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Objective To analyze vitamin D metabolism and response to ketoconazole, an imidazole derivative that inhibits the vitamin D-1-hydroxylase, in infants with idiopathic hypercalcemia, and hypercalciuria. Study design Twenty infants (4 days-17 months) with hypercalcemia, severe hypercalciuria, and low parathyroid hormone level, (10 had nephrocalcinosis), including 10 treated with ketoconazole (3-9 mg/kg/day), were followed to the age of 2 to 51 months. Vitamin D receptor expression (VDR), 24-hydroxylase activity, and functional gene polymorphisms of vitamin D metabolism regulators VDR(rs4516035), 1-hydroxylase(rs10877012), 24-hydroxylase(rs2248359), FGF23(rs7955866), Klotho(rs9536314, rs564481, rs648202), were evaluated. Results Serum calcium levels, which occurred faster in the ketoconazole group (0.7 ± 0.2 versus 2.4 ± 0.6 months; P = .0076), and urinary calcium excretion (2.5 ± 0.5 versus 4.2 ± 1.7 months) normalized in all patients. Serum 1,25-(OH)2D levels were high normal and positively correlated to 25-(OH)D levels. Serum 24,25-(OH)2D levels were low normal, and skin fibroblasts from 1 patient showed defective up-regulation of the 24-hydroxylase by 1,25-(OH)2D despite normal VDR binding ability. An abnormally low prevalence of haplotype CC/CC for H589H/A749A in Klotho gene was found in patients and family members. Conclusions Ketoconazole is a potentially useful and safe agent for treatment of infantile hypercalcemia. Abnormal vitamin D metabolism is suggested as the mechanism, possibly involving defective up-regulation of the 24-hydroxylase by 1,25-(OH)2D3, and the klotho-FGF23 axis.

We report here the first association between vitamin D–resistant rickets, alopecia, and type 1 diabetes in a child with compound heterozygous mutations in the VDR gene. Transfection studies suggest dissociated effects of VDR gene mutations on the regulation of genes involved in vitamin D metabolism and dendritic cell maturation. Introduction: Whereas vitamin D may play a role in the immune tolerance process, no patient has been reported to associate hereditary vitamin D–resistant rickets (HVDRR) and an autoimmune disease, and no attempt has been made to delineate the outcome of mutations of the vitamin D receptor (VDR) on the transcription of genes controlling immune tolerance. Materials and Methods: The VDR gene was analyzed in a child with vitamin D–resistant rickets, total alopecia, and early childhood–onset type 1 diabetes. Patient's fibroblasts and COS-7 cells transfected with wildtype or mutant VDRs were studied for ligand-binding capacity, transactivation activity using two gene promoters [CYP-24, a classical 1,25(OH)2D3-responsive gene, and relB, a critical NF-κB component for regulation of dendritic cell differentiation], VDR-RXR heterodimers association to CYP 24 VDREs by gel mobility shift assays, and co-activator binding by Glutathione-S-transferase pull-down assays. Results: Two novel compound heterozygous mutations (L263R and R391S) were identified in the VDR ligand-binding domain in this child. Both mutations significantly impaired VDR ligand-binding capacity but had dissociated effects on CYP-24 and RelB promoter responses to vitamin D. CYP 24 response binding to SRC-1 and RXR-heterodimer binding to CYP24 VDREs were abolished in L263R mutants but normal or partially altered in R391S mutants. In the opposite, RelB responses to vitamin D were close to normal in L263R mutants but abolished in R391S mutants. Conclusions: We report the first clinical association between HVDRR, total alopecia, and early childhood–onset type 1 diabetes. Mutations in the VDR ligand-binding domain may hamper the 1,25(OH)2D3–mediated relB responses, an effect that depends on the site of the VDR mutation and cannot be anticipated from VDR ligand-binding ability or CYP-24 response. Based on these results, we propose to survey the immune function in patients with HVDRR, including those with moderate features of rickets.

