24-25-dihydroxyvitamin-d-3 and Vitamin-D-Deficiency

Deficiency of 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and excessive fibroblast growth factor (FGF23) are suggested to be associated with increased mortality in patients with chronic kidney disease (CKD). Generally, 24-hydroxylation has been considered the first step in the degradation pathway of 1,25(OH)(2)D and 25(OH)D. 24,25-dihydroxyvitamin D [24,25(OH)(2)D] was believed to be a degradation product, with no important biological effects. However, some data have accumulated showing that 24,25(OH)(2)D has biological effects on its own. Under conditions of eucalcemia, the synthesis of 24,25(OH)(2)D is increased, and the synthesis of 1,25(OH)(2)D is decreased. In patients with CKD, both high parathyroid hormone levels, which decrease the activity of enzyme CYP24A1 (24-hydroxylase), and high FGF23 levels, which increase the activity of enzyme CYP24A1, were often detected. However, information about 24,25(OH)(2)D levels in these patients is very limited. Whether compensatory changes in levels of FGF23 and 24,25(OH)(2)D in CKD patients are protective or harmful remain unknown issues. Therefore, more studies are needed to identify the nature of the interactions between these molecules and to fully elucidate their clinical significance.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Humans; Hyperparathyroidism, Secondary; Kidney Failure, Chronic; Renal Dialysis; Vitamin D Deficiency; Vitamins

New knowledge of the biological and clinical importance of the steroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] and its receptor, the vitamin D receptor (VDR), has resulted in significant contributions to good bone health. However, worldwide reports have highlighted a variety of vitamin D insufficiency and deficiency diseases. Despite many publications and scientific meetings reporting advances in vitamin D science, a disturbing realization is growing that the newer scientific and clinical knowledge is not being translated into better human health. Over the past several decades, the biological sphere of influence of vitamin D(3), as defined by the tissue distribution of the VDR, has broadened at least 9-fold from the target organs required for calcium homeostasis (intestine, bone, kidney, and parathyroid). Now, research has shown that the pluripotent steroid hormone 1alpha,25(OH)(2)D(3) initiates the physiologic responses of >/=36 cell types that possess the VDR. In addition to the kidney's endocrine production of circulating 1alpha,25(OH)(2)D(3,) researchers have found a paracrine production of this steroid hormone in >/=10 extrarenal organs. This article identifies the fundamentals of the vitamin D endocrine system, including its potential for contributions to good health in 5 physiologic arenas in which investigators have clearly documented new biological actions of 1alpha,25(OH)(2)D(3) through the VDR. As a consequence, the nutritional guidelines for vitamin D(3) intake (defined by serum hydroxyvitamin D(3) concentrations) should be reevaluated, taking into account the contributions to good health that all 36 VDR target organs can provide.

Topics: 24,25-Dihydroxyvitamin D 3; Cholecalciferol; Humans; Nutrition Policy; Organ Specificity; Receptors, Calcitriol; Signal Transduction; Tissue Distribution; Vitamin D; Vitamin D Deficiency

Plasma 25(OH)D has emerged as a valuable biomarker for the many varied health-related effects of vitamin D in the clinic mainly because of the recognition of the importance of the enzyme, CYP27B1, or the 25(OH)D-alpha-hydroxylase in the extrarenal, target cell production of calcitriol. This review briefly assesses current methodology for plasma 25(OH)D assay focusing mainly on currrent controversies surrounding the definition of the normal range and performance characteristics of the assay, separate measurement of both 25(OH)D(2) and 25(OH)D(3), and quality assurance tesing of laboratories offering the test. Clinicians have two main types of 25(OH)D assay based on either high-performance liquid chromatography with UV or mass detection or higher throughput kits based on protein (competitive protein binding assay or radioimmunoassay) binding. Based on 30 yr of experience with measuring 25(OH)D levels, it is concluded that, in the hands of appropriately trained experts, both types of assay provide reliable and accurate results, but all laboratories providing 25(OH)D data need frequent external quality assurance service to ensure that this performance is maintained.

Topics: 24,25-Dihydroxyvitamin D 3; Biological Assay; Calcitriol; Humans; Quality Control; Reference Standards; Vitamin D Deficiency

Topics: 24,25-Dihydroxyvitamin D 3; Calcifediol; Calcitriol; Humans; Tissue Distribution; Ultraviolet Rays; Vitamin D; Vitamin D Deficiency

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone Resorption; Calcifediol; Calcitriol; Cholecalciferol; Chronic Kidney Disease-Mineral and Bone Disorder; Dihydroxycholecalciferols; Humans; Intestinal Absorption; Rickets; Vitamin D; Vitamin D Deficiency

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D 2; Animals; Calcitriol; Calcium; Chick Embryo; Dihydroxycholecalciferols; Dogs; Ergocalciferols; Female; Humans; Kidney Diseases; Liver Diseases; Pregnancy; Vitamin D; Vitamin D Deficiency

The physiologic role of free 25-hydroxyvitamin D [25(OH)D] in humans is unclear.. To assess whether rise in total vs free 25(OH)D is associated with change in downstream biomarkers of 25(OH)D entry into target cells in kidney and parathyroid: 24,25-dihyroxyvitamin D [24,25(OH)2D] and PTH, respectively.. 16-week randomized controlled trial.. 60 μg (2400 IU)/d of D3 or 20 μg/d of 25(OH)D3.. Academic medical center.. 35 adults age ≥18 years with 25(OH)D levels < 20 ng/mL.. 24,25(OH)2D, 1,25-dihyroxyvitamin D [1,25(OH)2D] and PTH.. At baseline, participants [D3 and 25(OH)D3 groups combined] were 35.1 ± 10.6 years. Mean total 25(OH)D, free 25(OH)D, 24,25(OH)2D, and PTH were 16.6 ng/mL, 4.6 pg/mL, 1.3 ng/mL, and 37.2 pg/mL, respectively. From 0 to 4 weeks, rise in only free 25(OH)D was associated with a concurrent 24,25(OH)2D increase [P = 0.03, adjusted for change in 1,25(OH)2D and supplementation regimen] and PTH decrease (P = 0.01, adjusted for change in calcium and supplementation regimen). Between 4 and 8 weeks, and again from 8 to 16 weeks, rises in free and total 25(OH)D were associated with 24,25(OH)2D increase; in contrast, rise in neither total nor free 25(OH)D was associated with PTH decrease during these time periods.. Early rise in free 25(OH)D during treatment of vitamin D deficiency was more strongly associated with changes in biomarkers of 25(OH)D entry into target kidney and parathyroid cells, suggesting a physiologic role of free 25(OH)D in humans.

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Biomarkers; Calcifediol; Calcium; Dietary Supplements; Female; Humans; Kidney; Male; Parathyroid Glands; Parathyroid Hormone; Time Factors; Vitamin D; Vitamin D Deficiency; Vitamins

Previous in vitro and in vivo studies indicate that enzymes that synthesize and metabolize vitamin D are magnesium dependent. Recent observational studies found that magnesium intake significantly interacted with vitamin D in relation to vitamin D status and risk of mortality. According to NHANES, 79% of US adults do not meet their Recommended Dietary Allowance of magnesium.. The aim of this study was to test the hypothesis that magnesium supplementation differentially affects vitamin D metabolism dependent on baseline 25-hydroxyvitamin D [25(OH)D] concentration.. The study included 180 participants aged 40-85 y and is a National Cancer Institute independently funded ancillary study, nested within the Personalized Prevention of Colorectal Cancer Trial (PPCCT), which enrolled 250 participants. The PPCCT is a double-blind 2 × 2 factorial randomized controlled trial conducted in the Vanderbilt University Medical Center. Doses for both magnesium and placebo were customized based on baseline dietary intakes. Subjects were randomly assigned to treatments using a permuted-block randomization algorithm. Changes in plasma 25-hydroxyvitamin D3 [25(OH)D3], 25-hydroxyvitamin D2 [25(OH)D2], 1,25-dihydroxyvitamin D3, 1,25-dihydroxyvitamin D2, and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] were measured by liquid chromatography-mass spectrometry.. The relations between magnesium treatment and plasma concentrations of 25(OH)D3, 25(OH)D2, and 24,25(OH)2D3 were significantly different dependent on the baseline concentrations of 25(OH)D, and significant interactions persisted after Bonferroni corrections. Magnesium supplementation increased the 25(OH)D3 concentration when baseline 25(OH)D concentrations were close to 30 ng/mL, but decreased it when baseline 25(OH)D was higher (from ∼30 to 50 ng/mL). Magnesium treatment significantly affected 24,25(OH)2D3 concentration when baseline 25(OH)D concentration was 50 ng/mL but not 30 ng/mL. On the other hand, magnesium treatment increased 25(OH)D2 as baseline 25(OH)D increased.. Our findings suggest that optimal magnesium status may be important for optimizing 25(OH)D status. This trial was registered at clinicaltrials.gov as NCT03265483.

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D 2; Aged; Calcifediol; Calcitriol; Dietary Supplements; Ergocalciferols; Female; Humans; Kidney; Magnesium; Magnesium Deficiency; Male; Middle Aged; Nutritional Status; Placebos; Vitamin D; Vitamin D Deficiency

Vitamin D insufficiency and secondary hyperparathyroidism (SHPT) are associated with increased morbidity and mortality in chronic kidney disease (CKD) and are poorly addressed by current treatments. The present clinical studies evaluated extended-release (ER) calcifediol, a novel vitamin D prohormone repletion therapy designed to gradually correct low serum total 25-hydroxyvitamin D, improve SHPT control and minimize the induction of CYP24A1 and FGF23.. Two identical multicenter, randomized, double-blind, placebo-controlled studies enrolled subjects from 89 US sites. A total of 429 subjects, balanced between studies, with stage 3 or 4 CKD, SHPT and vitamin D insufficiency were randomized 2:1 to receive oral ER calcifediol (30 or 60 µg) or placebo once daily at bedtime for 26 weeks. Most subjects (354 or 83%) completed dosing, and 298 (69%) entered a subsequent open-label extension study wherein ER calcifediol was administered without interruption for another 26 weeks.. ER calcifediol normalized serum total 25-hydroxyvitamin D concentrations (>30 ng/ml) in >95% of per-protocol subjects and reduced plasma intact parathyroid hormone (iPTH) by at least 10% in 72%. The proportion of subjects receiving ER calcifediol who achieved iPTH reductions of ≥30% increased progressively with treatment duration, reaching 22, 40 and 50% at 12, 26 and 52 weeks, respectively. iPTH lowering with ER calcifediol was independent of CKD stage and significantly greater than with placebo. ER calcifediol had inconsequential impact on serum calcium, phosphorus, FGF23 and adverse events.. Oral ER calcifediol is safe and effective in treating SHPT and vitamin D insufficiency in CKD.

Topics: 24,25-Dihydroxyvitamin D 3; Aged; Calcifediol; Calcium; Creatinine; Delayed-Action Preparations; Double-Blind Method; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Glomerular Filtration Rate; Humans; Hyperparathyroidism; Male; Middle Aged; Parathyroid Hormone; Phosphorus; Renal Insufficiency, Chronic; Vitamin D; Vitamin D Deficiency; Vitamins

Interactions between calcium and vitamin D may have implications for the regulation of serum 25-hydroxyvitamin D [25(OH)D] and its catabolism and, consequently, the vitamin D dietary requirement.. We investigated whether different calcium intakes influenced serum 25(OH)D and indexes of vitamin D activation and catabolism during winter and in the context of both adequate and inadequate vitamin D intakes.. A 15-wk winter-based, randomized, placebo-controlled, double-blind vitamin D₃ intervention (20 μg/d) study was carried out in free-living men and women aged ≥50 y (n = 125) who were stratified according to calcium intakes [moderate-low (<700 mg/d) or high (>1000 mg/d) intake]. The serum 25(OH)D concentration was the primary outcome, and serum calcium, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D [1,25(OH)₂D], 24,25-dihydroxyvitamin D [24,25(OH)₂D], the ratio of 24,25(OH)₂D to 25(OH)D, vitamin D-binding protein, and free 25(OH)D were exploratory outcomes.. A repeated-measures ANOVA showed there was no significant (P = 0.2) time × vitamin D treatment × calcium intake grouping interaction effect on the mean serum 25(OH)D concentration over the 15-wk intervention period. Serum 25(OH)D concentrations increased (P ≤ 0.005) and decreased (P ≤ 0.002) in vitamin D₃ and placebo groups, respectively, and were of similar magnitudes in subjects with calcium intakes <700 mg/d (and even <550 mg/d) compared with >1000 mg/d. The response of serum PTH, 1,25(OH)₂D, 24,25(OH)₂D, the ratio of 24,25(OH)₂D to 25(OH)D, and free 25(OH)D significantly differed in vitamin D₃ and placebo groups but not by calcium intake grouping.. We found no evidence of a vitamin D sparing effect of high calcium intake, which has been referred to by some authors as "vitamin D economy." Thus, recent dietary vitamin D requirement estimates will cover the vitamin D needs of even those individuals who have inadequate calcium intakes.

