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.     

Biological activity of 24,24-difluoro-25-hydroxyvitamin D3. Effect of blocking of 24-hydroxylation on the functions of vitamin D.

To examine the question of whether 24-hydroxylation plays and importance role in the physiological functions of vitamin D, the biological activity of 24,24-difluoro-25-hydroxyvitamin D was compared with that of 25-hydroxyvitamin D in vitamin D-deficient rats. These two compounds were found almost identically active in the stimulation of intestinal calcium transport, the mobilization of calcium from bone, the healing of rachitic epiphyseal plate cartilage, the elevation of serum inorganic phosphorus, the mineralization of rachitic bone, and in the prevention of rachitogenesis in rats. Little or no difference was detected in the time course of response of intestinal calcium transport or bone calcium mobilization to the two forms of vitamin D. Therefore, in the rat no support could be obtained for the idea that 24,25-dihydroxyvitamin D3 plays an important role in the known physiological responses to the vitamin.     

24,24-Difluoro-1,25-dihydroxyvitamin D3: In vitro production,isolation, and biological activity

24,24-Difluoro-1,25-dihydroxyvitamin D₃ has been synthesized by in vitro incubation of vitamin D-deficient chick kidney homogenates with 24,24-difluoro-25-dihydroxyvitamin D₃. The compound produced was isolated and purified by successive high-performance liquid chromatographic steps and then identified by means of ultraviolet absorption spectrophotometry and mass spectrometry. The difluoro analog of 1,25-dihydroxyvitamin D₃ is found to be highly active in stimulating intestinal calcium transport and bone calcium mobilization in vitamin D₃-deficient rats.     

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.     

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.     

Metabolism of 25-hydroxyvitamin D3 in kidney homogenates of chicks supplemented with vitamin D3.

Kidney homogenates from chicks fed a vitamin D-deficient diet for 10 days and supplemented with 6.5 nmol of vitamin D₃ 48 hr prior to sacrifice metabolized $\text{in}\text{vitro}\text{[}{}^3\text{H}\text{]}$-25-hydroxyvitamin D₃ (25-OH-D₃) to 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃] and 3 other metabolites (peaks A, C and E). When the homogenates were incubated with purified [³H]-24,25-(OH)₂-D₃, 3 similar metabolites (peaks A′, C′ and E′) were produced. On high pressure liquid chromatography, peaks A, C and E migrated to exactly the same respective positions as peaks A′, C′ and E′. Kidney homogenates from D-deficient chicks failed to produce these metabolites from [³H]-25-OH-D₃ or [³H]-24,25-(OH)₂-D₃. These results strongly suggest that the new metabolites reported here are synthesized via 24,25-(OH)₂-D₃ in the kidney of chicks supplemented with vitamin D₃.     

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.     

The stimulation of 25-hydroxyvitamin D3-1α-hydroxylase by estrogen

Homogenates of kidney from laying Japanese quail incubated in vitro with 25-hydroxy-[26,27-³H] vitamin D₃ produce more 1,25-dihydroxy-[26,27-³H]vitamin D₃ than do homogenates of kidney from mature nonlaying females or males maintained on the same diet and under identical conditions. Instead, the homogenates from male quail or nonlaying female quail convert 25-hydroxyvitamin D₃ to 24,25-dihydroxyvitamin D₃. The administration of 5 mg of estradiol to mature male quail 24 h prior to sacrifice suppressed the 25-hydroxyvitamin D₃-24-hydroxylase and markedly stimulated 25-hydroxyvitamin D₃-1-hydroxylase. The administration of estradiol to male quail caused hypercalcemia, which responded more slowly than did the 1-hydroxylase. As little as 0.1 mg of estradiol/quail was found effective in stimulating the 1-hydroxylase and suppressing the 24-hydroxylase. Other hormones such as follicle stimulating hormone (FSH), cortisone, testosterone, and progesterone, even at high dose levels, produced little or no change in the 25-hydroxyvitamin D₃-1-hydroxylase. Testosterone did, however, suppress the 25-hydroxyvitamin D₃-24-hydroxylase. The stimulation of the 25-hydroxyvitamin D₃-1-hydroxylase by parathyroid hormone was of a smaller magnitude than that of the estradiol, and the effects of the two hormones were additive, suggesting that they function by a different mechanism.     

A fluorinated vitamin D3 analog with biopotency greater than 1 alpha, 25-dihydroxy vitamin D3

Vitamin D metabolites and analogs induce $\text{de novo}$ synthesis of a specific calcium-binding protein in embryonic chick duodenum maintained in organ culture. Using calcium-binding protein biosynthesis as a specific and sensitive biochemical indicator of intrinsic biopotency, 24,24-difluoro-1α,25-dihydroxy vitamin D₃ was found to be approximately four times more potent on a molar basis than the most active, naturally occurring metabolite, 1α,25-dihydroxy vitamin D₃.     

Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ were synthesized using the Wittig–Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D₃, 1α,25-dihydroxyvitamin D₃ and 1α-hydroxylated vitamin D₃ analogues.     