In this study, two related young children, brother and sister, exhibited severe vitamin D-resistant rickets without alopecia. Sequence analysis of the total vitamin D receptor (VDR) cDNA from skin fibroblasts revealed a substitution of the unique tryptophan of the VDR by arginine at amino acid 286 (W286R). Cultured skin fibroblasts of the two patients expressed normal-size VDR protein (immunocytochemistry and Western blotting) and normal length VDR mRNA (Northern blotting). But, these fibroblasts, as well as COS-7 cells transfected with the W286R mutant, failed to bind 3H 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The tryptophan substitution did not affect VDR trafficking toward the nucleus but abolished the 24-hydroxylase gene response to 1,25(OH)2D3, even at 10(-6) M concentrations. In conclusion, this case report of a new family with hereditary vitamin D-resistant rickets (HVDRR) emphasizes the crucial role of the VDR tryptophan for ligand binding and for transactivation of 1,25(OH)2D3 target genes. It clearly shows the clinical significance of this VDR amino acid for calcium homeostasis and bone mineralization. This observation suggests further that the presence of a stable VDR-bound ligand may not be obligatory for normal hair follicle development.

To determine the biological criteria for neonatal vitamin D deficiency, serum 25-hydroxyvitamin D (calcidiol), parathyroid hormone (PTH), calcium, phosphates, and alkaline phosphatase (ALP) activity were measured during the winter-spring period in 80 healthy neonates and their mothers 3-6 d after delivery. A longitudinal 3-mo survey of the serum biology of 52 of these neonates consuming formula was also performed to test the influence of their neonatal vitamin D status on the effects of two oral ergocalciferol supplements (500 and 1000 IU or 12.5 and 25 micrograms/d). At birth, 63.7% of the infants had calcidiol concentrationsor = 30 nmol/L. Most of them had no other biological sign evocative of vitamin D deficiency, but 14 neonates had low calcidiol concentrations and serum PTH concentrations60 ng/L, the upper limit of the adult normal range. They also had a significantly lower mean serum calcium concentration than did neonates with calcidiol concentrations30 nmol/L. On the basis of the association of low calcidiol concentrations (or = 30 nmol/L) and high PTH concentrations (60 ng/L) as criteria for vitamin D deficiency, 24% of the neonates born to unsupplemented mothers were found to be vitamin D-deficient. Neonatal vitamin D status influenced the response of the infants to vitamin D supplements. Neonates with no sign of vitamin D deficiency showed similar changes in their serum calcidiol, calcium, phosphate, and PTH concentrations and ALP activity and no toxic effect (hypercalcemia or highly elevated calcidiol concentration) was observed whatever their vitamin D intake. In contrast, neonates with subclinical vitamin D deficiency had normalized serum PTH within 1 mo only when they were given 1000 IU ergocalciferol (25 micrograms)/d in addition to their formula.

Resume Le dosage de la 25-hydroxyvitamine D (25(OH)D), metabolite hepatique de reserve de la vitamine D, est important pour le depistage et le traitement d'eventuelles carences ou surcharges en vitamine D. Jusqu'a present, la plupart des differentes methodes de dosage utilisees necessitent toutes au moins 500 μl de serum, ce qui implique le plus souvent un macroprelevement veineux posant des problemes pour leur application en neonatalogie et pediatrie clinique. La nouveaute de notre technique reside dans le fait que l'etape d'extraction s'effectue a partir d'une quantite tres faible de serum ou plasma, 50 μl, ne necessitant pas obligatoirement de ponction veineuse et pouvant etre realisee a partir d'un prelevement capillaire, ce qui permet son utilisation en routine dans la pratique pediatrique et neonatalogique.

To compare extra-renal 1,25(OH)2D3 production in different types of granulomatous disease, and to identify the cell types responsible, we have evaluated the conversion of 25(OH)D3 in 1,25(OH)2D3 by uncultured cells recovered by bronchoalveolar lavage and blood mononuclear cells from normocalcemic patients with sarcoidosis and tuberculosis. 1,25(OH)2D3 was produced both by lavage cells (12/12 tuberculosis patients, 2/6 sarcoidosis patients) and blood mononuclear cells (3/5 tuberculosis patients, 0/3 sarcoidosis patients) from patients but not controls, but significantly greater amounts were produced by lavage cells from tuberculosis patients than those of sarcoidosis patients (P less than 0.001). 1,25(OH)2D3 production by lavage cells from tuberculosis patients correlated with the number of CD8+ T lymphocytes present but not other cell types. T lymphocytes appeared to be an important source of 1,25(OH)2D3 production, since purified T lymphocytes from all patients with tuberculosis produced 1,25(OH)2D3, and 1,25(OH)2D3 production by these cells correlated closely with that produced by unseparated lavage cells. Because 1,25(OH)2D3 can improve the capacity of macrophages to kill mycobacteria, our results support the conclusion that macrophage-lymphocyte interactions, mediated at least in part by 1,25(OH)2D3, may be an important component of a successful antituberculous immune response.