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D 2; Aged; Aged, 80 and over; Aging; Calcifediol; Calcitriol; Calcium; Calcium, Dietary; Cholecalciferol; Dietary Supplements; Double-Blind Method; Ergocalciferols; Female; Humans; Ireland; Male; Middle Aged; Nutritional Requirements; Seasons; Vitamin D Deficiency

Elevated concentrations of fibroblast growth factor 23 (FGF23) are postulated to promote 25-hydroxyvitamin D (25[OH]D) insufficiency in CKD by stimulating 24-hydroxylation of this metabolite, leading to its subsequent degradation; however, prospective human studies testing this relationship are lacking.. An open-label prospective study was conducted from October 2010 through July 2012 to compare the effect of 8 weeks of oral cholecalciferol therapy (50,000 IU twice weekly) on the production of 24,25(OH)2D3 in vitamin D-insufficient patients with CKD (n=15) and controls with normal kidney function (n=15). Vitamin D metabolites were comprehensively profiled at baseline and after treatment, along with FGF23 and other mineral metabolism parameters.. Vitamin D3 and 25(OH)D3 concentrations increased equivalently in the CKD and control groups following cholecalciferol treatment (median D3 change, 8.6 ng/ml [interquartile range, 3.9-25.6 ng/ml] for controls versus 12.6 ng/ml [6.9-41.2 ng/ml] for CKD [P=0.15]; 25(OH)D3 change, 39.2 ng/ml [30.9-47.2 ng/ml] for controls versus 39.9 ng/ml [31.5-44.1 ng/ml] for CKD [P=0.58]). Likewise, the absolute increase in 1α,25(OH)2D3 was similar between CKD participants and controls (change, 111.2 pg/ml [64.3-141.6 pg/ml] for controls versus 101.1 pg/ml [74.2-123.1 pg/ml] for CKD; P=0.38). Baseline and post-treatment 24,25(OH)2D3 concentrations were lower in the CKD group; moreover, the absolute increase in 24,25(OH)2D3 after therapy was markedly smaller in patients with CKD (change, 2.8 ng/ml [2.3-3.5 ng/ml] for controls versus 1.2 ng/ml [0.6-1.9 ng/ml] for patients with CKD; P<0.001). Furthermore, higher baseline FGF23 concentrations were associated with smaller increments in 24,25(OH)2D3 for individuals with CKD; this association was negated after adjustment for eGFR by multivariate analysis.. Patients with CKD exhibit an altered ability to increase serum 24,25(OH)2D3 after cholecalciferol therapy, suggesting decreased 24-hydroxylase activity in CKD. The observed relationship between baseline FGF23 and increments in 24,25(OH)2D3 further refutes the idea that FGF23 directly contributes to 25(OH)D insufficiency in CKD through stimulation of 24-hydroxylase activity.

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Aged; Bone Density Conservation Agents; Calcifediol; Calcitriol; Case-Control Studies; Cholecalciferol; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Glomerular Filtration Rate; Humans; Kidney Failure, Chronic; Male; Middle Aged; Prospective Studies; Vitamin D Deficiency

We previously showed that a single high dose of oral (po) cholecalciferol (D₃) sharply increases serum 25-hydroxyvitamin D [25(OH)D].. We evaluated the long-term bioavailability and metabolism of a single po or intramuscular (im) high dose of ergocalciferol (D₂) or D₃.. This was a prospective intervention study.. The study was conducted in an ambulatory care setting.. Participants were 24 subjects with hypovitaminosis D.. A single dose of 600,000 IU of po or im D₂ or D₃ was administered.. Serum 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)₂D] were measured at baseline and at days 30, 60, 90, and 120 by RIA. Serum 1,25(OH)₂D₂, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-hydroxyvitamin D₂ [24,25(OH)D₂], and 24,25-hydroxyvitamin D₃ [24,25(OH)D₃] were measured by liquid chromatography-tandem mass spectrometry in a subgroup of patients receiving the po formulations.. The areas under the curve of 25(OH)D after D₃ were significantly higher than those after D₂ (P < .0001). Serum 25(OH)D basal difference significantly increased at day 30 with po D₂ and D₃ (P < .01 and P < .0001) and up to day 90 with po D₃ (P < .01). The im formulations produced a slow increased, and values peaked at day 120 relative to the other time points (P < .0001). We found a decrease in 1,25(OH)₂D at day 30 (P < .05) and up to day 120 (P < .001) and an increase in 1,25(OH)₂D₂ at day 30 (P < .01) and up to day 120 (P < .01) after po D₂. Oral D₂ and D₃ produced increases in 24,25(OH)D₂ and 24,25(OH)D₃, respectively, at day 30 (P < .001).. A po dose of 600,000 IU of D₂ or D₃ is initially more effective in increasing serum 25(OH)D than the equivalent im dose and is rapidly metabolized. Our RIA assay for 1,25(OH)₂D may not recognize 1,25(OH)₂D₂.

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D 2; Administration, Oral; Aged; Biological Availability; Biotransformation; Calcifediol; Cholecalciferol; Chromatography, High Pressure Liquid; Ergocalciferols; Female; Humans; Injections, Intramuscular; Male; Middle Aged; Radioimmunoassay; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Vitamin D Deficiency

Vitamin D regulates calcium and immune function. While vitamin D deficiency has been associated with periodontitis, little information exists regarding its effect on wound healing and periodontal surgery outcomes. This longitudinal clinical trial assessed outcomes of periodontal surgery and teriparatide administration in vitamin-D-sufficient and -insufficient individuals. Forty individuals with severe chronic periodontitis received periodontal surgery, daily calcium and vitamin D supplements, and self-administered teriparatide or placebo for 6 wks to correspond with osseous healing time. Serum 25(OH)D was evaluated at baseline, 6 wks, and 6 mos post-surgery. Clinical and radiographic outcomes were evaluated over 1 yr. Placebo patients with baseline vitamin D deficiency [serum 25(OH)D, 16-19 ng/mL] had significantly less clinical attachment loss (CAL) gain (-0.43 mm vs. 0.92 mm, p < 0.01) and probing depth (PPD) reduction (0.43 mm vs. 1.83 mm, p < 0.01) than vitamin-D-sufficient individuals. Vitamin D levels had no significant impact on CAL and PPD improvements in teriparatide patients at 1 yr, but infrabony defect resolution was greater in teriparatide-treated vitamin-D-sufficient vs. -deficient individuals (2.05 mm vs. 0.87 mm, p = 0.03). Vitamin D deficiency at the time of periodontal surgery negatively affects treatment outcomes for up to 1 yr. Analysis of these data suggests that vitamin D status may be critical for post-surgical healing. (ClinicalTrials.gov number, CT00277706).

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Aged; Bone Density Conservation Agents; Chronic Periodontitis; Female; Humans; Linear Models; Longitudinal Studies; Male; Middle Aged; Teriparatide; Treatment Outcome; Vitamin D; Vitamin D Deficiency; Vitamins; Wound Healing

Background Simultaneous measurement of 25(OH)D and 24,25(OH)2D is a new tool for predicting vitamin D deficiency and allows evaluating CYP24A1 lack of function. Interpretation of 24,25(OH)2D should be performed according to 25(OH)D levels and a ratio, called the vitamin D metabolite ratio (VMR) has been proposed for such a purpose. Unfortunately, the VMR can be expressed in different ways and cannot be used if 24,25(OH)2D concentrations are undetectable. Here, we propose evaluating the enzyme activity taking into consideration the probability that a normal population presents undetectable 24,25(OH)2D concentrations according to 25(OH)D levels. We thus retrospectively measured 25(OH)D and 24,25(OH)2D in a population of 1200 young subjects to evaluate the 25(OH)D threshold above which the enzyme was induced. Methods Serum samples from 1200 infants, children, adolescent and young adults were used to simultaneously quantify 25(OH)D and 24,25(OH)2D by LCMS/MS. Results Median (interquartile range [IQR]) levels were 20.6 (14.4-27.2) ng/mL for 25(OH)D. 172 subjects (14.3%) presented 24,25(OH)2D values below the LOQ. When 25(OH)D values were <11 ng/mL, 63.1% of subjects presented undetectable 24,25(OH)2D concentrations. Percentage decreased with increasing 25(OH)D values to become 19.7% for 25(OH)D comprised between 12 and 15 ng/mL, 5.1% for 25(OH)D between 16 and 20 and 0.7% for 25(OH)D >21 ng/mL. Conclusions We suggest using a statistical approach to evaluate CYP24A1 function according to 25(OH)D concentrations. Our results also show that vitamin D deficiency, as defined biochemically, could be around 20 ng/mL in infants, children, adolescent and young adults and that vitamin D deficiency could be evaluated on a more individual basis.

Topics: 24,25-Dihydroxyvitamin D 3; Adolescent; Calcifediol; Child; Child, Preschool; Chromatography, High Pressure Liquid; Female; Humans; Infant; Limit of Detection; Male; Polymorphism, Single Nucleotide; Retrospective Studies; Tandem Mass Spectrometry; Vitamin D Deficiency; Vitamin D3 24-Hydroxylase; Young Adult

While vitamin D deficiency is common in patients with end stage renal disease on dialysis and treatment with Vitamin D. The subjects given Vitamin D. We conclude that the enzymatic activity of CYP24A1 is abnormal in end stage renal patients on dialysis. These trials were registered on clinicaltrials.govNCT00511225 on 8/1/2007; NCT01325610 on 1/17/2011; and NCT01675557 on 8/28/2012.

Topics: 24,25-Dihydroxyvitamin D 3; Aged; Cholecalciferol; Dietary Supplements; Ergocalciferols; Female; Humans; Male; Middle Aged; Renal Dialysis; Renal Insufficiency, Chronic; Vitamin D Deficiency; Vitamins

While disorders of impaired vitamin D activation and action have long been appreciated, the consequences of abnormalities in pathways leading to the inactivation of vitamin D metabolites have only recently been identified. Two recent articles have shed new light on this area of vitamin D biology. The report by Martineau et al., published in the JCI, describes a pathway in which binding of the vitamin D metabolite 24R,25(OH)2D3 to its effector molecule FAM57B2 plays an important role in endochondral ossification during bone repair. This work follows, and adds to, another recent JCI publication by Roizen et al., showing that rapid inactivation of vitamin D metabolites causes vitamin D deficiency, leading to vitamin D-dependent rickets.

Topics: 24,25-Dihydroxyvitamin D 3; Fractures, Bone; Humans; Vitamin D; Vitamin D Deficiency

Most studies examining the relationships between vitamin D and disease or health focus on the main 25-hydroxyvitamin D3 (25(OH)D3) metabolite, thus potentially overlooking contributions and dynamic effects of other vitamin D metabolites, the crucial roles of several of which have been previously demonstrated. The ideal assay would determine all relevant high and low-abundant vitamin D species simultaneously. We describe a sensitive quantitative assay for determining the chemotypes of vitamin D metabolites from serum after derivatisation and ultra-high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UHPLC-ESI-MS/MS). We performed a validation according to the 'FDA Guidance for Industry Bioanalytical Method Validation'. The proof-of-concept of the method was then demonstrated by following the metabolite concentrations in patients with chronic liver diseases (CLD) during the course of a vitamin D supplementation study. The new quantitative profiling assay provided highly sensitive, precise and accurate chemotypes of the vitamin D metabolic process rather than the usually determined 25(OH)D3 concentrations.

Topics: 24,25-Dihydroxyvitamin D 3; Calcifediol; Calcitriol; Chromatography, High Pressure Liquid; Dietary Supplements; Humans; Liver; Liver Cirrhosis; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Vitamin D Deficiency

Vitamin D insufficiency is prevalent in chronic kidney disease (CKD) and associated with secondary hyperparathyroidism (SHPT) and increased risk of bone and vascular disease. Unfortunately, supplementation of stage 3 or 4 CKD patients with currently recommended vitamin D2 or D3 regimens does not reliably restore serum total 25-hydroxyvitamin D to adequacy (≥30ng/mL) or effectively control SHPT. Preclinical and clinical studies were conducted to evaluate whether the effectiveness of vitamin D repletion depends, at least in part, on the rate of repletion. A modified-release (MR) oral formulation of calcifediol (25-hydroxyvitamin D3) was developed which raised serum 25-hydroxyvitamin D3 and calcitriol levels gradually. Single doses of either bolus intravenous (IV) or oral MR calcifediol were administered to vitamin D deficient rats. Bolus IV calcifediol produced rapid increases in serum 25-hydroxyvitamin D3, calcitriol and FGF23, along with significant induction of CYP24A1 in both kidney and parathyroid gland. In contrast, oral MR calcifediol produced gradual increases in serum 25-hydroxyvitamin D3 and calcitriol and achieved similar hormonal exposure, yet neither CYP24A1 nor FGF23 were induced. A 10-fold greater exposure to bolus IV than oral MR calcifediol was required to similarly lower intact parathyroid hormone (iPTH). Single doses of oral MR (450 or 900μg) or bolus IV (450μg) calcifediol were administered to patients with stage 3 or 4 CKD, SHPT and vitamin D insufficiency. Changes in serum 25-hydroxyvitamin D3 and calcitriol and in plasma iPTH were determined at multiple time-points over the following 42 days. IV calcifediol produced abrupt and pronounced increases in serum 25-hydroxyvitamin D3 and calcitriol, but little change in plasma iPTH. As in animals, these surges triggered increased vitamin D catabolism, as evidenced by elevated production of 24,25-dihydroxyvitamin D3. In contrast, MR calcifediol raised serum 25-hydroxyvitamin D3 and calcitriol gradually, and meaningfully lowered plasma iPTH levels. Taken together, these studies indicate that rapid increases in 25-hydroxyvitamin D3 trigger CYP24A1 and FGF23 induction, limiting effective exposure to calcitriol and iPTH reduction in SHPT. They also support further investigation of gradual vitamin D repletion for improved clinical effectiveness. This article is part of a Special Issue entitled "17th Vitamin D Workshop".

