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Abstract Background Vitamin D deficiency (VDD) is highly prevalent in the pediatric intensive care unit (ICU) and associated with worse clinical course. Trials in adult ICU demonstrate rapid restoration of vitamin D status using an enteral loading dose is safe and may improve outcomes. There have been no published trials of rapid normalization of VDD in the pediatric ICU. Methods We conducted a multicenter placebo-controlled phase II pilot feasibility randomized clinical trial from 2016 to 2017. We randomized 67 critically ill children with VDD from ICUs in Canada, Chile and Austria using a 2:1 randomization ratio to receive a loading dose of enteral cholecalciferol (10,000 IU/kg, maximum of 400,000 IU) or placebo. Participants, care givers, and outcomes assessors were blinded. The primary objective was to determine whether the loading dose normalized vitamin D status (25(OH)D > 75 nmol/L). Secondary objectives were to evaluate for adverse events and assess the feasibility of a phase III trial. Results Of 67 randomized participants, one was withdrawn and seven received more than one dose of cholecalciferol before the protocol was amended to a single loading dose, leaving 59 participants in the primary analyses (40 treatment, 19 placebo). Thirty-one/38 (81.6%) participants in the treatment arm achieved a plasma 25(OH)D concentration > 75 nmol/L versus 1/18 (5.6%) the placebo arm. The mean 25(OH)D concentration in the treatment arm was 125.9 nmol/L (SD 63.4). There was no evidence of vitamin D toxicity and no major drug or safety protocol violations. The accrual rate was 3.4 patients/month, supporting feasibility of a larger trial. A day 7 blood sample was collected for 84% of patients. A survey administered to 40 participating families showed that health-related quality of life (HRQL) was the most important outcome for families for the main trial (30, 75%). Conclusions A single 10,000 IU/kg dose can rapidly and safely normalize plasma 25(OH)D concentrations in critically ill children with VDD, but with significant variability in 25(OH)D concentrations. We established that a phase III multicentre trial is feasible. Using an outcome collected after hospital discharge (HRQL) will require strategies to minimize loss-to-follow-up. Trial Registration. Clinicaltrials.gov NCT02452762 Registered 25/05/2015.

Abstract Chronic kidney disease (CKD) results in calcitriol deficiency and altered vitamin D metabolism. The objective of this study was to assess the 24-hydroxylation-mediated metabolism of 25(OH)D3 and 1,25(OH)2D3 in a cross-sectional analysis of participants with a range of kidney function assessed by precise measured GFR (mGFR) (N = 143) and in rats with the induction and progression of experimental kidney disease. Vitamin D metabolites were assessed with LC–MS/MS. Circulating measures of 24-hydroxylation of 25(OH)D3 (24,25(OH)2D3:25(OH)D3) precisely decreased according to mGFR in humans and progressively in rats with developing CKD. In contrast, the 1,24,25(OH)3D3: 1,25(OH)2D3 vitamin D metabolite ratio increased in humans as the mGFR decreased and in rats with the induction and progression of CKD. Human participants taking cholecalciferol had higher circulating 1,24,25(OH)3D3, despite no increase of 1,25(OH)2D3. This first report of circulating 1,24,25(OH)3D3 in the setting of CKD provides novel insight into the uniquely altered vitamin D metabolism in this setting. A better understanding of the uniquely dysfunctional catabolic vitamin D profile in CKD may guide more effective treatment strategies. The potential that 24-hydroxylated products have biological activity of is an important area of future research.

Vitamin D has many physiological functions including upregulation of intestinal calcium and phosphate absorption, mobilization of bone resorption, renal reabsorption of calcium as well as actions on a variety of pleiotropic functions. It is believed that many of the hormonal effects of vitamin D involve a 1,25-dihydroxyvitamin D3-vitamin D receptor-mediated transcriptional mechanism involving binding to the cellular chromatin and regulating hundreds of genes in many tissues. This comprehensive historical review provides a unique perspective of the many steps of the discovery of vitamin D and its deficiency disease, rickets, stretching from 1650 until the present. The overview is divided into four distinct historical phases which cover the major developments in the field and in the process highlighting the: (a) first recognition of rickets or vitamin D deficiency; (b) discovery of the nutritional factor, vitamin D and its chemical structure; (c) elucidation of vitamin D metabolites including the hormonal form, 1,25-dihydroxyvitamin D3; (d) delineation of the vitamin D cellular machinery, functions and vitamin D-related diseases which focused on understanding the mechanism of action of vitamin D in its many target cells.

