Evidence for the metabolism of 24R-hydroxy-25-fluorovitamin D3 and 1alpha-hydroxy-25-fluorovitamin D3 to 1alpha,24R-dihydroxy-25-fluorovitamin D3.

High-pressure liquid chromatography capable of resolving all known vitamin D metabolites and a sensitive competitive binding protein assay specific for 1α,25-dihydroxyvitamin D₃ were used to assay the blood of rats dosed with ethanol, 1α-hydroxyvitamin D₃, 24R-hydroxy-25-fluorovitamin D₃, or 1α-hydroxy-25-fluorovitamin D₃. Compared to the ethanoldosed animals, the blood of rats dosed with 1α-hydroxyvitamin D₃ had increased levels of 1α,25-dihydroxyvitamin D₃; but those dosed with the fluorinated vitamins did not. Instead, their blood contained a compound that cochromatographs with 1α,24R-dihydroxyvitamin D₃ on high-pressure liquid chromatography and binds to the 1,25-dihydroxyvitamin D₃ receptor proteins. 1α,24R-Dihydroxyvitamin D₃ binds as well as 1α, 25-dihydroxyvitamin D₃ to the chick-intestinal cytosol receptor protein for 1α,25-dihydroxyvitamin D₃; whereas 1α,24S-dihydroxyvitamin D₃ binds only one-tenth as well as 1α,25-dihydroxyvitamin D₃. Thus it appears that in vivo, the fluorinated vitamin D compounds are converted to a compound likely to be 1α,24R-dihydroxy-25-fluorovitamin D₃ and that may rival the potency of 1α,25-dihydroxyvitamin D₃.     

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D₃, 24,24-difluoro-25-hydroxyvitamin D₃ has been synthesized and shown to be equally as active as 25-hydroxyvitamin D₃ in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D₃ was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D₃, while large amounts were found when 25-hydroxyvitamin D₃ was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D₃, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D₃ from 24,24-difluoro-25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D₃ is less active than 25-hydroxyvitamin D₃ and 24R,25-dihydroxyvitamin D₃. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D₃ is more active than 25-hydroxyvitamin D₃ which is itself more active than 24R,25-dihydroxyvitamin D₃. The 24,24-difluoro-1,25-dihydroxyvitamin D₃ is equal to 1,25-dihydroxyvitamin D₃, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D₃ in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D₃ cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.     

1 alpha, 25-difluorovitamin D3: an inert vitamin D analog

1α,25-Difluorovitamin D₃ has been synthesized by reacting 1,25-dihydroxyvitamin D₃-3-acetate with diethylaminosulfurtrifluoride followed by hydrolysis. Retention of configuration of the fluoro group in this reaction was demonstrated by physical studies using 1α-fluoro and 1β-fluorovitamin D₃ models. The 1,25-difluorovitamin D₃ compound possessed no vitamin D-like activity demonstrating the importance of 1α- and 25-hydroxylations of vitamin D for activity. However, 1,25-difluorovitamin D₃ had no anti-25-hydroxylation activity and no antivitamin D activity. Since 25-fluorovitamin D₃ has anti-25-hydroxylase activity, it appears the introduction of a fluoro group on the 1 position diminishes interaction of the vitamin D molecule with the 25-hydroxylase system.     

24,24-Difluoro-25-hydroxyvitamin D3-enhanced bone mineralization in rats. Comparison with 25-hydroxyvitamin3 and vitamin D3