This study examines the influence of chronic retroviral infection of mice with a LPBM5 virus mixture on the paracrine system involving immune cells and 1,25-(OH)2D3 in the spleen. Plasma ionized calcium, 25-(OH)D and 1,25-(OH)2D of infected mice were unchanged. In contrast, the specific binding of 1,25-(OH)2D3 to spleen cytosol and the number of monocyte/macrophages expressing 1,25-(OH)2D3 receptors (VDR) were markedly increased. The retroviral infection also influenced the local production of 1,25-(OH)2D3 in the spleen. It did not alter this production in monocyte/macrophages but increased that in isolated T cells. Isolated B cells in control mice did not produce 1,25-(OH)2D3, but they increased the ability of isolated T cells to produce this metabolite during coculture incubations. Infection altered this cell interaction as 1,25-(OH)2D3 production in infected T cells decreased when these cells were cocultured with infected B cells. Thus, chronic retroviral infection alters both the local vitamin D metabolism and VDR expression by immune cells in mice. These findings suggest close local interactions between 1,25-(OH)2D3 and immune system activation during retroviral infection.

The p17 matrix protein of the human immunodeficiency virus type 1 (HIV-1) plays a crucial role in AIDS pathogenesis. It orchestrates viral assembly and directs the preintegration complex to the nucleus of infected cells. Recently, the three-dimensional structure of p17 was shown to resemble that of interferon-gamma (IFN-gamma), suggesting that both proteins might share analogous functions. We demonstrate that in monocytes, p17 shares with IFN-gamma the ability to induce 1alpha-hydroxylase activity and to activate fructose 1,6-bisphosphatase gene expression in the presence of 25-hydroxyvitamin D3. However, p17 does not bind to the IFN-gamma cell membrane receptor and fails to increase expression of IFN-gamma-induced proteins, such as tryptophanyl-tRNA synthetase, Fc gammaRI, and HLA DR or B7/BB1 antigens. Altogether, our results raise the possibility that the structural resemblance between p17 and IFN-gamma causes the selective activation of a common pathway resulting in the production of 1,25-dihydroxyvitamin D3. We also found that unlike IFN-gamma, p17 increases the intracellular ATP content. Since transport of the HIV-1 preintegration complex through the nuclear membrane is an ATP-dependent process, our observation suggests that p17 plays a double role in this active transport, not only by acting as a chaperone molecule but also by recruiting the necessary energy for this process.

We have previously reported that in contrast to what has been described in adult lung, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has specific binding sites in rat fetal lung at the end of gestation, and it stimulates in vitro the phospholipid biosynthesis and surfactant release from fetal rat type II pneumocytes. In the present study an immunohistochemical technique using a rat monoclonal antibody (9A7 gamma) and binding studies were carried out on fresh lung tissues from fetal and newborn rats during the perinatal period to identify the cell(s) directly responsive to 1,25-(OH)2D3 in fetal lung and to look for a down-regulation of the 1,25-(OH)2D3 receptors in the perinatal period. We also searched for a regulation of 1,25-(OH)2D3 binding to fetal lung by 1,25-(OH)2D3 itself and by factors known to affect lung maturation or be involved in parturition. Our results suggest that 1) fetal type II pneumocytes are target cells for 1,25-(OH)2D3; 2) a physiological down-regulation of the 1,25-(OH)2D3 receptors in rat lung occurs in the perinatal period, starting a few hours before birth and lasting at least up to the fifth day of life; and 3) the capacity of rat fetal lung to bind 1,25-(OH)2D3 can be modulated in vitro by different hormones; a small inhibitory effect is observed with oxytocin (100 microU/ml), while PRL (10(-8) M), T4 (10(-6)-10(-10) M), 1,25-(OH)2D3 (10(-9)-10(-10) M), and, to a lesser extent, dexamethasone (10(-7) M) induce a 2- to 4-fold increase in the number of 1,25-(OH)2D3 receptors without altering the binding affinity of receptor for 1,25-(OH)2D3.