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Administration, Oral; Animals; Calcifediol; Calcium; Dietary Supplements; Drug Liberation; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Gene Expression Regulation; Humans; Male; Parathyroid Hormone; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; RNA, Messenger; Vitamin D Deficiency; Vitamin D3 24-Hydroxylase; Vitamins

24,25-Dihydroxyvitamin D [24,25(OH)2D] is a metabolite of 25-hydroxyvitamin D (25D). Blacks frequently have low total 25D without manifestations of vitamin D deficiency, suggesting that total serum 25D may incorrectly reflect vitamin D status in different racial groups. The ratio of serum 24,25(OH)2D to 25D [vitamin D metabolite ratio (VMR)] represents a new candidate biomarker for vitamin D status.. We measured 24,25(OH)2D3 and 25D3 by mass spectrometry in a random community cohort of black (n = 212) and white (n = 164) Americans to evaluate VMR as a marker for vitamin D status. We measured parathyroid hormone concentrations by immunoassay to compare VMR and 25D3 against a physiological indicator of vitamin D deficiency.. Serum 24,25(OH)2D3 strongly correlated with 25D3 in both black and white study participants (r = 0.90, P < 0.001 and r = 0.86, P < 0.001 respectively). Blacks had lower mean 25D3 than whites [17.0 (7.8) vs 27.5 (11.3) ng/mL; 42.4 (19.5) vs 68.6 (28.2) nmol/L, P < 0.001] and lower mean 24,25(OH)2D3 [2.1 (1.3) vs 3.6 (2.0) ng/mL; 5.1 (3.1) vs 8.7 (4.8) nmol/L, P < 0.001]. In contrast to total 25D3 concentrations, mean VMR values were similar in blacks and whites [11.9 (4.0) vs 12.5 (3.4), P = 0.16, respectively] and were negatively correlated with parathyroid hormone concentrations in both races (rs = -0.26, P < 0.001, and rs = -0.25, P < 0.001, respectively).. Our results provide further evidence that measurement of total 25D for assessment of vitamin D status in patients of African descent deserves reevaluation and suggest that alternative measures such as VMR should be considered.

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Biomarkers; Black or African American; Cross-Sectional Studies; Female; Humans; Male; Middle Aged; Parathyroid Hormone; Reproducibility of Results; Tandem Mass Spectrometry; Vitamin D; Vitamin D Deficiency; White People

Vitamin D insufficiency is highly associated with cardiovascular morbidity and mortality. We have demonstrated enhanced vascular calcification in LDL receptor knockout (LDLR(-/-)) mice fed a diet low in vitamin D. This study aimed to investigate the impact of a diet low in vitamin D on vascular calcification in wild-type (WT) mice lacking atherosclerotic plaques and the effects of a persistent and discontinuous vitamin D insufficiency on atherosclerotic plaque composition in LDLR(-/-) mice. The study was performed with 4-wk-old male WT and LDLR(-/-) mice that were fed a normal calcium/phosphate Western diet (210 g/kg fat, 1.5 g/kg cholesterol) containing either adequate (+D; 1000 IU/kg) or low (-D; 50 IU/kg) amounts of vitamin D-3 for 16 wk. Four groups of LDLR(-/-) mice received 1 of the 2 diets for additional 16 wk (total 32 wk) and were compared with mice fed the diets for only 16 wk. WT and LDLR(-/-) mice that were fed the -D diet for 16 wk tended to develop more calcified spots in the aortic valve than mice fed the +D diet (+50% and +56%, respectively; P < 0.10). In LDLR(-/-) mice, the extent of calcification increased from week 16 to week 32 and was higher in the -D than in the +D group (P < 0.05). The calcification, owing to the -D diet, was accompanied by highly expressed osteoblast differentiation factors, indicating a transdifferentiation of vascular cells to osteoblast-like cells. Feeding the +D diet subsequent to the -D diet reduced the vascular calcification (P < 0.05). LDLR(-/-) mice fed the -D diet for 32 wk had higher plaque lipid depositions (+48%, P < 0.05) and a higher expression of cluster of differentiation 68 (+31%, P < 0.05) and tumor necrosis factor α (+134%, P < 0.001) than the +D group. Collectively, the findings imply low vitamin D status as a causal factor for vascular calcification and atherosclerosis.

Topics: 24,25-Dihydroxyvitamin D 3; Animal Feed; Animals; Aorta; Calcitriol; Calcium; Cholecalciferol; Genotype; Male; Mice, Inbred C57BL; Osteoblasts; Phosphates; Plaque, Atherosclerotic; Receptors, LDL; Vascular Calcification; Vitamin D; Vitamin D Deficiency; Vitamins

The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)2D].. We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)2D using 100 μL of serum with a 5-minute run time.. The assay uses a newly synthesized internal standard d6-24,25-(OH)2D3 enabling the quantitation of 24,25-(OH)2D3 as well as the determination of the ratio of 25-OH-D3 to 24,25-(OH)2D3, a physiologically useful parameter.. We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models.. The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D2 and 25-OH-D3) determination (-2% to -5% bias) and exhibited low inter- and intraassay coefficients of variation (4%-7%) and lower limits of quantitation of 0.25-0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D3 and 24,25-(OH)2D3 (r(2) = 0.80) in subjects over a broad range of 25-OH-D3 values and a marked lack of production of 24,25-(OH)2D3 below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D3 to 24,25-(OH)2D3, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80-100) in patients with idiopathic infantile hypercalcemia.. The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Blood Chemical Analysis; Chromatography, Liquid; Dietary Supplements; Female; Humans; Hypercalcemia; Mice; Mice, Knockout; Predictive Value of Tests; Quinoxalines; Tandem Mass Spectrometry; Triazoles; Vitamin D; Vitamin D Deficiency

The optimal circulating concentration of 25(OH) vitamin D is controversial.. The aim was to investigate if FGF-23 and 24,25(OH)2D can guide cholecalciferol replacement.. Oral cholecalciferol (10,000 IU weekly) administered to subjects with 25(OH)D levels < 20 ηg/mL and eGFR > 60 mL/min/1.73 m(2) (n = 25), chronic kidney disease (CKD) (n = 27), or end stage renal disease (ESRD) (n = 14).. The study was conducted at the Veterans Affairs clinics.. Serum FGF-23, PTH, 25(OH)D, 1,25(OH)2D, 24,25(OH)2D, calcium, and phosphorous concentrations, and urinary excretion of calcium and phosphorus at baseline and after 8 weeks of treatment.. Cholecalciferol treatment increased concentrations of serum 25(OH)D by (19.3 ± 8 ηg/mL, P = .001; 12.2 ± 9 ηg/mL, P = .0001) and 24,25(OH)2D (1.14 ± 0.89 ηg/mL, P = .0024; 1.0 ± 0.72 ηg/mL P = .0002), and reduced serum PTH (-11 ± 21 pg/mL, P = .0292; -42 ± 68 pg/mL, P = .0494) in normal and CKD subjects, respectively. Cholecalciferol increased serum FGF-23 levels only in normal subjects (44 ± 57 ηg/mL, P = .01). Increments in serum 25(OH)D positively correlated with serum FGF-23 and 24,25(OH)2D and negatively correlated with PTH. In ESRD, cholecalciferol administration increased 25(OH)D by (16.6 ± 6.6 ηg/mL P ≤ .05) without changing 24,25(OH)2D, FGF-23 or PTH levels.. Modest elevations of serum 25(OH)D levels after cholecalciferol treatment are sufficient to induce compensatory degradative pathways in patients with sufficient renal reserves, suggesting that optimal circulating 25(OH)D levels are approximately 20 ηg/mL. In addition, catabolism of 25(OH)D may also contribute to the low circulating vitamin D levels in CKD, since elevations of FGF-23 in CKD are associated with increased 24,25(OH)2D after cholecalciferol administration.

Topics: 24,25-Dihydroxyvitamin D 3; Aged; Cholecalciferol; Drug Monitoring; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Humans; Kidney; Male; Middle Aged; Multivariate Analysis; Parathyroid Hormone; Prospective Studies; Renal Insufficiency, Chronic; Vitamin D; Vitamin D Deficiency; Vitamins

Parathyroid hormone (PTH) is used as a marker of vitamin D (VD) status. However, PTH depends on many other factors. The 24,25-dihydroxy VD (24,25VD) concentration may be a sensitive marker because its production is reduced in VD deficiency. The relationship between VD metabolites, their ratio and PTH was investigated in adolescents from the UK and The Gambia with different calcium intakes and VD status. In the UK, there was a significant positive (+ve) association between 25VD and both 1,25-dihydroxy VD (1,25VD) and 24,25VD and a negative (-ve) association with PTH. The 24,25:25VD ratio was consistent across the 25VD concentration range. There was a +ve association between PTH and 1,25:25VD, (1,25+24,25):25VD or 1,25:24,25VD, a -ve association with 24,25VD and none with 1,25VD or 24,25:25VD. Using LnPTH and 1,25:25VD ratio (but not 1,25VD:24,25VD or 25VD:24,25VD) increased uniformity between groups and strength of relationships compared to PTH and 1,25 or 25VD alone. In The Gambia, there was a significant -ve relationship between 25VD and PTH and none with 1,25VD. There was a +ve association between 1,25VD or 1,25:25VD and PTH. The more uniform prediction of PTH by the 1,25VD:25VD ratio may be because this better reflects the extent to which PTH-induced 1,25VD production can be met by VD supply. Further validation is needed.

Topics: 24,25-Dihydroxyvitamin D 3; Adolescent; Adult; Biomarkers; Calcium; Dose-Response Relationship, Drug; Female; Gambia; Humans; Male; Models, Biological; Parathyroid Hormone; United Kingdom; Vitamin D; Vitamin D Deficiency

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Aged; Cohort Studies; Critical Illness; Female; France; Humans; Incidence; Intensive Care Units; Male; Middle Aged; Prospective Studies; Risk Factors; Severity of Illness Index; Vitamin D; Vitamin D Deficiency

Rickets in infants attributable to inadequate vitamin D intake and decreased exposure to sunlight continues to be reported in the United States. There are also concerns for vitamin D deficiency in older children and adolescents. Because there are limited natural dietary sources of vitamin D and adequate sunshine exposure for the cutaneous synthesis of vitamin D is not easily determined for a given individual and may increase the risk of skin cancer, the recommendations to ensure adequate vitamin D status have been revised to include all infants, including those who are exclusively breastfed and older children and adolescents. It is now recommended that all infants and children, including adolescents, have a minimum daily intake of 400 IU of vitamin D beginning soon after birth. The current recommendation replaces the previous recommendation of a minimum daily intake of 200 IU/day of vitamin D supplementation beginning in the first 2 months after birth and continuing through adolescence. These revised guidelines for vitamin D intake for healthy infants, children, and adolescents are based on evidence from new clinical trials and the historical precedence of safely giving 400 IU of vitamin D per day in the pediatric and adolescent population. New evidence supports a potential role for vitamin D in maintaining innate immunity and preventing diseases such as diabetes and cancer. The new data may eventually refine what constitutes vitamin D sufficiency or deficiency.

