     

Role of carbonic anhydrase in bone resorption induced by 1,25 dihydroxyvitamin D3 in vitro

Summary The present investigation was undertaken to study the role of carbonic anhydrase in 1,25 dihydroxyvitamin D₃-induced bone resorption. Calvaria were removed from 5- to 6-day-old mice and cultured for periods up to 96 h in Dulbecco's Modified Eagle Medium (high glucose, 4,500 mg/dl) supplemented with antibiotics and either heat-inactivated horse and fetal calf sera or bovine serum albumin. The experimental cultures contained 1×10⁻⁸ M 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃). All cultures were incubated at 37°C in 5% CO(in2)/95% air. Bone resorption was assessed by release of stable calcium into the medium. Bone enzymes (acid and alkaline phosphatases and carbonic anhydrase) were determined following homogenization in 0.25 M sucrose. The effects of 1,25(OH)₂D₃ were studied in the presence and absence of the carbonic anhydrase inhibitor acetazolamide and its analogue (CL 13,850), which lacks inhibitory activity. Acetazolamide inhibited 1,25(OH)₂D₃-induced calcium release in a dose-dependent fashion from 10⁻⁵–10⁻⁴ M. When added to the cultures at a concentration of 1×10⁻⁴ M, acetazolamide completely blocked the 1,25(OH)₂D₃-induced calcium release, a phenomenon not seen with an equimolar concentration of CL 13,850. The most significant finding was that 1,25(OH)₂D₃-induced calcium release was accompanied by a significant increase in the carbonic anhydrase activity of bone at both 48 (treated/control ratio=2.05) and 96 (treated/control ratio=2.59) hours. Bone alkaline phosphatase activity decreased and acid phosphatase activity increased in response to 1,25(OH)₂D₃. These findings support the concept that carbonic anhydrase is involved in bone resorption inducedin vitro by certain calcemic hormones and related compounds.     

Chronological changes of3H-1,25(OH)2D3 in tissue and serum after consecutive oral doses of active vitamin D in rats : Comparative study of 1αOHD3 and 1,25(OH)2D3

To determine the effect of consecutive oral administration of 1αOHD₃ or 1,25(OH)₂D₃ on the metabolism of 1,25(OH)₂D₃, seven-month-old female rats were given 1αOHD₃ (0.4 µg/kg/day) or 1,25(OH)₂D₃ (0.2 µg/kg/day) for 14 days. After the oral administration of 2 μCi of³H-1αOHD₃ or³H-1,25(OH)₂D₃, the rats were sacrificed at 2,6 or 24 h, and the distribution of these tracers and their metabolites in the serum, intestines, liver, kidneys and bone were studied. The consecutive treatment with 1,25(OH)₂D₃ or 1αOHD₃ did not basically alter the elution patterns of³H labeled metabolites on HPLC. The tissue levels of³H-1,25(OH)₂D₃, administered or converted from³H-1αOHD₃, were lower in the treated rats than those in the controls at 24 h, indicating the accelerated disappearance of 3H-1,25 (OH)₂D₃ following the treatment with 1αOHD₃ or 1,25(OH)₂D₃. The degree of acceleration, however, was less following the treatment with 1αOHD₃ than that after treatment with 1,25(OH)₂D₃. The degree ofacceleration, however, was less following the treatment with 1αOHD₃ than that after treatment with 1,25(OH)₂D₃. This finding might indicate that, when 1αOHD₃ or 1,25(OH)₂D₃ is consecutively administered, the 1,25(OH)₂D₃ converted from 1αOHD₃ by the liver remains longer in the tissues including bone than 1,25(OH)₂D₃ absorbed directly from the intestine.

     

Regulation of Osteogenic Differentiation of Human Bone Marrow Stromal Cells: Interaction Between Transforming Growth Factor-β and 1,25(OH)2 Vitamin D3 In Vitro

Bone marrow stromal cells are believed to play a major role in bone formation as a major source of osteoprogenitor cells, however, very little is known about how the osteogenic differentiation of these cells is regulated by systemic hormones and local growth factors. We examined the effects of TGF-β and its interaction with 1,25(OH)₂ Vitamin D₃ [1,25(OH)₂D₃] on the differentiation and proliferation of human bone marrow stromal cells (hBMSC) in secondary cultures. Alkaline phosphatase (ALP) activity was inhibited by TGF-β (0.1–10 ng/ml) and increased by 1,25(OH)₂D₃ (50 nM), however, co-treatment of TGF-β and 1,25(OH)₂D₃ synergistically enhanced ALP activity with maximal stimulation occurring at about 8 days after treatment. This synergistic effect was independent of proliferation because, in contrast to TGF-β alone, combined treatment with TGF-β and 1,25(OH)₂D₃ had no effect on hBMSC proliferation. As no synergistic effect was seen with combinations of 1,25(OH)₂D₃ and other osteotrophic growth factors, including BMP-2, IGF-I, and basic fibroblast growth factor (bFGF), it would seem likely that the synergistic interaction is specific for TGF-β. The increased ALP activity was due to an enhancement of 1,25(OH)₂D₃-induced ALP activity by TGF-β, rather than vice versa. In contrast, TGF-β inhibited 1,25(OH)₂D₃-induced osteocalcin production. Taken together, these results indicate that TGF-β and 1,25(OH)₂D₃ act synergistically to stimulate the recruitment of BMSC to the osteoblast lineage. This interaction may play an important role in bone remodeling. Received: 24 March 1998 / Accepted: 1 February 1999     

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.     

1,25-Dihydroxyvitamin D3 Promotes Vitamin K2 Metabolism in Human Osteoblasts

It has been reported that vitamin K₂ (menaquinone-4) promoted 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃)-induced mineralization and enhanced γ-carboxyglutamic acid (Gla)-containing osteocalcin accumulation in cultured human osteoblasts. In the present study, we investigated whether menaquinone-4 (MK-4) was metabolized in human osteoblasts to act as a cofactor of γ-glutamyl carboxylase. Both conversions of MK-4 to MK-4 2,3-epoxide (epoxide) and epoxide to MK-4 were observed in cell extracts of cultured human osteoblasts. The effect of 1,25(OH)₂D₃ and warfarin on the vitamin K cycle to cultured osteoblasts were examined. With the addition of 1 nM 1,25(OH)₂D₃ or 25 μM warfarin in cultured osteoblasts, the yield of epoxide from MK-4 increased. However, the conversion of epoxide to MK-4 was strongly inhibited by the addition of warfarin (2.5–25 μM), whereas it was almost not inhibited by 1,25(OH)₂D₃ (0.1–10 nM). To clarify the mechanism for this phenomenon, a cell-free assay system was studied. Osteoblast microsomes were incubated with 10 μM epoxide in the presence or absence of warfarin and 1,25(OH)₂D₃. Epoxide reductase, one of the enzymes in the vitamin K cycle was strongly inhibited by warfarin (2.5–25 μM), whereas it was not affected by 1,25(OH)₂D₃ (0.1–1 nM). Moreover, there was no effect of pretreatment of osteoblasts with 1 nM 1,25(OH)₂D₃ on the activity of epoxide reductase. However, the activity of epoxidase, that is the γ-glutamyl carboxylase was induced by the pretreatment of osteoblasts with 1 nM 1,25(OH)₂D₃. In the present study, it was demonstrated that the vitamin K metabolic cycle functions in human osteoblasts as well as in the liver, the post-translational mechanism, by which 1,25(OH)₂D₃ caused mineralization in cooperation with vitamin K₂ was clarified. Received: 20 September 2000 / Accepted: 19 February 2001