Abstract Background Vitamin D status of pregnant women is associated with body composition of the offspring. The objective of this study was to assess whether the association between maternal vitamin D status and neonatal adiposity is modified by maternal adiposity preconception. Methods Healthy mothers and their term appropriate weight for gestational age (AGA) infants (n = 142; 59% male, Greater Montreal, March 2016-2019) were studied at birth and 1 month postpartum (2-6 weeks). Newborn (24-36 h) serum was collected to measure total 25-hydroxyvitamin D [25(OH)D] (immunoassay); maternal pre-pregnancy BMI was obtained from the medical record. Anthropometry, body composition (dual-energy X-ray absorptiometry) and serum 25(OH)D were measured at 2-6 weeks postpartum in mothers and infants. Mothers were grouped into 4 categories based on their vitamin D status (sufficient 25(OH)D ≥ 50 nmol/L vs. at risk of being insufficient

Abstract Background The vast majority of children undergoing cardiac surgery have low vitamin D levels post-operative, which may contribute to greater illness severity and worse clinical outcomes. Prior to the initiation of a large phase III clinical trial focused on clinical outcomes, studies are required to evaluate the feasibility of the study protocol, including whether the proposed dosing regimen can safely prevent post-operative vitamin D deficiency in this high-risk population. Methods We conducted a two-arm, double-blind dose evaluation randomized controlled trial in children requiring cardiopulmonary bypass for congenital heart disease. Pre-operatively, participants were randomized to receive cholecalciferol representing usual care (< 1 year = 400 IU/day, > 1 year = 600 IU/day) or a higher dose approximating the Institute of Medicine tolerable upper intake level (< 1 year = 1600 IU/day, > 1 year = 2400 IU/day). The feasibility outcomes were post-operative vitamin D status (primary), vitamin D-related adverse events, accrual rate, study withdrawal rate, blinding, and protocol non-adherence. Results Forty-six children were randomized, and five withdrew prior to surgery, leaving 41 children (21 high dose, 20 usual care) in the final analysis. The high dose group had higher 25-hydroxyvitamin D concentrations both intraoperatively (mean difference + 25.9 nmol/L; 95% CI 8.3–43.5) and post-operatively (mean difference + 17.2 nmol/L; 95% CI 5.5–29.0). Fewer participants receiving high-dose supplementation had post-operative serum 25-hydroxyvitamin D concentrations under 50 nmol/L, compared with usual care (RR 0.31, 95% CI 0.11–0.87). Post-operative vitamin D status was associated with the treatment arm and the number of doses received. There were no cases of hypercalcemia, and no significant adverse events related to vitamin D. While only 75% of the target sample size was recruited (limited funding), the consent rate (83%), accrual rate (1.5 per site month), number of withdrawals (11%), and ability to maintain blinding support feasibility of a larger trial. Conclusions Pre-operative daily high-dose supplementation improved vitamin D status pre-operatively and at time of pediatric ICU admission. The protocol for a more definitive trial should limit enrollment of children with at least 30 days between randomization and surgery to allow adequate duration of supplementation or consider a loading dose. Trial registration ClinicalTrials.gov, NCT01838447 . Registered on April 24, 2013

Background/Objective: Cyp24a1-null mice deficient in 24,25(OH)2D3 display impaired callus formation during the endochondral phase of bone fracture repair. The 24,25(OH)2D3 metabolite acted by binding to the TLC domain containing 3B isoform 2 (TLCD3B2, previously named FAM57B2) effector protein, which then synthesizes lactosylceramide (LacCer). Treatment with 24,25(OH)2D3 or LacCer restored callus size and mechanical properties in Cyp24a1-null mice. Methods: To assess the safety of these molecules and test their efficacy for bone healing in wild-type, non-genetically modified mice, we treated 12-week-old, osteotomized C57BL/6 female mice with each compound for up to 21 days post-osteotomy. Control cohorts were injected with vehicle. Results: Neither compound was found to exhibit any nephro- nor hepato-toxicity. Calcemia remained stable throughout the experiment and was unaffected by either treatment. Supplementation with 24,25(OH)2D3 increased circulating levels of this metabolite about 8-fold, decreased 1,25(OH)2D3 levels, and significantly increased circulating 1,24,25(OH)3D3 levels, suggesting 1?-hydroxylation of 24,25(OH)2D3. TLCD3B2 was found to be expressed in fracture callus at the surface of unmineralized or pre-mineralized cartilage on day 10 and day 12 post-osteotomy and to progressively recede to become undetectable by day 18. Treatment with 24,25(OH)2D3 or LacCer reduced the number of TLCD3B2-positive cells. Both treatments also significantly increased stiffness and elastic modulus of the healing bone callus. Conclusion: Exogenous administration of 24,25(OH)2D3 or LacCer improved the biomechanical properties of repaired bones in wild-type animals without affecting circulating calcium levels or other blood parameters, demonstrating preclinical safety and efficacy. Translational potential: Our data suggest the use of 24R,25-dihydroxyvitamin D3 or lactosylceramide for ameliorating fracture healing in clinical practice.