Topics: 24,25-Dihydroxyvitamin D 3; Adolescent; Animals; Breast Feeding; Child; Cholecalciferol; Fetus; Humans; Infant; Infant Food; Milk; Nutrition Policy; Rickets; Sunlight; Vitamin D; Vitamin D Deficiency

Children and adolescents with the high bone turnover comprise a high risk population for vitamin D insufficiency. A sample of 178 clinically healthy children aged 3 to 18 years who came from public schools and lived in North West of Greece participated in the study. They were grouped into three age groups (I: 3-10, II: 11-14 and III: 15-18 years of age). Blood samples were taken during winter and summer months for determining calciotropic hormones, calcium, phosphate and biochemical markers of bone synthesis.A high percentage (47%) of the subjects aged 15-18 years was found to have 25OHD <10 ng/ml in winter but much less (13-14%) of the younger ages (13-14 years), while in the summer they were all >10 ng/ml. The prevalence was even higher in the girls of the older group accompanied by lower Pi concentrations again in winter (win:1.19+/-0.03, sum:1.93+/-0.03 mmol/l, p < 0.001). The 24,25(OH)(2)D levels were changing in parallel to 25OHD, but again in the older subjects, during winter, they were by 2/3 lower than the summer ones (0.73+/-0.10 vs. 2.41+/-0.20 ng/ml, p < 0.001). No significant differences were found between seasons and groups in the 1,25(OH)(2)D levels. The biochemical markers of bone synthesis, osteocalcin (OC) and total alkaline phosphatase (ALP), were found significantly lower in the girls of the older group both in winter and summer respectively. Even in a sunny country like Greece the adolescents living in an urban area are in high risk for vitamin D deficiency during winter. Supplementation with vitamin D of milk, of popular beverages and perhaps some foods would be of help.

Topics: 24,25-Dihydroxyvitamin D 3; Adolescent; Adolescent Development; Alkaline Phosphatase; Calcium; Child; Child Development; Child, Preschool; Female; Greece; Humans; Male; Osteocalcin; Phosphates; Seasons; Vitamin D; Vitamin D Deficiency

The effect(s) of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] on fracture healing was studied in a vitamin D-depleted chick model. 24R,25(OH)2D3, together with another hormonally active vitamin D metabolite, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], improved bone mechanical strength parameters (torsional strength, angular deformation, and stiffness) and the ash content. The synthetic epimer 24S,25-dihydroxyvitamin D3 [24S,25(OH)2D3] was not as potent as the natural 24R,25(OH)2D3. In light of the ability of the fracture-healing callus to discriminate between 24R,25(OH)2D3 and 24S,25(OH)2D3, a search was initiated in fracture-healing callus tissue for the presence of a specific 24R,25(OH)2D3 receptor. No evidence was obtained for a classical nuclear/cytosol receptor for 24R,25(OH)2D3 in the fracture-healing callus. A specific receptor/binding protein for 24R,25(OH)2D3 was found in the callus membrane fraction, which showed different ligand binding affinities [KD = 18.3 +/- 1.9 nmol/L, Bmax = 43.9 +/- 6.0 fmol/mg; relative competitive index (RCI) for 24R,25(OH)2D3/24S,25(OH)2D3/25(OH)D3/1alpha,25(OH)2D3 = 100/37/401/2.0] compared with the ubiquitous serum vitamin D-binding protein (RCI = 100/99/219/5). Also, a callus membrane-binding protein/receptor for 1alpha,25(OH)2D3 was detected with a KD = 0.83 +/- 0.35 nmol/L and a Bmax = 35.5 +/- 5.2 fmol/mg. Thus, we have demonstrated a biological role for 24R,25(OH)2D3 in fracture healing and described the presence of its receptor/binding protein in a callus membrane fraction.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bony Callus; Calcifediol; Calcitriol; Chickens; Fracture Healing; Male; Receptors, Calcitriol; Stereoisomerism; Structure-Activity Relationship; Tibial Fractures; Vitamin D Deficiency

Matrix remodeling plays a prominent role in growth plate calcification. Since interleukin-1 (IL-1) has been implicated in stimulating proteinase production and inhibiting matrix synthesis in articular cartilage, we examined whether IL-1 was present in growth plate and whether the vitamin D metabolites, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 1,25) and 24,25(OH)2D3 (24,25), regulate the level of IL-1 found in this tissue. Sprague-Dawley rats were placed on normal (Normal rats) or rachitogenic diet (-VDP rats). The -VDP rats were either left untreated, injected 24 h prior to euthanasia with 24,25 (-VDP+24,25 rats) or 1,25 (-VDP+1,25 rats), or were given ergocalciferol (Ergo rats) orally, 48 h prior to euthanasia. Growth plates were harvested and extracted in buffer containing 1 M guanidine. IL-1 activity was measured by adding authentic cytokine or growth plate extracts to cultures of lapine articular cartilage and assaying release of glycosaminoglycans (GAGs) and changes in collagenase and neutral metalloproteinase activity. Neutralization of activity in the extracts was performed using polyclonal antisera to IL-1alpha or IL-1beta. An ELISA was used to determine levels of IL-1alpha and beta in the extracts. All extracts contained IL-1alpha and beta, as determined by ELISA. Levels of IL-1beta, but not IL-1alpha, were affected by the vitamin D status of the animal. Extracts from -VDP+24,25 animals contained significantly more IL-1beta than any of the other treatment groups, with the level found in these animals being 3-fold higher than normal and 2-fold higher than -VDP. Extracts were also tested in the bioassay to determine the level of active cytokine present. All growth plate extracts contained activity which altered GAG and proteinase release by lapine articular cartilage. Extracts from -VDP-, -VDP+1,25-, and -VDP+Ergo-treated rats stimulated a 40% increase in glycosaminoglycan release compared with extracts from normal rats. In contrast, extracts from -VDP+24,25-treated rats stimulated a 300% increase in glycosaminoglycan release. Both collagenase and neutral metalloproteinase activity of lapine cartilage were increased after incubation with the growth plate extracts. Collagenase activity was significantly increased 8- to 13-fold by the addition of extracts from -VDP-, -VDP+24,25-, or -VDP+1,25-treated animals. Neutral metalloproteinase activity was similarly increased by 4- to 10-fold. To characterize this activity further, growth plate extracts were in

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcitriol; Collagenases; Enzyme-Linked Immunosorbent Assay; Ergocalciferols; Glycosaminoglycans; Growth Plate; Interleukin-1; Male; Metalloendopeptidases; Rats; Rats, Sprague-Dawley; Tissue Extracts; Vitamin D Deficiency

The serum levels of the active Vitamin D metabolites 25-hydroxyvitamin D[25(OH)D], 1,25-dihydroxyvitamin D[1,25(OH)2D] and 24,25 dihydroxyvitamin D [24,25(OH)2D], were studied in 21 children with Down's syndrome (DS) in Cantabria, a northern region of Spain, located at 44 degrees N latitude. Serum calcium, magnesium, phosphate, alkaline phosphatase, parathormone and osteocalcine were also determined. In the DS group, the average values of the three Vitamin D metabolites were comparable to those of an age-matched group both in winter and summer times. No child with DS showed values below the normal range, either in Vitamin D metabolites, or in the other parameters of calcium metabolism. The normal increment of 25(OH)D and 24,25(OH)2 values from March to October was not observed in five children. This anomaly was corrected in three, after adequate rules of sun exposure during summer time were followed. In the other two, the 25(OH)D levels were high throughout the study. This investigation shows that children with DS do not require Vitamin D prescription when appropriate periods of sunlight exposure are provided.

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D 2; Calcitriol; Child; Child, Preschool; Cross-Cultural Comparison; Down Syndrome; Female; Humans; Male; Reference Values; Spain; Sunlight; Vitamin D; Vitamin D Deficiency

The steady state serum concentration of 1,25-dihydroxyvitamin D [1,25-(OH)2D] is determined by the relative rates of its biosynthesis via the renal mitochondrial 1-hydroxylase and catabolism via renal and target cell 24-hydroxylases. It is not yet known whether the two catalytic activities are mediated by the product of a single gene or products of distinct genes. To address this question, we undertook to assess 24-hydroxylase function in patients with vitamin D-dependency rickets type I (VDDR-I), a Mendelian disorder of 1,25-(OH)2D synthesis attributable to a defect in renal 1-hydroxylase activity. To assess renal 24-hydroxylase activity, we measured the serum concentration of 24,25-dihydroxyvitamin D [24,25-(OH)2D] and its 25-hydroxyvitamin D (25OHD) precursor. We also measured target cell, 1,25-(OH)2D3-inducible 24-hydroxylase activity and calcitroic acid production in skin fibroblasts from VDDR-I patients and age- and sex-matched controls. Serum levels of 24,25-(OH)2D and 25OHD were similar in VDDR-I patients and controls [ratio of product to substrate, 0.062 +/- 0.013 (n = 5) vs. 0.067 +/- 0.005 (n = 10), mean +/- SEM, for patients and controls, respectively]. Circulating levels of 1,25-(OH)2D were also comparable in both groups [80.6 +/- 15.5 (n = 5) vs. 86.1 +/- 5.2 (n = 10) pmol/L, for patients and controls, respectively], presumably indicative of compliance with calcitriol therapy. Skin fibroblasts from VDDR-I patients exhibited 24-hydroxylase activity which was indistinguishable from that observed in control fibroblasts [108 +/- 14 (n = 5) vs. 96 +/- 25 fmol/10(6) cells.min (n = 6), for patients and controls, respectively]. Similarly, calcitroic acid production was comparable in fibroblast cultures derived from the two groups of subjects [31 +/- 6 vs. 33 +/- 3 fmol/10(6) cells.min (n = 3), for patients and controls, respectively]. Our data demonstrate that renal and target cell 24-hydroxylase activities are normal in patients with VDDR-I and suggest that the renal 1- and 24-hydroxylases likely represent, or contain, distinct polypeptides encoded by different genes.

Topics: 24,25-Dihydroxyvitamin D 3; Adolescent; Adult; Calcitriol; Cells, Cultured; Child; Cytochrome P-450 Enzyme System; Female; Fibroblasts; Humans; Hydroxylation; Male; Rickets; Skin; Steroid Hydroxylases; Vitamin D; Vitamin D Deficiency; Vitamin D3 24-Hydroxylase

We have found that carp and bastard halibut contain 25-hydroxyvitamin D3 (25-D3)-1 alpha-hydroxylase in the liver besides in the kidney by the following in vivo and in vitro experiments. When [3H]-25-D3 was intraperitoneally injected to vitamin D(D)-deficient carp and normal bastard halibut (D-deficient bastard halibut could not be raised because they died during farming), the profiles of high-performance liquid chromatography (HPLC) of the plasma lipid extract showed the formation of a peak corresponding to [3H]-1 alpha,25-dihydroxyvitamin D3 (1,25-D3). When [3H]-25-D3 was incubated with liver homogenates of the fish, a peak corresponding to [3H]-1,25-D3 was also observed in the profile of HPLC. The formation of the metabolite was confirmed by the thermal isomerization into the pre-isomer and mass fragmentography. Although the 1 alpha-hydroxylase was also observed in the kidney, the activity of the enzyme was lower than that in the liver. The results suggest that 25-D3-1 alpha-hydroxylase exists in the liver of carp and bastard halibut and the 25-D3 formed from D3 in the liver is immediately metabolized into 1,25-D3 in the same tissue. The suggestion is supported by the fact that D3 is a major circulating compound with small amounts of 1,25-D3 in the fish while the plasma levels of 25-D3 are under the limit of detection.

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Animals; Calcifediol; Carps; Cholecalciferol; Cholestanetriol 26-Monooxygenase; Chromatography, High Pressure Liquid; Fishes; Injections, Intraperitoneal; Kidney; Liver; Mitochondria; Mitochondria, Liver; Steroid Hydroxylases; Subcellular Fractions; Tritium; Vitamin D Deficiency

Type X collagen is a significant component of the extracellular matrix of the hypertrophic zone of physeal cartilage, but its precise role in endochondral ossification has not been determined. The concentration of type X collagen increases in physeal cartilage in chicks with vitamin D deficiency. The purpose of our study was to determine whether defective endochondral ossification due to vitamin D deficiency was associated with abnormalities in the distribution of type X collagen in the proximal tibiotarsus of chicks. To accomplish this, we induced vitamin D deficiency in broiler chicks and sequentially evaluated the pattern of type X collagen immunoreactivity in the proximal tibiotarsus using a monoclonal antibody specific for chicken type X collagen. Type X collagen immunoreactivity was present in the matrix of the prehypertrophic zone, hypertrophic zone, cartilage cores of the primary spongiosa, and within the chondrocytes of the prehypertrophic and early hypertrophic zones in vitamin D-deficient and D-replete chicks. However, rachitic chicks exhibited two consistent differences in type X collagen immunoreactivity: hypertrophic chondrocytes in the late hypertrophic zone and primary spongiosa contained intracellular type X collagen; and type X collagen was concentrated into laminated aggregates in the pericellular and territorial matrices in the late hypertrophic zone and primary spongiosa.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Antibodies, Monoclonal; Bone and Bones; Calcitriol; Calcium; Chickens; Collagen; Immunohistochemistry; Male; Osteogenesis; Phosphorus; Vitamin D Deficiency