The biological activity of 24,24-difluoro-25-hydroxyvitamin D₃ was assessed using elevation of serum phosphorus and healing of rickets of vitamin D-deficient rats. Various levels of 24,24-difluoro-25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₃ were administered daily for 2 weeks in the dose range of 6.5 to 3250 pmol after feeding rats a low phosphorus, vitamin D-deficient diet for 3 weeks. Vitamin D₃ was concurrently tested at dose levels of 650 and 3250 pmol. 24,24-Difluoro-25-hydroxyvitamin D₃ is approximately equipotent with 25-hydroxyvitamin D₃ in stimulation of growth, mineralization of rachitic bone, and elevation of serum inorganic phosphorus. Radiological manifestations of rickets were also equally improved by 24,24-difluoro-25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₃. Compared with vitamin D₃, these compounds were approximately 5 to 10 times more active in mineralization using rats on a low phosphorus, vitamin D-deficient diet. The functional role, if any, for 24-hydroxylated vitamin D compounds, such as 24,25-dihydroxyvitamin D₃, therefore remains obscure. It appears that vitamin D compounds that cannot be 24-hydroxylated evoke no disorder in bone mineralization.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

25,26-dihydroxyvitamin D3 is not a major intermediate in 25-hydroxyvitamin D3-26,23-lactone formation

Structural similarities between 25S,26-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃-26,23-lactone and their concomitant multifold increase in the plasma of animals treated with pharmacological doses of vitamin D₃ suggest a precursor-product relationship. However, a single dose of 25S,26-[³H]dihydroxyvitamin D₃ given to rats treated chronically with pharmacological amounts of vitamin D₃ did not result in detectable plasma 25-[³H]hydroxyvitamin D₃-26,23-lactone. Multiple doses of synthetic 25S,26-dihydroxyvitamin D₃ given to vitamin D₃-deficient rats treated chronically with pharmacological amounts of vitamin D₂ also did not result in detectable plasma 25-hydroxyvitamin D₃-26,23-lactone. Furthermore, homogenates prepared from vitamin d-deficient chickens, dosed with 1,25-dihydroxyvitamin D₃, converted 25-[³H]hydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. But these same homogenates did not convert 25S,26-[³H]dihydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. These data indicate that 25,26-dihydroxyvitamin D₃ is not an intermediate in 25-hydroxyvitamin D₃26, 23-lactone formation.     

Rat plasma 25-hydroxyvitamin D3 binding protein: An inhibitor of the 25-hydroxyvitamin D3-1 α-hydroxylase

An antibody was prepared from serum of rabbits injected with a pure inhibitor protein obtained from rat serum for chick renal 25-hydroxyvitamin D₃-1α-hydroxylase. The antibody was separated from the endogenous inhibitor in rabbit serum. The antibody shows a single precipitin line with the rat serum antigen and with crude calf serum. Furthermore, the antibody removes the 4.0 S 25-hydroxyvitamin D₃ binding protein from rat serum. The removal of the 25-hydroxyvitamin D₃ binding protein from rat serum with antibody brings about a proportionate removal of inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase. The pure inhibitor binds 25-hydroxyvitamin D₃, as demonstrated by sucrose density gradient sedimentation, and shows specificity of binding identical to the serum transport globulin for 25-hydroxyvitamin D₃. Thus, the previously reported inhibitor of the 25-hydroxyvitamin D₃-1α-hydroxylase in rat preparations is the serum 25-hydroxyvitamin D₃ transport protein or some derivative thereof. The antibody added to rat renal mitochondrial preparations does increase the activity of the 1- and 24-hydroxylases slightly but not markedly.     

Treatment with 50000 IU Vitamin D2 Every Other Week and Effect on Serum 25-Hydroxyvitamin D2, 25-Hydroxyvitamin D3, and Total 25-Hydroxyvitamin D in Aclinical Setting