Abstract The assay of vitamin D that began in the 1970s with the quantification of one or two metabolites, 25‐OH‐D or 1,25‐(OH)2D, continues to evolve with the emergence of liquid chromatography tandem mass spectrometry (LC‐MS/MS) as the technique of choice. This highly accurate, specific, and sensitive technique has been adopted by many fields of endocrinology for the measurement of multiple other components of the metabolome, and its advantage is that it not only makes it feasible to assay 25‐OH‐D or 1,25‐(OH)2D but also other circulating vitamin D metabolites in the vitamin D metabolome. In the process, this broadens the spectrum of vitamin D metabolites, which the clinician can use to evaluate the many complex genetic and acquired diseases of calcium and phosphate homeostasis involving vitamin D. Several examples are provided in this review that additional metabolites (eg, 24,25‐(OH)2D3, 25‐OH‐D3‐26,23‐lactone, and 1,24,25‐(OH)3D3) or their ratios with the main forms offer valuable additional diagnostic information. This approach illustrates that biomarkers of disease can also include metabolites devoid of biological activity. Herein, a case is presented that the decision to switch to a LC‐MS/MS technology permits the measurement of a larger number of vitamin D metabolites simultaneously and does not need to lead to a dramatic increase in cost or complexity because the technique uses a highly versatile tandem mass spectrometer with plenty of reserve analytical capacity. Physicians are encouraged to consider adding this rapidly evolving technique aimed at evaluating the wider vitamin D metabolome toward streamlining their approach to calcium‐ and phosphate‐related disease states. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Mutations in CYP24A1 (vitamin D 24-hydroxylase) and SLC34A1 (renal phosphate transporter NPT2a) cause autosomal recessive Infantile Hypercalcemia type 1 and 2, illustrating links between vitamin D and phosphate metabolism. Patients may present with hypercalciuria and alternate between chronic phases with normal serum calcium but inappropriately high 1,25-(OH)2D and appropriately low PTH, and acute phases with hypercalcemia with suppressed PTH. Mutations in SLC34A3 and SLC9A3R1 have been associated with phosphate wasting without hypercalcemia. The aims of this study were to evaluate the frequency of mutations in these genes in patients with a medical history suggestive of CYP24A1 mutation to search for a specific pattern. Using next generation sequencing, we screened for mutations in 185 patients with PTH levels < 20 pg/mL, hypercalcemia and/or hypercalciuria, and relatives. Twenty-eight (15%) patients harbored biallelic mutations in CYP24A1 (25) and SLC34A3 (3), mostly associated with renal disease (lithiasis, nephrocalcinosis) (86%). Hypophosphatemia was found in 7 patients with biallelic mutations in CYP24A1 and a normal phosphatemia was reported in 2 patients with biallelic mutations in SLC34A3. Rare variations in SLC34A1 and SLC34A3 were mostly of uncertain significance. Fifteen patients (8%) carried only one heterozygous mutation. Heterozygous relatives carrying SLC34A1 or SLC34A3 variation may present with biochemical changes in mineral metabolism. Two patients’ genotype may suggest digenism (heterozygous variations in different genes). No variation was found in SLC9A3R1. As no specific pattern can be found, patients with medical history suggestive of CYP24A1 mutation should benefit from SLC34A1 and SLC34A3 analysis.