Vitamin D-deficient chicken embryos were obtained by feeding laying hens diets in which 3-7 micrograms calcitriol replaced the vitamin D3 supplement. A large proportion of the D-deficient embryos failed to complete the prehatching positional changes required to start pulmonary respiration. For this reason most of them became cyanotic and had subcutaneous edema and hemorrhages in the head and neck and died without hatching. Total as well as leg-bone and muscle weights were significantly lower in the deficient embryos than in the controls and these changes probably explain the inability of the embryos to complete the movements required to place the beak in contact with the air chamber and start pulmonary respiration. The histological study of the tibiae showed decreased mineralization with narrower trabeculae and enlarged osteoid seams; bone resorption at the inner surface was also significantly decreased. The ultrastructural study of parathyroid glands showed increased functional activity reflected by increased number and size of cisternae of rough endoplasmic reticulum. Injection of 10 ng calcitriol, 1 microgram 24,25-(OH)2D3, or 2 micrograms 25OHD3 to deficient embryos on the 14th day of incubation improved hatchability, bone and muscle weights, and both bone mineralization and resorption.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Calcifediol; Calcitriol; Chick Embryo; Dihydroxycholecalciferols; Movement; Muscles; Parathyroid Glands; Poultry Diseases; Vitamin D Deficiency

Previous studies revealed that administration of 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] to calcium (Ca)-deficient rats causes a dose-dependent reduction in markedly elevated serum 1,25-(OH)2D3 level. Although the results suggested that the metabolism of 1,25-(OH)2D3 was accelerated by 24,25-(OH)2D3, those experiments could not define whether the enhanced metabolism of 1,25-(OH)2D3 played a role in the reduction in the serum 1,25-(OH)2D3 level. In the present study, in order to address this issue more specifically, serum 1,25-(OH)2D3 was maintained solely by exogenous administration through miniosmotic pumps of 1,25-(OH)2D3 into vitamin D-deficient rats. Thus, by measuring the serum 1,25-(OH)2D3 concentration, the effect of 24,25-(OH)2D3 on the MCR of 1,25-(OH)2D3 could be examined. Administration of 24,25-(OH)2D3 caused a dose-dependent enhancement in the MCR of 1,25-(OH)2D3, and 1 microgram/100 g rat.day 24,25-(OH)2D3, which elevated serum 24,25-(OH)2D3 to 8.6 +/- 1.3 ng/ml, significantly increased MCR and suppressed serum levels of 1,25-(OH)2D3. The effect of 24,25-(OH)2D3 on 1,25-(OH)2D3 metabolism developed with a rapid time course, and the recovery of iv injected [1 beta-3H]1,25-(OH)2D3 in blood was significantly reduced within 1 h. In addition, there was an increase in radioactivity in the water-soluble fraction of serum as well as in urine, suggesting that 1,25-(OH)2D3 is rapidly degraded to a water-soluble metabolite(s). Furthermore, the reduction in serum 1,25-(OH)2D3 was associated with a reduction in both serum and urinary Ca levels. Because the conversion of [3H]24,25-(OH)2D3 to [3H]1,24,25-(OH)2D3 or other metabolites was minimal in these rats, 24,25-(OH)2D3 appears to act without being converted into other metabolites. These results demonstrate that 24,25-(OH)2D3 rapidly stimulates the metabolism of 1,25-(OH)2D3 and reduces its serum level. It is suggested that 24,25-(OH)2D3 plays a role in modifying serum 1,25-(OH)2D3 concentrations by affecting the metabolism of 1,25-(OH)2D3 and may have a therapeutic values in the treatment of hypercalcemia or hypercalciuria caused by 1,25-(OH)2D3 excess.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcitriol; Calcium; Dihydroxycholecalciferols; Male; Metabolic Clearance Rate; Phosphates; Rats; Rats, Inbred Strains; Tritium; Vitamin D Deficiency

The mechanical properties of the rat femur treated with a large dose of 24R,25(OH)2D3 were examined and their relationship with the mass and mineral contents of the bone were investigated. Male Wistar rats were fed diet containing 0, 0.025, 1.25, 4.0, or 12.5 ppm 24R,25(OH)2D3 for two years starting six weeks after birth. The rats were killed and their right femurs were removed. The adhering soft tissue were stripped off. Radiographs were made of the femur and its bone mineral content was measured by single photon absorptiometry. Then a three-point bending test was done with pressure exerted in the plane of natural extension. After mechanical testing, non-decalcified cross-sections of the femur were prepared at the mid diaphysis as close to the test fracture site as possible. On x-ray images, the cortical thickness was clearly increased in groups treated with larger doses, and the mid-cortical segmental mineral content of the femur increased dose-dependently to about 150%. The mechanical parameters in the treated animals also increased significantly, strength to 120% (p less than 0.01), energy-absorption capacity to 124% (p less than 0.05), and structural stiffness to 183% (p less than 0.01). Segmental bone mineral contents showed the positive correlations with strength and structural stiffness in both the control and the 24R,25(OH)2D3 treated groups as well. We concluded that the increase in the bone density in animals administered high doses of 24R,25(OH)2D3 was accompanied by an increase in mechanical strength of the bone.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Biomechanical Phenomena; Dihydroxycholecalciferols; Femur; Male; Minerals; Rats; Rats, Inbred Strains; Stress, Mechanical; Vitamin D Deficiency

To assess the correlation between osteoid and vitamin D in patients with a proximal femoral fracture, bone biopsies of the fracture site and the iliac crest were studied; and vitamin-D levels were measured in fasting blood taken on the day of admission. No osteomalacia was found at either site in any of the 95 patients investigated. In 65/95 patients, levels of 25-hydroxy-vitamin D (25-OHD) and 24,25-dihydroxy-vitamin D (24, 25-OH2D) were within the normal range, whereas 30/95 patients were deficient. Because there was no correlation between the amount of osteoid and vitamin-D metabolites in our patients, we concluded that osteomalacia was not a contributory factor in the pathogenesis of the hip fracture.

Topics: 24,25-Dihydroxyvitamin D 3; Aged; Aged, 80 and over; Biopsy; Evaluation Studies as Topic; Female; Femur Neck; Hip Fractures; Humans; Ilium; Male; Middle Aged; Osteomalacia; Vitamin D; Vitamin D Deficiency

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Calcium; Chickens; Cholecalciferol; Dihydroxycholecalciferols; Dose-Response Relationship, Drug; Duodenum; Hydroxycholecalciferols; Kidney; Organ Size; Parathyroid Glands; Ultimobranchial Body; Vitamin D Deficiency

Chickens were raised for 6 weeks from the date of hatch under red light on a vitamin D-free diet; controls were given an oral vitamin D supplement. Vitamin D-deficient animals showed decreased total serum calcium concentration and decreased DNA content in epiphysis and kidney homogenates. In calcifying epiphysis, total carbonic anhydrase (CA) activity was decreased, but activity per microgram DNA was slightly increased and specific activity was double that of the controls. Polyacrylamide gel isoelectric focusing after preparation of the enzyme showed a picture similar to that seen after parathyroid hormone (PTH) administration in chicks; therefore, this could be considered a secondary hyperparathyroidism. The CA activation was not seen in the kidney which can be explained by induction of an endogenous inhibitor protein of the cyclic AMP-dependent protein kinase exclusively in the kidney in vitamin D deficiency. In an additional experiment, chickens were raised for 3 weeks from the date of hatch under red light on a vitamin D-free diet. Daily oral substitution by different vitamin D metabolites (1,25(OH)2D3, 25OHD3, 24,25(OH)2D3) over 7 days led to CA activation compared with controls probably by restoring protein kinase activity in the kidney. Our results show that CA activity is inversely correlated with serum calcium concentrations which is in agreement with a regulatory mechanism recently proposed by us.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcifediol; Calcitriol; Calcium; Carbonic Anhydrases; Chickens; Dihydroxycholecalciferols; DNA; Epiphyses; Kidney; Vitamin D; Vitamin D Deficiency

A large dose of 24R,25(OH)2D3 was administered to the vitamin D-repleted rat to examine its effect on the bone. Male Wistar rats were fed a diet containing 0, 0.025, 1.25, 4.0, and 12.5 ppm 24R, 25(OH)2D3 for 2 years starting at age 6 weeks. The estimated amounts of daily intake of 24R,25(OH)2D3 were 0, 93, 4640, 14680, and 49580 ng/100 g body weight, respectively. No notable difference was found in either the weight or the death rate of the animal. The long-term administration of massive doses of 24R,25(OH)2D3 did not lead to hypercalcemia nor did it affect the blood phosphorus, alkaline-phosphatase, or creatinine levels. Radiographs revealed a striking increase in the bone density on the bones from the animals treated with 1.25 ppm or more 24R,25(OH)2D3. Direct single photon absorptiometry revealed a dose-dependent increase in total bone minerals of both the femur and coccyx. Histological examination revealed a marked increase in the cortical thickness of the femur as well as in the cancellous bone volume of the coccyx. Polarizing microscopy demonstrated the lamellar structure of the bone, and undecalcified sections confirmed the increase of mineralized bone. Ash weight, calcium, phosphorus, and magnesium contents on the tibia and fibula also indicated the ascending dose-dependent increase up to 150% of the control. The parameters of bone size were not altered in any group. These results clearly suggest that 24R,25(OH)2D3 given in massive doses has the pharmacological action of increasing bone volume in the rat without causing remarkable hypercalcemia.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Dihydroxycholecalciferols; Male; Rats; Rats, Inbred Strains; Vitamin D Deficiency

In a prior study, we reported vitamin D metabolite profiles in the plasma of healthy children. In view of these findings, we investigated these same profiles in children with moderate renal insufficiency. Specimens were obtained in both summer and winter in untreated patients, and before and after treatment for up to 1 year, with either 25(OH)D3 or 1,25(OH)2D3. Mean pretreatment 1,25(OH)2D levels were normal. Levels of 25(OH)D3 were also normal and continued to vary with season. Mean pretreatment 24,25(OH)2D3 levels were significantly lower in patients and, interestingly, did not show the normal seasonal variation. Treatment with 25(OH)D3 resulted in consistent and sustained rises in 25(OH)D3 and 24,25(OH)2D3 levels, but no increase in 1,25(OH)2D levels. After 1,25(OH)2D3 treatment, 25(OH)D3 levels were unchanged but still showed seasonal variation in some patients, suggesting a lack of feedback control by 1,25(OH)2D3. Levels of 24,25(OH)2D3 were not significantly different from baseline values. Levels of 1,25(OH)2D increased initially then dropped to pretreatment levels or lower. Normal 1,25(OH)2D levels and reduced 24,25(OH)2D3 levels with the loss of seasonal variation suggests in our patients that the kidney was able to maintain 1,25(OH)2D levels at the expense of 24,25(OH)2D3 levels, presumably to preserve calcium homeostasis.

Topics: 24,25-Dihydroxyvitamin D 3; Adolescent; Calcifediol; Calcitriol; Child; Child, Preschool; Glomerular Filtration Rate; Humans; Kidney Failure, Chronic; Seasons; Vitamin D; Vitamin D Deficiency

The purpose of the present work was to study the effect of vitamin D and its metabolites in correcting hypovitaminosis D in the elderly. Thirty elderly people (mean age 78.4 years) participated in this study. They all had low serum levels of 25-hydroxyvitamin D (25-OH-D), of 24,25-dihydroxyvitamin D [24,25(OH)2D] and of 1,25-dihydroxyvitamin D [1,25(OH)2D]. These low levels did not increase in nine subjects after oral administration of vitamin D2 (3,000 IU/day for 12 weeks). However, administration of 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3) to 12 other subjects (0.5 micrograms/day for 8 weeks) led to a significant increase in the serum levels of 1,25(OH)2D. The other vitamin D metabolite levels remained unchanged. A significant increase in the levels of all three main vitamin D metabolites was obtained following administration of 25-hydroxyvitamin D3 (25-OH-D3) to a third group of nine subjects (25 micrograms/day for 1 week). These results suggest that vitamin D nutrition in elderly people insufficently exposed to the sun could be maintained by regular administration of 25-OH-D, whereas the administration of native vitamin D (ergocalciferol) in the doses used was inadequate for vitamin D nutrition.