ObjectiveTo examine the effect of 50 000 IU-vitamin D₂ supplementation in a clinical setting on serum total 25-hydroxyvitamin D (25[OH]D), 25-hydroxyvitamin D₂ (25[OH]D₂), and 25-hydroxyvitamin D₃ (25[OH]D₃).MethodsThis retrospective cohort study was performed in an urban tertiary referral hospital in Boston, Massachusetts. Patients who had been prescribed 50 000 IU vitamin D₂ repletion and maintenance programs were identified through a search of our electronic medical record. Baseline and follow-up total serum 25(OH)D, 25(OH)D₂, and 25(OH)D₃ levels were compared.ResultsWe examined the medical records of 48 patients who had been prescribed 50 000 IU vitamin D₂ in our clinic. Mean ± standard deviation baseline total 25(OH) D was 31.0 ± 10.6 ng/mL and rose to 48.3 ± 13.4 ng/mL after treatment (P <.001). 25(OH)D₂ increased from 4.2 ± 4.3 ng/mL to 34.6 ± 12.3 ng/mL after treatment (P <.001), for an average of 158 days (range, 35-735 days). Serum 25(OH)D3 decreased from 26.8 ± 10.8 ng/mL to 13.7 ± 7.9 ng/mL (P <.001).ConclusionsFifty thousand IU vitamin D₂ repletion and maintenance therapy substantially increases total 25(OH)D and 25(OH)D2 despite a decrease in serum 25(OH)D3. This treatment program is an appropriate and effective strategy to treat and prevent vitamin D deficiency.(Endocr Pract. 2012;18:399-402)     

The role of 1,25-dihydroxyvitamin D3 and parathyroid hormone in the regulation of chick renal 25-hydroxyvitamin D3-24-hydroxylase.

A single 325-pmol dose of 1,25-dihydroxyvitamin D₃ given to chicks fed a vitamin D-deficient diet containing 3% calcium and 0.6% phosphorus suppresses renal mitochondrial 25-hydroxyvitamin D₃-1α-hydroxylase and stimulates the 25-hydroxyvitamin D₃-24-hydroxylase as measured by in vitro assay. This alteration in the enzymatic activity takes place over a period of hours. The administration of parathyroid hormone rapidly suppresses the 25-hydroxyvitamin D₃-24-hydroxylase. The alterations in the hydroxylases by parathyroid hormone or 1,25-dihydroxyvitamin D₃ are not related to changes in serum clacium or phosphate but could be related to changes in intracellular levels of these ions. Actinomycin D or cycloheximide given in vivo reduces the 25-hydroxyvitamin D₃-24-hydroxylase activity rapidly which suggests that the turnover of the enzyme and its messenger RNA is rapid (1- and 5-h half-life, respectively). The half-lives of the hydroxylases are sufficiently short to permit a consideration that the regulation by 1,25-dihydroxyvitamin D₃ and parathyroid hormone may involve enzyme synthesis and degradation.     

Quantitation of vitamin D and its metabolites and their plasma concentrations in five species of animals

Chromatographic methods suitable for the resolution of 24,25-dihydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂, 25-hydroxyvitamin D₃-26,23 lactone, and 25,26-dihydroxyvitamin D₂ are described. These four metabolites comigrated in high-pressure liquid chromatography on silicic acid columns developed in 11:89 isopropanol:hexane. Adequate resolution was achieved by subjecting the four-metabolite complex to high-pressure liquid chromatography column developed in 2:98 isopropanol:methylene chloride. This additional chromatographic step, coupled with modifications of assay procedures previously described, allowed for the estimation of plasma concentrations of vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂, 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₂, 24,25-dihydroxyvitamin D₃, 25,26 dihydroxyvitamin D₂, 25,26-dihydroxyvitamin D₃, 25-hydroxyvitamin D₃-26,23 lactone, and 1,25-dihydroxyvitamin D (1,25-dihydroxyvitamin D₂ plus 1,25-dihydroxyvitamin D₃). The samples automatically were introduced onto the high-pressure liquid chromatography columns with a Waters 710A “intelligent” processor. The metabolites were automatically collected with the aid of a programmable timer that advanced a fraction collector at predetermined intervals. The assays were used to determine the plasma vitamin D and vitamin D metabolite concentrations in five species of adult farm animals.     