Abstract Vitamin D receptor agonist (VDRA) therapy for PTH suppression is a mainstay for patients with severe CKD. Calcitriol (1,25‐(OH)2D3) is a former first‐line VDRA in CKD treatment. However, a consequence of its use in CKD is accelerated vascular calcification (VC). An experimental CKD model was used to determine whether altering the calcitriol delivery profile to obtain different PTH suppression levels could improve vascular health outcomes. High adenine diet (0.25%) was used to generate experimental CKD in rats. CKD rats were treated using different calcitriol dosing strategies: (a) 20 ng/kg SD (n = 8), (b) 80 ng/kg SD (n = 8), (c) 5 ng/kg QID (n = 9), or (d) 20 ng/kg QID (n = 9). Multiple targets of calcitriol were assessed which include arterial calcium and phosphate as well as circulating calcium, phosphate, PTH, FGF‐23, VWF, and vitamin D metabolome. PTH suppression occurred dose‐dependently after 1‐week calcitriol treatment (P 10× in all calcitriol‐treated rats (P

ABSTRACT: Objective: Hypercalcemia can be caused by a variety of pathologies and factors. Vitamin D plays a central role in calcium homeostasis, where tight control of its metabolism is necessary. Inadequate 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) enzyme activity leads to failure of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D catabolism, resulting in hypercalcemia.Methods: This study consists of a case report and brief review of the literature. A young woman with persistent hypercalcemia of unknown origin was investigated by measuring phosphate, parathyroid hormone, and vitamin D metabolites, as well as by evaluating kidney and bone involvement.Results: The patient was asymptomatic and had a cystitis-like syndrome during childhood. The patient exhibited hypercalcemia and hypercalciuria with suppressed PTH. Liquid chromatography with tandem mass spectrometry analysis of vitamin D metabolites revealed elevated 1,25-dihydroxyvitamin D3 (301 pg/mL) and an elevated ratio of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 of 390. Screening for CYP24A1 gene mutations was performed. Bi-allelic mutations in the coding region of CYP24A1 were identified (Arg396Trp/Leu409S), confirming the diagnosis of hypercalcemia due to CYP24A1 mutation. The patient's family members were also studied.Conclusion: This particular case of hypercalcemia due to a mutation in CYP24A1 emphasizes 2 main concerns: vitamin D-related hypercalcemia should be considered at any age in patients with hypercalcemia, and asymptomatic patients with CYP24A1 mutations are at risk of developing hypercalcemia arising from vitamin D supplementation.Abbreviations: 1,25-(OH)2D3 1,25-dihydroxyvitamin D3 25-OH-D3 25-hydroxyvitamin D3 24,25-(OH)2D3 24,25-dihydroxyvitamin D3 CYP24A1 25-hydroxyvitamin D-24-hydroxylase FGF23 fibroblast growth factor 23 IIH idiopathic infantile hypercalcemia LC-MS/MS liquid chromatography with tandem mass spectrometry PTH parathyroid hormone

Introduction. Loss of function mutations of CYP24A1 gene, which is involved in vitamin D catabolism, cause vitamin D-mediated PTH-independent hypercalcemia. The phenotype varies from life-threatening forms in the infancy to milder forms in the adulthood. Case Presentation. We report a case of a 17-year-old woman with a history of nephrolithiasis, mild PTH-independent hypercalcemia (10,5mg/dL), and high serum 1,25(OH)2D concentrations (107pg/mL). Other causes of hypercalcemia associated with the above biochemical signature were excluded. Family history revealed nephrolithiasis in the sister. Blood testing in first-degree relatives showed serum PTH in the low-normal range and 1,25(OH)2D at the upper normal limit or slightly elevated. The CYP24A1 gene analysis revealed a known homozygous loss-of-function pathogenic variant (c.428_430delAAG, rs777676129, p.Glu143del). The panel of vitamin D metabolites evaluated by liquid chromatography showed the typical profile of CYP24A1 mutations, namely, low 24,25(OH)2D3, elevated 25(OH)D3:24,25(OH)2D3 ratio, and undetectable 1,24,25(OH)3D3. The parents and both the siblings harbored the same variant in heterozygosis. We decided for a watchful waiting approach and the patient remained clinically and biochemically stable over a 24-month followup. Conclusion. CYP24A1 gene mutations should be considered in cases of PTH-independent hypercalcemia, once that more common causes (hypercalcemia of malignancy, granulomatous diseases, and vitamin D intoxication) have been ruled out.