Topics: 24,25-Dihydroxyvitamin D 3; Aged; Aged, 80 and over; Calcifediol; Calcitriol; Dihydroxycholecalciferols; Ergocalciferols; Female; Humans; Hydroxycholecalciferols; Male; Vitamin D Deficiency

The influence of vitamin D metabolites (at 1 X 10(-10) M) on the calcification of cartilage matrix (measured by 45Ca2+ uptake) and the C-propeptide of type II collagen (measured by radioimmunoassay) has been studied using organ cultures and chondrocytes isolated from growth plates of vitamin D-deficient and -sufficient 11-day-old rats. Vitamin D-deficient rats had reduced amounts of C-propeptide in their serum and freshly isolated growth plate chondrocytes. In all chondrocytes cultured from vitamin D-deficient animals, the C-propeptide content was maximal at 24 hr whereas calcification continued to increase for up to 72 hr. In organ and chondrocyte cultures of tissue from vitamin D-sufficient rats, both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) were required for maximal stimulation of calcification and maximal increases in C-propeptide content. In these D-replete tissues, 24,25-(OH)2D3 had a less stimulatory effect on both calcification (organ and cell cultures) and C-propeptide (organ cultures only), while 1,25(OH)2D3 alone had no effect in cell cultures but an inhibitory effect in organ cultures. Studies of cells or tissue from growth plates of vitamin D-deficient rats demonstrated that 24,25(OH)2D3 alone produced maximal calcification and maximal increases in the C-propeptide content. 1,25(OH)2D3 generally had an inhibitory effect on both calcification and C-propeptide when used alone. In the presence of 1,25(OH)2D3, the stimulatory effect of 24,25(OH)2D3 was partly abrogated. Maximal stimulation of calcification and increases in C-propeptide by 24,25(OH)2D3 were observed at 1 X 10(-9) M and 1 X 10(-10) M. In none of these studies was there any effect on proteoglycan content.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcification, Physiologic; Calcitriol; Calcium; Calcium-Binding Proteins; Cells, Cultured; Collagen; Collagen Type II; Dihydroxycholecalciferols; Growth Plate; Organ Culture Techniques; Protein Precursors; Rats; Rats, Inbred Strains; Reference Values; Uronic Acids; Vitamin D Deficiency

Abnormalities in plasma vitamin D metabolites and an increased prevalence of osteomalacia have been described in elderly patients sustaining a fracture of the femoral neck. In order to investigate whether the plasma concentrations of the vitamin D metabolites are normal, and whether vitamin D deficient osteomalacia in patients with femoral fracture can be diagnosed using biochemical criteria alone, we have studied before and after 7 days of 40 micrograms oral 25-hydroxyvitamin D3 elderly patients admitted to hospital with a femoral fracture, elderly patients undergoing elective replacement of the femoral head and elderly control patients in hospital with no clinical evidence of bone disease. Plasma 25-hydroxyvitamin D (25(OH)D) and 24,25-dihydroxyvitamin D increased after 7 days of oral 25-hydroxyvitamin D3 to the same levels in the three groups, but in contrast to the controls there was no significant increase in plasma 1,25-dihydroxyvitamin D or radiocalcium absorption in femoral fracture and hip replacement patients. However, when femoral fracture patients were restudied 6-12 months after fracture, plasma 1,25-dihydroxyvitamin D increased after oral 25-hydroxyvitamin D3 to the same extent as it had in the control patients. We conclude that reduced calcium absorption due to low plasma 25(OH)D levels, i.e., vitamin D insufficiency is common in all elderly patients. Furthermore biochemical criteria for diagnosis of vitamin D-deficient osteomalacia are of very limited use at the time of fracture in elderly patients since there is a failure of production of 1,25 dihydroxyvitamin D which resolves within 6-12 months of the fracture. This failure makes the 1,25-dihydroxyvitamin D response to oral 25(OH)D an unreliable guide to the presence of vitamin D-deficient osteomalacia at the time of fracture. The abnormality in 1,25-dihydroxyvitamin D is also present in patients undergoing hip replacement surgery, and is therefore unlikely to be involved in the aetiology of femoral neck fracture. It may, however, contribute to the morbidity after fracture.

Topics: 24,25-Dihydroxyvitamin D 3; Absorption; Aged; Calcifediol; Calcitriol; Calcium; Creatinine; Cyclic AMP; Dihydroxycholecalciferols; Female; Femoral Neck Fractures; Hip Prosthesis; Humans; Osteomalacia; Parathyroid Hormone; Vitamin D; Vitamin D Deficiency

Phosphate accumulation by muscle in vitro and the effects of vitamin D3 metabolites thereupon were studied in cultures of chick embryo skeletal muscle myoblasts and intact chick soleus muscles. A significant proportion of phosphate accumulation by the cells was Na+-dependent, saturable with respect to phosphate, energy-dependent and inhibited by ouabain and arsenate, in agreement with the operation of a Na+-phosphate cotransport system in the muscle cell plasma membrane as has been described for intestine and kidney. This was further supported by the demonstration of substrate-saturable phosphate uptake in sarcolemma vesicles isolated from chick skeletal muscle. Preincubation of myoblast and soleus muscle cultures with physiological levels of 25-hydroxy-vitamin D3 resulted in a significant stimulation of phosphate accumulation by cultures. 1,25-dihydroxy-vitamin D3 had no effects on the differentiated tissue whereas it markedly increased phosphate accumulation by embryonic muscle cells. In addition, it could be shown that 25-hydroxy-vitamin D3 affects the Na+-linked component of cell phosphate uptake through a mechanism dependent on de novo protein synthesis.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Biological Transport; Calcifediol; Calcitriol; Cells, Cultured; Chickens; Dihydroxycholecalciferols; Kinetics; Muscles; Phosphates; Sodium; Vitamin D Deficiency

Vitamin D-deficient chicken embryos were obtained by feeding laying hens a diet in which 5 micrograms 1,25(OH)2D3/kg feed were substituted for the vitamin D3 supplement in the control diet. Hatchability, total Ca and inorganic P concentration in blood, and tibial ash/dry weight ratio were determined in the vitamin D-deficient embryos and in embryos obtained from hens fed the control diet supplemented with 1100 IU vitamin D3/kg feed. After 5 weeks on the substituted diet the hens laid eggs that showed decreased hatchability in spite of excellent shell quality. All determinations in blood and bones were made on embryos of eggs laid after 6-12 weeks on the diets. On the 17th day of incubation the embryos derived from hens fed the substituted diet showed significant hypocalcemia and hyperphosphatemia and a low tibial ash/dry weight ratio. Injection of 1,25(OH)2D3 3 days before killing corrected the hypocalcemia of the deficient embryos. Those chicks that managed to hatch had normal levels of calcium and inorganic phosphate 1 day after hatching. These findings support previous suggestions by us and other authors that vitamin D metabolites are required by the embryo in order to mobilize calcium from the shell, and decreased hatchability in vitamin D-deficient embryos is related to a defect in calcium mobilization from the shell. While in previous studies a decrease in hatchability was the only parameter used to judge D deficiency of the embryos in our present studies, the deficiency is confirmed by demonstrating a deficit in mineral metabolism which is a more specific sign of D deficiency.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcitriol; Calcium; Chick Embryo; Dihydroxycholecalciferols; Egg Shell; Minerals; Phosphates; Tibia; Vitamin D Deficiency

Kidneys from both normal and vitamin D-deficient rats were found to show changes in responsiveness to vitamin D metabolites during postnatal development, correlated with the concentrations of the specific receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or the specific binding protein for 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3]. Cytosol preparations from kidneys of vitamin D-deficient rats, in the second week of life, contained specific binding proteins for 24,25-(OH)2D3. From the fourth week of life, specific receptors for 1,25(OH)2D3 were predominant. In the third week after birth, both the receptor for 1,25(OH)2D3 and the 24,25(OH)2D3 binding protein were present. We have used a sensitive parameter for vitamin D action, the stimulation of creatine kinase BB (CKBB) activity, to measure the response of kidneys from vitamin D-deficient or normal rats. In the first days of life of vitamin D-deficient rats, the kidneys did not respond to either vitamin D metabolite; in the second week of life, there was stimulation of renal CKBB only by 24R,25(OH)2D3; beginning in the fourth week of life, only 1,25(OH)2D3 stimulated renal CKBB. However, during the third week of life, CKBB activity was increased by both metabolites. In normal animals, which showed a lower CK activity at all ages, the response was similar to that in vitamin D-deficient animals but the peak was achieved a few days later. The stimulation of CKBB by vitamin D metabolites occurred in all the zones of the kidneys. An increase in renal CKBB by 1,25(OH)2D3 was also detected immunohistochemically. The increase of CKBB activity caused by the two vitamin D metabolites at different stages of development, closely correlated with changes in the presence of the 1,25(OH)2D3 receptor or the 24,25(OH)2D3 binding protein, suggests a specific role for each metabolite during renal development.

Topics: 24,25-Dihydroxyvitamin D 3; Aging; Animals; Calcitriol; Creatine Kinase; Dihydroxycholecalciferols; Female; Isoenzymes; Kidney; Male; Rats; Rats, Inbred Strains; Receptors, Calcitriol; Receptors, Steroid; Tissue Distribution; Vitamin D Deficiency

The linear rate of bone mineral apposition (BMAR) was measured in vitamin D-deficient and vitamin D-sufficient adult rats before and during treatment with either 25-hydroxyvitamin D3 (25OHD3), 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3]. Dietary vitamin D restriction caused a fall in BMAR which began after 1 week and fell progressively to a value of 35-50% of control values by 4 weeks. The fall in BMAR was related to a fall in the serum concentrations of 25(OH)D3 and 24,25-(OH)2D3, without a fall in the 1,25-(OH)2D3 concentration. Dietary supplementation of the D-deficient animals with either 25OHD3 or 24,25-(OH)2D3 at doses of 200 ng/day restored BMAR. If vitamin D-deficient animals were thyroparathyroid-ectomized before supplementation with vitamin D metabolites, 24,25-(OH)2D3 administration was without effect on BMAR. The combined administration of PTH and 24,25-(OH)2D3 to such animals led to a restoration of the BMAR to normal. In vitamin D-sufficient animals, parathyroidectomy led to a 50% reduction in BMAR, which could be restored by treatment with PTH alone but not with 24,25-(OH)2D3. Simultaneous treatment of these animals with PTH and 24,25-(OH)2D3 led to a greater than normal increase in BMAR (130% of control) in these animals. These data support the concept that 24,25-(OH)2D3 has a role in the regulation of bone formation and/or mineralization, and demonstrate the interrelation between the effects of PTH and 24,25-(OH)2D3 on bone.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Calcifediol; Calcitriol; Dihydroxycholecalciferols; Male; Minerals; Parathyroid Glands; Parathyroid Hormone; Rats; Rats, Inbred Strains; Vitamin D; Vitamin D Deficiency

When added to primary cultures of chick kidney cells, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) decreased the basal rate of production of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and increased that of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). The normal stimulatory effect of parathyroid hormone and forskolin on 1,25(OH)2D3 production was abolished or blunted by the presence of TPA and TPA overcame the inhibitory effect of PTH and forskolin on 24,25(OH)2D3 production. The evidence suggests that protein kinase C may be involved in the regulation of 25(OH)D3 metabolism by chick kidney cells.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcifediol; Calcitriol; Cells, Cultured; Chickens; Colforsin; Dihydroxycholecalciferols; Dose-Response Relationship, Drug; Kidney; Parathyroid Hormone; Tetradecanoylphorbol Acetate; Vitamin D Deficiency

We investigated the stimulation of creatine kinase (CK) and ornithine decarboxylase (ODC) by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] in doses ranging from 1.625 to 6500 pmol in 4-week-old vitamin D-deficient chicks. Enzyme activities were monitored for 72 h. 1,25(OH)2D3 but not 24R,25(OH)2D3 enhanced the activity of ODC in duodenum and bone. The time course of ODC activity in bone was biphasic, with an increase after 1 h and a higher peak after 24 h. Diaphyses and epiphyses responded equally well after a dose of 6500 pmol. The kidney, liver, and lung showed 1.5-3.8-fold increase in CK activity following 1,25(OH)2D3, reaching a maximum between 3-5 h. However, sustained stimulation of CK activity could still be demonstrated after 72 h, and the 48-h levels in the lung even exceeded the 5-h values. No change of activity of either enzyme was noted in heart and brain after application of 1,25(OH)2D3. There was no coincidence of stimulation of ODC and CK by 1,25(OH)2D3 in the same tissue, and the dose-responsiveness of both enzymes differed considerably. Near maximum activities of ODC were achieved with 19.5 pmol 1,25(OH)2D3 in duodenum and pancreas, while maximum responses of CK occurred in the liver at 195 pmol and in lung and kidney at 6500 pmol. 24R,25(OH)2D3 failed to produce any consistent effects of either enzyme in all tissues examined. These results, particularly the lack of response to 24R,25(OH)2D3, are different from those reported in rats.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Calcitriol; Chickens; Creatine Kinase; Dihydroxycholecalciferols; Male; Ornithine Decarboxylase; Viscera; Vitamin D Deficiency

Evidence to date has failed to show a consistent effect of vitamin D metabolites on PTH secretion. This study was undertaken to assess the possible direct, acute effects of vitamin D metabolites on PTH secretion in vitro. Ethanol has been used in several published studies as the vehicle for vitamin D metabolites. We found that 0.2-1.0% ethanol inhibited PTH release from dispersed bovine parathyroid cells (PTC). Our experiments with vitamin D metabolites used ethanol as a vehicle at a concentration less than 0.1%. When compared to ethanol treatment, 10-100 nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 25 and 100 nM 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 100 nM 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3) had no effect on PTH release from PTC incubated for up to 4 h. A combination of 1,25(OH)2D3 and 24,25(OH)2D3 (each 25 or 100 nM) was without effect. Also, 100 nM 1,25(OH)2D3 had no effect on PTH release from either bovine parathyroid gland slices or from parathyroid glands from either vitamin D-replete (+D) or vitamin D-deficient (-D) rats incubated for up to 4 h. The i.v. injection of 1 microgram 1.25(OH)2D3 into -D rats had no effect on either serum PTH or calcium (Ca), either 0.5 or 1.0 h after treatment. Parathyroid glands from -D rats incubated with 0.75 mM Ca secreted more PTH than glands of similar weight from rats given 25 micrograms vitamin D3 3 days earlier, suggesting that vitamin D or a metabolite of vitamin D may modulate the sensitivity of the parathyroid gland to medium Ca. In summary, we found no evidence for a direct, acute effect of vitamin D metabolites on PTH secretion under diverse experimental conditions.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcitriol; Calcium; Cattle; Dihydroxycholecalciferols; Ethanol; In Vitro Techniques; Male; Parathyroid Glands; Parathyroid Hormone; Rats; Rats, Inbred Strains; Vitamin D; Vitamin D Deficiency