Inhibition of vitamin D metabolism by ethane-1-hydroxyl-1, 1-diphosphonate

The administration of disodium-ethane-1-hydroxy-1,1-diphosphonate (20 mg/kg body weight subcutaneously) to chicks given adequate amounts of vitamin D₃ causes a hypercalcemia, inhibits bone mineralization, and inhibits intestinal calcium transport. The administration of 1,25-dihydroxyvitamin D₃, a metabolically active form of vitamin D₃, restores intestinal calcium absorption to normal but does not restore bone mineralization in disodium-ethane-1-hydroxy-1,1-diphosphonate-treated chicks. In rachitic chicks, the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment does not further reduce the low intestinal calcium transport values while it nevertheless further reduces bone ash levels and increases serum calcium concentration.These observations prompted a more detailed study of the relationship between disodium-ethane-1-hydroxy-1,1-diphosphonate treatment and vitamin D metabolism. A study of the hydroxylation of 25-hydroxyvitamin D₃ in an in vitro system employing kidney mitochondria from chicks receiving disodium-ethane-1-hydroxy-1,1-diphosphonate treatment demonstrates a marked decrease in 1,25-dihydroxyvitamin D₃ production and a marked increase in the 24,25-dihydroxyvitamin D₃ production. In addition, the in vivo metabolism of 25-hydroxy-[26,27-³H]vitamin D₃ in disodium-ethane-1-hydroxy-1,1-diphosphonate treated chicks supports the in vitro observations. In rachitic chicks the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment markedly reduces the 25-hydroxyvitamin D₃-1-hydroxylase activity of kidney, but does not increase the 25-hydroxyvitamin D₃-24-hydroxylase.These results provide strong evidence that large doses of disodium-ethane-1-hydroxy-1,1-diphosphonate produce a marked effect on calcium metabolism via alterations in the metabolism of vitamin D as well as the expected direct effect on the bone.     

Studies on the mode of action of calciferol. X. 24-Nor-25-hydroxyvitamin D3, an analog of 25-hydroxyvitamin D3 having "anti-vitamin" activity.

24-Nor-25-hydroxyvitamin D₃, an analog of 25-hydroxyvitamin D₃, has been chemically synthesized in six steps. This steroid was tested in chicks, $\text{in vivo}$, for its ability to generate the classic vitamin D mediated responses of stimulation of intestinal calcium transport and bone calcium mobilization. Although the 24-nor-25-OH-vitamin D₃ itself exhibited no biological activity in these assays, the analog was found to inhibit the normal responses produced by a physiological dose of vitamin D₃. These results suggest that 24-nor-25-OH-vitamin D₃ may satisfy certain requirements expected of a calciferol “anti-vitamin.”     

Synthesis of 24S and 24R-hydroxy-[24-3H]vitamin D3 and their metabolism in rachitic rats.

An epimeric mixture of 24-hydroxy-[24-₃H]vitamin D₃ was synthesized by the reduction of 24-ketovitamin D₃ by sodium borotritide. The epimeric mixture was converted to the trimethylsilylether derivatives and subjected to high-pressure liquid chromatography using silica gel columns to separate the 24-hydroxy-[24-₃H]vitamin D₃ isomers. The 24R-hydroxy-[24-₃H] vitamin D₃ induced calcification in rachitic rats while the 24S-hydroxy-[24-₃H] vitamin D₃ had little or no such activity. As both isomers of 24-hydroxy-vitamin D₃ are metabolized to 24,25-dihydroxyvitamin D³, it appears that the 24-hydroxyvitamin D₃-25-hydroxylase does not discriminate between the isomers. Only the R-isomer of 24-hydroxyvitamin D₃ is metabolized to 1,24-dihydroxyvitamin D₃, although only trace amounts of this compound were found 2 days after the administration of 24-hydroxyvitamin D₃. The striking difference in the metabolism of the isomers is the high selectivity of the 1-hydroxylase for R-isomer. It is suggested that the high specificity of biological activity for the R-isomer of 24-hydroxyvitamin D₃ is because of the specificity of the 1-hydroxylation of 24,25-dihydroxyvitamin D₃ for the R configuration.     

Biological activity of 1 alpha-hydroxyvitamin D2 in the rat.