The in vitro effects of vitamin D3 metabolites, parathyroid extract (PTE), purified parathyroid hormone (bPTH), vitamin A, and heparin on acid and alkaline phosphatases in rat or mouse calvaria in culture were investigated. Results show that: (a) when compared to values found in half calvaria incubated for 24 h in control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with PTE (L USP/ml), bPTH (4 x 10(-8)M), heparin (5 USP/ml), vitamin A (23 USP/ml), 25-(OH)D3 (2.5 x 10(-11) to 2.5 x 10(-8)M), 24,25-(OH)2D3, and 1,25-(OH)2D3 (2.5 x 10(-12) to 2.5 x 10(-7M); (b) the presence of 24,25-(OH)2D3 at low concentrations in the incubation medium decreases significantly the PTE, bPTH, vitamin A, or heparin induced stimulation of the phosphatase activities. This interaction is also observed when measuring beta glucuronidase and glucose-6-phosphatase activities and 45Ca release from previously labeled mouse calvaria; (c) a similar activity could not be found with 1,25-(OH)2D3 suggesting that 24,25-(OH)2D3 may have a specific role in bone metabolism.

Summary: The present method for the measurement of 24,25-dihydroxyvitamin D (24,25–(OH)2D) in serum associates the highly sensitive competitive protein binding assay of Preece et al. and a double-step chromatography of the serum lipid extract (Sephadex LH 20 column and high pressure liquid chromatograph). With this assay, values observed in 16 normal children, 10 adolescents and 5 neonates, were not significantly different from those found in 14 normal adults (25–(OH)D: 20 ± 7.4 ng/ml, 24,25–(OH)2D: 1.4 ± 0.8 ng/ml). No correlation was found between 25–(OH)D and 24,25–(OH)2D serum concentrations. Measurements of 2S-(OH)D and 24,25–(OH)2D serum concentrations in some pathologic states demonstrated the existence of a 25–(OH)D-24-hydroxylase in children with three types of vitamin D resistant rickets (hereditary hypophosphatemia, pseudodeficiency rickets, and vitamin D resistance associated with Recklinghausen's neurofibromatosis). Finally, results observed during vitamin D administration suggest a regulation of the 24,25–(OH)2D concentration in human serum: 1) 24,25–(OH)2D serum concentration increased after vitamin D administration, yet it was not found higher in a 3-wk-old child with vitamin D2 intoxication (25–(OH)D: 900 ng/ml, 24,25–(OH)2D: 6 ng/ml) than in three children with vitamin D deficiency rickets in the 2nd wk after administration of vitamin D2 and calcium (25–(OH)D: 25.5 ± 9.2 ng/ml, 24,25–(OH)2D: 13.4 ± 3.7 ng/ml; 2) In a child with pseudodeficiency rickets, 24,25–(OH)2D concentrations were found elevated before treatment. They decreased to normal values during treatment with la-(OH) cholecalciferol, whereas 25–(OH)D concentrations were not significantly different before and after treatment.

This report concerns a 13 year old girl with the clinical and radiological features of mild idiopathic juvenile osteoporosis. In this patient, no alteration was detected in serum calcium (total + ionized) and phosphorus concentration, serum alkaline phosphatase activity, nor in urinary calcium and phosphorus excretions. Plasma concentrations of cortisol were normal during daytime and sleep. Circulating immunoreactive parathyroid hormone was normal or low. The serum 25-(OH)D and 24,25-(OH)2D concentrations were below the normal range, and the 1,25-(OH)2D concentrations were above the normal range (720 pmol/l) at the beginning of the investigation. All vitamin D metabolites concentrations returned to normal values at the time of radiological recovery and after calcium and 25-(OH)D3 supplementation. A possible relationship between alterations of bone and of circulating vitamin D metabolites is discussed.