It has previously been shown that vitamin D deficiency impairs arginine-induced insulin secretion from the isolated, perfused rat pancreas (Science 1980; 209:823-25). Since vitamin D is known to be metabolized to 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) and 24R,25-dihydroxyvitamin D3 (24,25[OH]2D3), it is essential to clarify which vitamin D metabolite has the important role of enhancing insulin secretion. In this report, a comparison is made of the relative efficacy of 3-wk repletion with vitamin D3 (980 pmol/day), 1,25(OH)2D3 (39 pmol/day or 195 pmol/day), and 24,25(OH)2D3 (650 pmol/day) on arginine-induced insulin secretion from the isolated, perfused rat pancreas; in this experiment, the daily caloric intake of the animals receiving vitamin D or its metabolites was controlled by pair feeding to the caloric intake of the vitamin D-deficient rats. 1,25(OH)2D3 repletion was found to completely restore insulin secretion to the levels seen in vitamin D3-replete, pair-fed controls in both the first and second phases, while 24R,25(OH)2D3 only partially improved insulin secretion, and then only in the first phase. Changes of both serum calcium levels and dietary caloric intake after vitamin D metabolite administration are concluded to play a lesser role on the enhancement of insulin secretion, since, in a separate experiment, vitamin D-deficient rats with normal serum calcium levels did not show recovery of insulin secretion equivalent to the vitamin D-replete animals under conditions of dietary pair feeding. These results suggest that 1,25(OH)2D3 but not 24,25(OH)2D3 plays an essential role in the normal insulin secretion irrespective of the dietary caloric intake and prevailing serum calcium levels.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Arginine; Body Weight; Calcitriol; Calcium; Cholecalciferol; Dihydroxycholecalciferols; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Male; Perfusion; Rats; Vitamin D Deficiency

The biochemical nature of the physiological defect found in chick embryos from hens supported on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D is described. Vitamin D-deficient hens (44-wk-old) were divided into six groups of five and dosed daily for 19 wk with either 2.0 micrograms of 25-hydroxyvitamin D3, 2.0 micrograms of 24,24-difluoro-25-hydroxy-vitamin D3, 0.4 micrograms of 1,25-dihydroxyvitamin D3, 2.0 micrograms of 24,25-dihydroxyvitamin D3, 0.4 micrograms of 1,25-dihydroxyvitamin D3 plus 2.0 micrograms of 24,25-dihydroxyvitamin D3, or vehicle only. Normal embryonic development was found in eggs from hens given 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, whereas embryos from hens given 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, or their combination were abnormal and failed to hatch. Embryos from hens fed 1,25-dihydroxyvitamin D3 and/or 24,25-dihydroxyvitamin D3 had vitamin D deficiency: low bone ash, low plasma calcium, low total body calcium, and extremely high plasma phosphorus. Because the shell is the major source of calcium for the developing embryo, calcium transport from the shell to the embryos across the chorioallantoic membrane apparently fails, giving rise to the observed defects in embryonic development.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Biological Transport; Bone and Bones; Calcifediol; Calcitriol; Calcium; Chick Embryo; Chickens; Cholecalciferol; Dihydroxycholecalciferols; Egg Shell; Female; Phosphorus; Vitamin D Deficiency

The role of vitamin D metabolites in the regulation of hepatic 25-hydroxyvitamin D production was investigated by examining the effects of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and 24,25-dihydroxyvitamin D on the synthesis of [25-3H]hydroxyvitamin D by rachitic rat liver homogenates. Production of [25-3H]hydroxyvitamin D was inhibited by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, but not by 24,25-dihydroxyvitamin D. 25-Hydroxyvitamin D increased the Km of the vitamin D-25-hydroxylase enzyme(s), while 1,25-dihydroxyvitamin D decreased the Vmax with a Ki of 88.7 ng/ml. Inhibition of hepatic 25-hydroxyvitamin D production by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D may be another control mechanism to regulate circulating vitamin D levels.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcifediol; Calcitriol; Dihydroxycholecalciferols; Hydroxycholecalciferols; Kinetics; Liver; Male; Rats; Rats, Inbred Strains; Rickets; Tritium; Vitamin D Deficiency

Topics: 24,25-Dihydroxyvitamin D 3; Aged; Calcium; Dihydroxycholecalciferols; Female; Humans; Hyperparathyroidism; Osteomalacia; Phosphorus; Vitamin D Deficiency

Chemical synthesis of (24R)-24,25-dihydroxy-[26,27-3H]vitamin D3, and its 24-epimer has been devised that allows introduction of 3H at the terminal step of the synthesis. The epimeric mixture is derivatized as the tris(trimethylsilyl) ethers and resolved by high-performance liquid chromatography. The product has a specific activity of 178 Ci/mmol and is fully active in binding to the rat plasma vitamin D binding protein and in the elevation of serum calcium levels of vitamin D deficient rats. The synthesis begins with the readily available 3 beta-hydroxy-5-cholenic acid methyl ester and involves a Pummerer rearrangement, introduction of the delta 7, irradiation, and isolation of the 26,27-dinor-25-carboxylic acid methyl ester of vitamin D3. This compound is then treated with a Grignard reagent containing 3H (80 +/- 10 Ci/mmol).

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Biological Assay; Calcium; Dihydroxycholecalciferols; Female; Indicators and Reagents; Isomerism; Isotope Labeling; Magnetic Resonance Spectroscopy; Rats; Spectrophotometry, Ultraviolet; Tritium; Vitamin D Deficiency

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 from 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 less than 0.01) and initial mineralization rate (r = 0.63, P less than 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.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Blood Urea Nitrogen; Body Weight; Bone and Bones; Bone Development; Bone Matrix; Bone Resorption; Calcium; Dihydroxycholecalciferols; Injections, Intraperitoneal; Male; Osteogenesis; Phosphorus; Rats; Vitamin D Deficiency

The effect of the X-linked Hyp mutation on 25-hydroxyvitamin D3 (25-OH-D3) metabolism in mouse renal cortical slices was investigated. Vitamin D replete normal mice and Hyp littermates fed the control diet synthesized primarily 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3); only minimal synthesis of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was detected in both genotypes and 1,25-(OH)2D3 formation was not significantly greater in Hyp mice relative to normal littermates, despite hypophosphatemia and hypocalcemia in the mutants. Calcium-deficient diet fed to normal mice reduced serum calcium (p less than 0.01), increased renal 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) activity (p less than 0.05), and decreased 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) activity (p less than 0.05). In contrast, Hyp littermates on the calcium-deficient diet had decreased serum calcium (p less than 0.01), without significant changes in the renal metabolism of 25-OH-D3. Both normal and Hyp mice responded to the vitamin D-deficient diet with a fall in serum calcium (p less than 0.01), significantly increased renal 1-OHase, and significantly decreased renal 24-OHase activities. In Hyp mice, the fall in serum calcium on the vitamin D-deficient diet was significantly greater than that observed on the calcium-deficient diet. Therefore the ability of Hyp mice to increase renal 1-OHase activity when fed the vitamin D-deficient diet and their failure to do so on the calcium-deficient diet may be related to the resulting degree of hypocalcemia. The results suggest that although Hyp mice can respond to a disturbance of calcium homeostasis, the in vivo signal for the stimulation of renal 1-OHase activity may be set at a different threshold in the Hyp mouse; i.e. a lower serum calcium concentration is necessary for Hyp mice to initiate increased synthesis of 1,25(-OH)2D3.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcifediol; Calcitriol; Calcium; Diet; Dihydroxycholecalciferols; Female; Hypophosphatemia, Familial; In Vitro Techniques; Kidney; Kidney Cortex; Male; Mice; Mice, Inbred C57BL; Phosphorus; Vitamin D Deficiency; X Chromosome

The conversion of 25-hydroxyvitamin D3 (25 OH D3) to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and 1,24,25-trihydroxyvitamin D3 (1.24,25-(OH)3D3) was studied in renal tubules prepared from chicks raised on a vitamin D deficient diet with or without vitamin D supplementation. As described previously, in tubules from vitamin D deficient chicks, cyclic AMP caused an increase in the net accumulation of 1,25-(OH)2D3, the major metabolite formed under these circumstances. This stimulation was shown to be due to an increased maximum velocity of the hydroxylation reaction. There was also a significant inhibition of the net accumulation of 24,25-(OH)2D3. Cyclic GMP caused a significant inhibition of 1,25-(OH)2D3 formation and stimulation of the net accumulation of 24,25-(OH)2D3. In chicks supplemented with high doses of vitamin D, 24,25-(OH)2D3 was the major metabolite of 25 OH D3 detected and 1-hydroxylase activity was negligible. Under these circumstances, neither cyclic AMP nor cyclic GMP affected net accumulation of 24,25(OH)2D3. This suggested that the apparent effect of the nucleotides on formation of 24,25-(OH)2D3 may have been due to further metabolism of 24,25-(OH)2D3 when 1-hydroxylase activity was high. It is concluded that cyclic AMp and cyclic GMP have reciprocal effects on renal 25 OH D3-1-hydroxylase activity, and both should be considered potential intracellular regulators of 25 OH D3 metabolism.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcifediol; Calcitriol; Chickens; Cyclic AMP; Cyclic GMP; Dihydroxycholecalciferols; Hydroxycholecalciferols; Kidney Tubules; Male; Vitamin D Deficiency

Previously we have shown that the isolated perfused kidney from vitamin D-deficient rats converts [3H]25(OH)D3 into [3H]1 alpha,25(OH)2D3. When certain vitamin D metabolites were added to perfusate the same kidney began to synthesize [3H]24R,25(OH)2D3. In this study we investigated the structural requirements of the vitamin D molecule necessary to stimulate synthesis of [3H]24R,25(OH)2D3 in a 1-hydroxylating kidney. Kidneys were perfused with tracer [3H]25(OH)D3 (450 pM) alone and in the presence of a variety of hydroxylated metabolites and fluorinated analogues of vitamin D3 at concentrations of 450 pM to 25 microM. Tracer [3H]25(OH)D3 alone resulted in synthesis of only [3H]1 alpha,25(OH)2D3 during the 6-h perfusion period. 25-Hydroxylated metabolites [25(OH)D3, 25 nM; 1 alpha,25(OH)2D3, 25 nM; 24R,25(OH)2D3, 25 nM; 24(F)2,25(OH)D3, 50 nM] stimulated [3H]24R,25(OH)2D3 production at 2 h of perfusion. On the other hand, analogues without the 25-hydroxyl group [D3; 1 alpha(OH)D3; 25(F)D3; 1 alpha(OH),25(F)D3; 1 alpha(F)D3; 1 beta(F)D3]; did not stimulate [3H]24R,25(OH)2D3 synthesis. We conclude that the 25-hydroxyl group is an essential determinant of 24-hydroxylation.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Cholecalciferol; Dihydroxycholecalciferols; Kidney; Kinetics; Male; Perfusion; Rats; Rats, Inbred Strains; Structure-Activity Relationship; Tritium; Vitamin D; Vitamin D Deficiency

The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Bone Development; Calcitriol; Carboxy-Lyases; Dihydroxycholecalciferols; DNA; Dose-Response Relationship, Drug; Duodenum; Enzyme Activation; Epiphyses; Female; Ornithine Decarboxylase; Rats; Rats, Inbred Strains; Vitamin D Deficiency

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Animals; Calcifediol; Calcitriol; Calcium; Cytochrome P-450 Enzyme System; Dihydroxycholecalciferols; Kidney; Male; Parathyroid Hormone; Rats; Steroid Hydroxylases; Vitamin D Deficiency; Vitamin D3 24-Hydroxylase

Vitamin D metabolites were measured in sera of normal, vitamin D deficient and nonazotemic nephrotic rats. The concentrations of all metabolites were reduced in nephrotic and vitamin D deficient animals although 1,25-dihydroxyvitamin D values remained relatively normal in the nephrotic group. Twenty-four hours after the intravenous injection of tritiated 25-hydroxycholecalciferol, approximately 34% of the injected radioactivity appeared in the urine of the nephrotic animals compared with 0.4% in the controls. In extracts from nephrotic sera subjected to high performance liquid chromatography, the percentage of radioactive counts comigrating with 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol was significantly increased. The various metabolites were present in urine in approximately the same ratios as in serum. Dynamic histomorphometry of tibial metaphyses showed no abnormality. Urinary losses of vitamin D metabolites constitute the major cause for low serum values in nephrotic rats. The apparent synthetic rates are not impaired.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Calcifediol; Calcitriol; Calcium; Carrier Proteins; Dihydroxycholecalciferols; Male; Nephrotic Syndrome; Rats; Rats, Inbred Strains; Vitamin D; Vitamin D Deficiency; Vitamin D-Binding Protein