The biological activity of 1α-hydroxyvitamin D₂ has been determined in vitamin D-deficient rats. In the calcification of the rachitic epiphyseal plate, 1α-hydroxyvitamin D₂ is more active than 25-hydroxyvitamin D₃, while it is equally active in stimulating intestinal calcium absorption. On the other hand, it is much less active (one-third to one-fifth) than 25-hydroxyvitamin D₃ in the mobilization of calcium from bone. In both the intestinal and bone responses, 1α-hydroxyvitamin D₂ (312 pmol) is active in nephrectomized rats while 25-hydroxyvitamin D₃ is not.     

The vitamin D system in iguanian lizards

The apparent plasma concentration of vitamin D binding protein (DBP) in an iguanian lizard, Pogona barbata, and the affinity of this protein for 25-hydroxyvitamin D₃ (25(OH)D₃), 25-hydroxyvitamin D₂ (25(OH)D₂), and 1,25-dihydroxyvitamin D₃ (1,25(OH)D₃) was found to resemble more closely that of the domestic hen than that of the human. The characteristics of Pogona DBP, the pattern of vitamin D metabolites derived from injected radioactive vitamin D₃ and the plasma concentrations of endogenous 25-hydroxyvitamin D (25(OH)D) in a range of iguanian lizards have been examined. The findings suggest that 25-hydroxyvitamin D (25(OH)D) is the major metabolite of vitamin D, and that it may represent the storage form of vitamin D in these species in the same way as in mammals. High concentrations of vitamin D within iguanian embryos and egg yolks suggest a role for this compound in embryogenesis in these species, and perhaps indicates that there is a mechanism for vitamin D delivery to eggs comparable to that found in the domestic chicken.     

Acute Erythemal Ultraviolet Radiation Causes Systemic Immunosuppression in the Absence of Increased 25-Hydroxyvitamin D3 Levels in Male Mice

Vitamin D is synthesised by ultraviolet (UV) irradiation of skin and is hypothesized to be a direct mediator of the immunosuppression that occurs following UV radiation (UVR) exposure. Both UVR and vitamin D drive immune responses towards tolerance by ultimately increasing the suppressive activities of regulatory T cells. To examine a role for UVR-induced vitamin D, vitamin D₃-deficient mice were established by dietary vitamin D₃ restriction. In comparison to vitamin D₃-replete mice, vitamin D₃-deficient mice had significantly reduced serum levels of 25-hydroxyvitamin D₃ (25(OH)D₃, <20 nmol.L⁻¹) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃, <20 pmol.L⁻¹). Following either acute erythemal UVR, or chronic sub-erythemal UVR (8 exposures over 4 weeks) treatment, serum 25(OH)D₃ levels significantly increased in vitamin D₃-deficient female but not male mice. To determine if UVR-induced vitamin D was a mediator of UVR-induced systemic immunosuppression, responses were measured in mice that were able (female) or unable (male) to increase systemic levels of 25(OH)D₃ after UVR. Erythemal UVR (≥4 kJ/m²) suppressed contact hypersensitivity responses (T helper type-1 or -17), aspects of allergic airway disease (T helper type-2) and also the in vivo priming capacity of bone marrow-derived dendritic cells to a similar degree in female and male vitamin D₃-deficient mice. Thus, in male mice, UVR-induced 25(OH)D₃ is not essential for mediating the immunosuppressive effects of erythemal UVR.     

The influence of substitution at C24 on the calcium-binding protein-stimulating activity of vitamin D metabolites in chick embryonic duodenum

The production of calcium-binding protein, in vitro, by embryonic chick duodenum has been used to assess the potency of vitamin D compounds. The introduction of an hydroxyl on 1-, 25-, or 24R-position enhanced biological activity while the introduction of both 1α- and 25-hydroxyls produced maximal activity. However 24R-hydroxylation of 1,25-dihydroxyvitamin D₃ diminished activity. The vitamin D₂ side chain on 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D did not greatly diminish activity in contrast to the fact that the vitamin D₂ compounds are 10% as active as the vitamin D₃ compounds in vivo in the chick. These results support the idea that the target organs of the chick do not discriminate against the vitamin D₂ side chain and that the discrimination in this species is at the level of metabolism.