Confusion, apathy, recurrent vomiting, abdominal pain, polyuria, polydipsia, and dehydration are the most often noted clinical symptoms of vitamin D toxicity (VDT; also called vitamin D intoxication or hypervitaminosis D). VDT and its clinical manifestation, severe hypercalcemia, are related to excessive long-term intake of vitamin D, malfunctions of the vitamin D metabolic pathway, or the existence of coincident disease that produces the active vitamin D metabolite locally. Although VDT is rare, the health effects can be serious if it is not promptly identified. Many forms of exogenous (iatrogenic) and endogenous VDT exist. Exogenous VDT is usually caused by the inadvertent or improper intake of extremely high doses of pharmacological preparations of vitamin D and is associated with hypercalcemia. Serum 25-hydroxyvitamin D [25(OH)D] concentrations higher than 150 ng/ml (375 nmol/l) are the hallmark of VDT due to vitamin D overdosing. Endogenous VDT may develop from excessive production of an active vitamin D metabolite – 1,25(OH)2D in granulomatous disorders and in some lymphomas or from the reduced degradation of that metabolite in idiopathic infantile hypercalcemia. Endogenous VDT may also develop from an excessive production of 25(OH)D and 1,25(OH)2D in congenital disorders, such as Williams–Beuren syndrome. Laboratory testing during routine clinical examinations may reveal asymptomatic hypercalcemia caused by the intake of vitamin D even in doses recommended for the general population and considered safe. That phenomenon, called hypersensitivity to vitamin D, reflects dysregulated vitamin D metabolism. Researchers have proposed many processes to explain VDT. Those processes include elevated activity of 1α-hydroxylase or inhibited activity of 24-hydroxylase, both leading to increased concentration of 1,25(OH)D; increased number of vitamin D receptors; and saturation of the capacity of vitamin D binding protein. Increased public awareness of vitamin D–related health benefits might increase the risk of VDT due to self-administration of vitamin D in doses higher then recommended for age and body weight or even higher than the established upper limit intake values. Consequently, the incidence of hypercalcemia due to hypervitaminosis D might increase.

The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field.

Recommendations for vitamin D supplementation for preterm infants span a wide range of doses. Response to vitamin D supplementation and impact on outcomes in preterm infants is not well understood.Evaluate serum 25(OH)D3 concentration changes after 4 weeks in response to two different doses of vitamin D3 supplementation in a population of premature infants and quantify the impact on NICU outcomes.32 infants born at 24-32 weeks gestation were prospectively randomized to receive 400 or 800 IU/day vitamin D3 supplementation. Serum 25(OH)D3 levels were measured every 4 weeks. The Wilcoxon signed rank test was used to compare serum levels of 25(OH)D3 at 4 weeks and each subsequent time point. A p-value of

Two mammalian hCYP26A expression systems have been used to analyze the metabolic products of CYP26A. Through the use of extensive HPLC, UV spectroscopy, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) methodology, we have conclusively demonstrated that the complex mixture of products comprises 4-OH-all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid, and 18-OH-all-trans-retinoic acid, and more polar products, partially identified as dihydroxy and mono-oxo, mono-hydroxy derivatives. These more polar products are presumed to result from multiple hydroxylations on the β-ionone ring. The inter-relationship of initial and polar metabolites was inferred from both gene-dose and time-course experiments.Both initial and secondary metabolic steps after 4-oxo-all-trans-retinoic acid are ketoconazole-sensitive, suggesting that steps in the production of water-soluble metabolites are cytochrome P450-dependent.

Many new analogues of the vitamin-D hormone, 1 alpha,25-dihydroxy-vitamin D(3) [1 alpha,25-(OH)(2)D(3); calcitriol], have emerged that can mimic its various actions in classic calcium transport systems and/or in the regulation of cell proliferation and cell differentiation. Though some of these analogues have accentuated activity in cell differentiation assays in vitro, they lack appreciable "calcemic" activity in vivo, leading to the name "noncalcemic analogues." Several of these analogues are promising candidates for use in treatment of psoriasis and in tumor suppression, one of them, calcipotriol, being already widely approved for the former indication. New generations of calcemic analogues with altered pharmacokinetics are appearing for use in secondary hyperparathyroidism and osteoporosis. We believe that the selective properties of both types of analogues stem from altered receptor binding, blood protein binding, and rate of catabolism.