The milk from cows fed normal levels of vitamin D has been found to contain approximately 40 IU per liter of vitamin D activity. A 14-fold increase in dietary vitamin D intake causes only a doubling of the amount of vitamin D in milk. This was determined by measuring stimulation of intestinal calcium transport in the vitamin D-deficient rat. Four vitamin D compounds were then isolated from cow's milk using a combination of conventional chromatography on Sephadex LH-20 and Lipidex 5000 followed by high-performance liquid chromatography. 24,25-Dihydroxycholecalciferol and 1,25-dihydroxycholecalciferol were measured using binding protein assays. One liter of milk contained 27 ng and 4.9 ng, respectively, of these two metabolites. Together these account for about 15% of the vitamin D activity. Cholecalciferol was found to be present at a concentration of 281 ng/liter or 11 IU/liter of biological activity. The milk contained 145 ng/liter 25-hydroxycholecalciferol or 29 IU/liter of activity. Therefore the known vitamin D compounds fully account for the biological activity observed in milk. It is therefore clear that no evidence could be found for the existence of a highly active water-soluble form of vitamin D in milk.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Biological Transport; Calcifediol; Calcitriol; Calcium; Cattle; Cholecalciferol; Dihydroxycholecalciferols; Female; Hydroxycholecalciferols; Male; Milk; Pregnancy; Rats; Vitamin D; Vitamin D Deficiency

Chicks were depleted of vitamin D, divided into groups, and treated daily with (a) cholecalciferol, (b) 1 alpha-hydroxycholecalciferol [1 alpha (OH)-D3], (c) 24R, 25-dihydroxycholecalciferol [24R,25-(OH)2D3], or (d) 1 alpha (OH)D3 and 24R,25(OH)2D3. Two additional groups of chicks were studied, one that was continuously depleted of vitamin D, and another that was continuously supplemented with the vitamin, since day 1. After killing, the tibiae were removed and tested for their mechanical properties. Bending load was applied to the midshaft, and the intrinsic properties of this site, its quantity and geometry were analyzed. From a mechanical point of view, the weakest bones found were of birds depleted of vitamin D, whereas the strongest were of those treated with 1 alpha (OH)D3. Only the bones of the 24R,25(OH)2D3-treated or the 1 alpha (OH)D3 and 24R,25(OH)2D3-treated groups of birds showed mechanical properties comparable to those obtained with vitamin D-replete chicks.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Bone and Bones; Calcium; Chickens; Cholecalciferol; Dihydroxycholecalciferols; Hydroxycholecalciferols; Phosphates; Stress, Mechanical; Vitamin D Deficiency

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcifediol; Calcitriol; Chickens; Chromatography, High Pressure Liquid; Dihydroxycholecalciferols; Hydroxycholecalciferols; Hydroxylation; Kidney; Tritium; Vitamin D Deficiency

The serum levels of the three major vitamin D metabolites [25-hydroxyvitamin D (25-OHD), 1,25-dihydroxyitamin D (1,25-(OH)2D), 24,25-dihydroxyvitamin D (24,25-(OH)2 D)] and immunoreactive parathyroid hormone (iPTH) were measured in 14 morbid obese patients, who later on were subjected to jejunoileal bypass surgery. The preoperative median values of 25-OHD and 24,25-(OH)2D were reduced compared with controls (P less than 0.001), whereas elevated concentrations were found of 1,25-(OH)2D (P less than 0.005). Median levels of iPTH in the obese group were significantly higher than those found in normal subjects (P less than 0.001). A decrease was observed in serum concentrations of all three vitamin D metabolites following jejunoileal bypass (P less than 0.005). An increase in the serum levels of iPTH and alkaline phosphatase was seen postoperatively (P less than 0.002), probably indicating a secondary hyperparathyroidism. The results show that the vitamin D metabolism is slightly abnormal in severely obese patients. Jejunoileal bypass is followed by severe disturbances of vitamin D metabolism.

Topics: 24,25-Dihydroxyvitamin D 3; Adult; Alkaline Phosphatase; Calcifediol; Calcitriol; Dihydroxycholecalciferols; Female; Humans; Ileum; Jejunum; Male; Middle Aged; Obesity; Parathyroid Hormone; Vitamin D; Vitamin D Deficiency

The concentration of vitamin D was determined in human and bovine plasma samples under various physiological and nonphysiological conditions using a nonequilibrium ligand binding assay. Prior to ligand binding analysis the vitamin D in the plasma organic extracts was purified using chromatographic procedures involving Lipidex-5000 and high performance liquid chromatography. The use of a nonequilibrium assay system greatly increased the sensitivity of our assay allowing for a minimum volume of the initial plasma sample. The vitamin D levels in plasma responded to increased sun exposure as well as to the intoxication with vitamin D3. Analysis of a plasma sample from a vitamin D-deficient patient revealed that lipid interference was not a factor in this ligand binding assay.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Binding, Competitive; Calcifediol; Carrier Proteins; Cattle; Cholecalciferol; Chromatography, High Pressure Liquid; Dihydroxycholecalciferols; Ergocalciferols; Humans; Hydroxycholecalciferols; Microchemistry; Vitamin D; Vitamin D Deficiency; Vitamin D-Binding Protein

The levels of vitamin D metabolites were measured in three children with a decreased dietary intake of calcium and vitamin D and sun exposure. All three children had hypocalcemia, hypophosphatemia, and elevated alkaline phosphatase activities. Two children had rickets, aminoaciduria, and elevated immunoreactive parathyroid hormone (iPTH) concentrations. The concentrations of vitamins D2 and D3, 25-hydroxyvitamins D2 and D3 (25-OH-D2 and 25-OH-D3), and 24,25-dihydroxyvitamin D (24,25-[OH]2D) were reduced. Nonetheless, the levels of calcitriol (1,25-[OH]2D) were normal. The combination of hypocalcemia, hypophosphatemia, and increased iPTH concentrations should result in supranormal calcitriol concentrations. Moreover, the ratio of PTH to calcitriol is significantly higher than in normal subjects. Accordingly, in patients with vitamin D deficiency and "normal" calcitriol values, the synthesis of this compound may be reduced. The evaluation of vitamin D deficiency should include the measurement of all metabolites.

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D 2; Adolescent; Calcifediol; Calcitriol; Child; Cholecalciferol; Dihydroxycholecalciferols; Ergocalciferols; Humans; Hydroxycholecalciferols; Parathyroid Hormone; Reference Values; Vitamin D Deficiency

Isolated renal cortical slices were used to study the conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25](OH2)D3] by the rat kidney. Production of 1,25-(OH)2D3 and 24,25-(OH)2D3 was linear with time (30-90 min) and tissue weight (40-250 mg). Production of 1,25-(OH)2D3 was greatest (134 +/- 17 pg/mg tissue.h) in animals fed a low calcium, vitamin D-deficient diet. The greatest 24,25-(OH)2D3 production (106 +/- 17 pg/mg tissue.h) was seen in animals fed a high calcium, vitamin D-replete diet, 1,25-(OH)2D3 production was reduced to 23% of maximum by the addition of 1.2% calcium or 0.8% strontium to the vitamin D-deficient, low calcium diet. Production of 1,25-(OH)2D3 and 24,25-(OH)2D3 was greatly reduced in renal cortical slices that had been heated before incubation. Slices of renal medulla produced only small amounts of 1,25-(OH)2D3 compared to slices of renal cortex. These studies provide direct evidence for the production of 1,25-(OH)2D3 and 24,25-(OH)2D3 by the mammalian renal cortex. They also demonstrate that this production may be modulated by dietary calcium, strontium, and vitamin D.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Calcifediol; Calcitriol; Calcium; Dihydroxycholecalciferols; Hydroxycholecalciferols; In Vitro Techniques; Kidney; Kinetics; Male; Rats; Strontium; Tritium; Vitamin D Deficiency

An in vitro assay of mammalian 25-hydroxyvitamin D3 1 alpha- and 24R-hydroxylases in kidney has been developed. It had been suggested that 25-hydroxyvitamin D binding protein present in mammalian blood and tissues inhibits the enzyme activities in cell-free preparations by binding the substrate 25-hydroxyvitamin D3 more strongly than the hydroxylases bind it. This inhibitory effect is overcome by the addition of substantial amounts of unlabeled 25-hydroxyvitamin D3 to saturate the binding sites of this protein. The resulting metabolites produced in vitro by rat kidney homogenates were isolated and firmly identified by ultraviolet absorption spectrometry and mass spectrometry as 1,25-dihydroxyvitamin D3 and (24R)-24,25-dihydroxyvitamin D3. Maximal 1 alpha-hydroxylation of 25-hydroxyvitamin D3 could be demonstrated in kidney homogenates prepared from vitamin D-deficient rats. Thyroparathyroidectomy of these rats resulted in total suppression of the 1 alpha-hydroxylase. Homogenates of kidney from rats given vitamin D showed little or no 1 alpha-hydroxylase and substantial 24R-hydroxylase activity. Thyroparathyroidectomy of these rts markedly increased the 24R-hydroxylase activity.

Topics: 24,25-Dihydroxyvitamin D 3; 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Animals; Calcifediol; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Dihydroxycholecalciferols; Hydroxycholecalciferols; Kidney; Male; Mass Spectrometry; Rats; Receptors, Calcitriol; Receptors, Steroid; Spectrophotometry, Ultraviolet; Steroid Hydroxylases; Vitamin D Deficiency; Vitamin D3 24-Hydroxylase

Metabolism of 25-hydroxyvitamin D3 (25-OH-D3) was examined in chicks supplemented with vitamin D3. Kidney homogenates metabolized in vitro [3H]-25-OH-D3 to 3 new metabolites (peaks A, C and E) by way of 24,25-dihydroxyvitamin D3. The enzymes responsible for the synthesis of these metabolites appeared to be induced by 1 alpha,25-dihydroxyvitamin D3. Production of these metabolites was increased in parallel with the increase of the supplemented levels of vitamin D3, while recovery of the radioactivity in the chloroform phase was sharply decreased. The production of peak C was considered to be closely related to the transfer of the radioactive metabolites to the water-soluble phase. These results may indicate that 24-hydroxylation is a degradation step in the 25-OH-D3 metabolism.

Topics: 24,25-Dihydroxyvitamin D 3; Animals; Chickens; Cholecalciferol; Chromatography, High Pressure Liquid; Cytosol; Dihydroxycholecalciferols; Hydroxycholecalciferols; Kidney; Male; Microsomes; Mitochondria; Vitamin D Deficiency

The adenylate cyclase activation by bovine synthetic parathyroid hormone (bPTH) (1-34) was studied in vitro in kidney plasma membranes from D-deficient (D-Mb) or normal (D+Mb) rats. In D-Mb, the apparent affinity of parathyroid hormone (PTH) for membranes (170 +/- 30 nM) was significantly higher than that measured in D+Mb (55 +/- 5 nM). The maximum velocity of the PTH-stimulated adenylate cyclase was significantly higher in D+Mb than in D-Mb (163.0 +/- 13.7 and 93.4 +/- 6.7 pmol of cAMP/mg of protein/min, respectively). The action of vitamin D metabolites on the adenylate cyclase stimulation by PTH was then studied in vitro in D-Mb and D+Mb. In D-Mb, 25-hydroxyvitamin D3, 24,25-, and 1, 25-dihydroxyvitamin D3 significantly inhibited cAMP production in the presence of 0.87 microM of bPTH. Vitamin D3 had no effect. Maximal inhibition (86%) was observed for 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 decreased the maximum velocity of PTH-stimulated adenylate cyclase but did not modify the bPTH apparent affinity for D-Mb. The vitamin D3 metabolites tested did not modify the cyclase stimulation by isoproterenol, sodium fluoride, or 5'-guanylylimidodiphosphate. The presence of 1,25-dihydroxyvitamin D3 or 25-hydroxyvitamin D3 did not increase the (Na-K)-ATPase or the phosphodiesterase activities. In the presence of 1,25-dihydroxyvitamin D3 and bPTH, the apparent affinity of ATP for the catalytic moiety was not modified. The maximum velocity was decreased. These results suggest an in vitro interaction between hydroxylated vitamin D metabolites and kidney membranes PTH receptor.

Topics: 24,25-Dihydroxyvitamin D 3; Adenylyl Cyclases; Animals; Calcitriol; Cell Membrane; Cholecalciferol; Dihydroxycholecalciferols; Kidney; Kinetics; Male; Parathyroid Hormone; Rats; Vitamin D Deficiency