The hypotheses that bone is a target organ for somatostatin and that vitamin D3 metabolites interact with this peptide on bone were tested in vitro, using calvaria from newborn rats with the following results. 1) [125I]iV-Tyr1 -somatostatin bound specifically to intact calvaria tissue and cells (0.1–7.5% at the 30th–50th min of incubation) and to the crude cytosol fraction (23.6 ± 4.8% of the total radioactivity incubated with cytosol). Three binding sites with different affinities could be detected; the Kd of one of them was 1.17 ± 0.17 × 10-10 M, with a binding site capacity of 0.34 ± 0.05 pmol/mg protein. 2) Low doses of somatostatin (6 × 10-9 to 3 × 10-7 M) affected the cAMP content of calvaria incubated in a protein-free medium for 5–60 min. The somatostatin-induced change was calcium dependent; there was an increase in cAMP with 0 or 1 mM calcium and a decrease with 3 mM calcium. 3) Somatostatin and vitamin D3 metabolites influenced the effects of one another on cAMP content and phosphatase activiti...

Thirteen children, 6 females, 7 males, aged 2 to 13 years were studied. At the time of study they were on continuous ambulatory peritoneal dialysis (CAPD) for 1 to 22 months. 25-(OH)D loss in daily dialysate fluids represented 2 to 22 micrograms/day. A significant correlation was found between 25-(OH)D plasma concentration and 25-(OH)D dialysate concentration. 25-(OH)D clearance was correlated to 25-(OH)D binding protein clearance (p less than 0.001). These findings of important 25-(OH)D losses in the dialysate fluid of children on CAPD demonstrate the necessity of carefully adapted vitamin D intakes with such a treatment.

Circulating vitamin D metabolite concentrations, i.e. 25-(OH)D, 24,25-(OH)2D, 1,25-(OH)2D have been assayed in 14 hypercalcemic children. Results are as follows: a) Children with vitamin D intoxication (n = 2) had elevated serum 25-(OH)D and 24,25-(OH)2D concentrations but their 1,25-(OH)2D concentrations were similar to those found in normocalcemic children (10-110 pg/ml); b) Children with familial idiopathic hypercalcemia and hypocalciuria (n = 5), children with hypercalcemia and either Bartter's syndrome (n = 1), hemangiomatosis (n = 1), osteopetrosis after medullary graft (n = 1), also had 1,25-(OH)2D concentrations in the normal range; c) In contrast, 1,25-(OH)2D were elevated (160-470 pg/ml) in the four children with severe idiopathic hypercalcemia and elfin facies.

Results from in vitro works suggest that 1,25- and 24,25-dihydroxyvitamin D3 (1,25-(OH)2D3 and 24,25-(OH)2D3) act on bone via different mechanisms. The present investigation was performed to study the effect of these two metabolites and of their precursor 25-hyxdroxyvitamin D3 (25-(OH)D3) on bone cAMP content in vitro. Rats' paired half calvaria were incubated under sterile conditions with one vitamin D3 derivative (10−13 to 10−9 M) or with ethanol (0.005 ml for 15 min to 24 h in 1 ml medium containing 0, 0.2, 1, 2, or 3 mM calcium. In some experiments: (a) cycloheximide (10−5M) was added simultaneously with the vitamin D3 metabolites; (b) 1–84 bPTH (5 × 10−8 M) was added for 5 or 15 min at the end of the 24 h incubation. Calvaria were immersed in 1 ml TCA 5% 4°C and homogenized. The cAMP was extracted with diethylether and measured by a competitive protein binding assay. Results bring further evidence for a particular effect of low doses of 24,25-(OH)2D3 (10−9 to 10−12M) and of 25-(OH)D3 (10−9 to 10−11M) on bone, different from that of 1,25-(OH)2D3: cAMP content was higher in 24,25-(OH)2D3- or 25-(OH)D3-treated and lower in 1,25-(OH)2D3-treated calvaria than in ethanol-treated ones with 1 mM calcium. The 1,25-(OH)2D3 effect persisted in calcium-free medium whereas 25-(OH)D3 and 24,25-(OH)2D3 effects could not be observed with 0 mM nor with 3 mM calcium. The required duration of the preincubation (over 1 h) as well as the inhibitory action of cycloheximide may suggest an involvement of protein synthesis in the vitamin D3 metabolites effects. Neither 1,25-(OH)2D3 nor 24,25-(OH)2D3 affected the PTH-induced increase in bone cAMP content.

The in vitro formation of 24, 25-(OH) 2D3 during incubation of rat calvarium with 25-(OH) D3 could be demonstrated. The in vitro synthesis of 24, 25-(OH) 2D3 has been localized in the mitochondrial fraction of the calvarium tissue. It could be detected also in calvarium cells in culture, but results indicate that the ability to synthetize 24, 25-(OH) 2D3 may be specific of some cell types only. The in vitro formation of 24, 25-(OH) 2D3 was observed with calvarium from rat neonates as well as from younger fetal rats, yet, in this latter period the in vitro formation of 24, 25-(OH) 2D3 was not found at all days of pregnancy tested in our experimental conditions (17th to 21st day).

Two unrelated patients, aged 22 months and 31 months, with alopecia and rickets resistant to 1,25-dihydroxyvitamin D (1,25-(OH)2D] (vitamin D-dependency type II) presented with similar biochemical and radiologic features. They were treated with large doses of vitamin D3 derivatives [25-hydroxyvitamin D3 (25-(OH)D3), 1,25-(OH)2D3, and 1 alpha-hydroxyvitamin D3] for 28 months and 6 yr, respectively. In both patients, serum 1,25-(OH)2D levels remained high (approximately 10- to 100-fold normal) during the different therapeutic regimens. Circulating 1,25-(OH)2D and 24,25-dihydroxyvitamin D levels at various stages of the disease suggested in these children disturbances in the regulation of 25-hydroxyvitamin D (25(OH)D) 1 alpha- and 24-hydroxylase systems. In one child, all therapeutic trials were unsuccessful. Studies of her cultured skin fibroblasts showed low capacity (10% normal) for saturable (presumably receptor mediated) nuclear uptake of tritiated 1,25-(OH)2D3; the uptake process of nucleus associated 1,25-(OH)2D3 was normal in apparent affinity for 1,25-(OH)2D3 and in sedimentation velocity of nucleus-associated hormone. In the second child, correction of biochemical abnormalities, healing of rickets, and catch-up growth were obtained during similar therapeutic trials up to the age of 6 yr when a relapse occurred. This relapse has persisted for 2 yr in spite of similar or higher circulating concentrations of 25-(OH)D and 1,25-(OH)2D than those obtained previously when she was responsive to therapy. In her cultured skin fibroblasts, saturable high affinity nuclear uptake of 1,25(OH)2D was unmeasurable.1) distinct patterns of clinical response can occur in patients with the syndrome of vitamin D-dependency type II, and can be associated with differing abnormalities in interaction of 1,25-(OH)2D3 with cultured skin fibroblasts; 2) aggravation of the resistance to 1,25-(OH)2D3 may occur during long term therapy in some patients.

The effect of thyroparathyroidectomy (TPTX) on the plasma concentrations of the vitamin D metabolites (25-(OH)D, 24,25-(OH)2D and 1,25-(OH)2D) has been studied in pregnant rats and their fetuses during the last quarter of gestation. Maternal and fetal vitamin D metabolites were not significantly affected by TPTX. A significant increase in plasma 1,25-(OH)2D concentrations was observed in both TPTX and control mothers and fetuses from days 19 to 21. Fetal and maternal plasma 25-(OH)D were positively correlated in both control and TPTX groups. Such a correlation was also found for 24,25-(OH)2D in the two groups. In contrast, a positive correlation between maternal and fetal plasma concentrations of 1,25-(OH)2D was found in TPTX but not in control rats. These data suggest that major alterations in calcium metabolism, such as that produced by maternal TPTX, are insufficient to affect the changes in maternal and fetal plasma 1,25-(OH)2D during late pregnancy significantly. They also suggest that parathyroid hormone, thyroxine, and/or calcitonin may control a possible placental transfer of 1,25-(OH)2D in the rat. J. Endocr. (1988) 116, 381–385

This study of the three main vitamin D metabolites namely 25-(OH) D, 24, 25-(OH) 2D and 1, 25-(OH) 2D includes (1) a summary of the usual assay techniques, (2) a discussion about the concentrations and the role of these metabolites during the neonatal and childhood periods, (3) a report of the results obtained during the past seven years in our laboratory with comments on the interest and limits of these assays for the diagnosis of different types of rickets.