Biological activities and binding properties of 23,23-difluoro-25-hydroxyvitamin D3 and its 1 alpha-hydroxy derivative

23,23-Difluoro-25-hydroxyvitamin D3 is 5-10 times less active than 25-hydroxyvitamin D3 in stimulating intestinal calcium transport, bone calcium mobilization, increasing serum phosphorus, mineralization of rachitic bone, and binding to the plasma transport protein in rats. It is converted to 23,23-difluoro-1 alpha, 25-dihydroxyvitamin D3 by chick renal 25-hydroxyvitamin D-1-hydroxylase. This compound is one-seventh as active as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3. Thus, fluoro substitution on carbon-23 of vitamin D has an unexpected and unexplained suppressive action on plasma binding and biological activity. However, since this substitution does not block the biological response of 25-hydroxyvitamin D3, these results provide additional evidence that 23-hydroxylation of vitamin D is not involved in biological function.     

Inhibitors of the 25-hydroxylation of vitamin D3 in the rat

Two new sidechain-modified analogs of vitamin D₃, 25-azavitamin D₃ and 25-fluorovitamin D₃, were prepared; both compounds were found to inhibit the in vivo 25-hydroxylation of vitamin D₃ in the rat. 25-Azavitamin D₃ was chemically synthesized from a degradation product of stigmasterol by a six-step process. The desired carbon skeleton was efficiently assembled by alkylation of a suitably protected C-20 bromomethylpregnane with the enolate of N,N-dimethylacetamide (70%). The completion of the synthesis utilized the known photochemistry of steroidal 5,7-dienes to prepare the vitamin D triene system. In contrast, 25-fluorovitamin D₃ was prepared by direct vitamin modification. 25-Hydroxyvitamin D₃ 3-acetate was fluorinated with diethylaminosulfur trifluoride to give 25-fluorovitamin D₃ 3-acetate (59%); saponification provided the desired analog. When vitamin D-deficient rats on a low calcium diet were dosed with [3-³H]vitamin D₃ (0.05 μg), 10% of the dose was found in serum as 25-hydroxyvitamin D₃ 4 hr after administration. If 25-azavitamin D₃ (50 or 200 μg) was given 2 hr before the radiolabeled vitamin D₃, however, serum 25-hydroxyvitamin D₃ concentration was markedly reduced. 25-Fluorovitamin D₃ caused similar reduction when administered at much lower doses.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

Kidney microsomal metabolism of 25-hydroxyvitamin D3+.

Vitamin D₃-deficient chick kidney microsomes $\text{in vitro}$ metabolize 25-hydroxyvitamin D₃ to two polar metabolites by a pathway which may involve side-chain modification. Molecular oxygen and a source of reduced nicotinamide adenine dinucleotide phosphate are required for this metabolism. Kidney cytosol obtained from deficient chicks or kidney microsomes of vitamin D₃-repleted chicks do not metabolize 25-hydroxyvitamin D₃. The two products are tentatively designated MIC-I and MIC-II.     

Biotransformations of 25-hydroxyvitamin D3 by kidney microsomes.

Vitamin D₃-deficient chick kidney microsomes $\text{in}\text{vitro}$ metabolize 25-hydroxy-[26(27)-methyl-³H]-vitamin D₃ to yet structurally unidentified polar metabolites previously designated MIC-I and MIC-II. Kidney microsomes of vitamin D₃-repleted chicks could not be demonstrated to produce these metabolites when ³H was the radioactive isotope in positions C-26 and C-27 of the substrate. However, when 25-hydroxy-[26,27-¹⁴C]-vitamin D₃ was the radioactive substrate, MIC-I and MIC-II production was independent of the vitamin D₃ status of the chicks. These results suggest that under conditions of vitamin D₃-sufficiency, there is augmented sequential kidney metabolism of 25-hydroxyvitamin D₃ to products with modified side-chains involving C-26 and/or C-27. It is possible that this metabolism is responsible for the regulation of kidney cellular concentrations of 25-hydroxyvitamin D₃.     

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.     

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.”     

Effect of alcohol on renal vitamin D metabolism in chickens.

Intraperitoneal administration of ethanol to young chickens (both vitamin D-replete and vitamin D-deficient) produced a significant impairment of renal 25 hydroxyvitamin D₃ 1α-hydroxylase (EC 1.14.13.13) activity with no significant change in serum calcium or phosphorus. In ethanol treated D-replete chicks the renal 25 hydroxyvitamin D₃ 24-hydroxylase activity was enhanced, and serum 25 hydroxyvitamin D₃ was significantly increased. The alkaline phosphatase levels in the D-deficient ethanol treated chicks were significantly less than the controls. Our data suggest that the impairment of the metabolic effects of vitamin D due to ethanol occurs chiefly via a renal, rather than a hepatic mechanism. Furthermore, 1α -hydroxylated metabolites of vitamin D would appear to be the logical treatment of choice for the bone disease of alcoholism.     

Control of kidney 25-hydroxyvitamin D3 metabolism. Strontium and the involvement of parathyroid hormone.

The action of parathyroid extract (PTE) on the renal metabolism of 25-hydroxyvitamin D₃ (25-OHD₃) was evaluated in rat models for strontium rickets and hypoparathyroidism. PTE elevated the production of 1α,25-(OH)₂D₃ and suppressed the synthesis of 24,25-(OH)₂D₃ in both animal models. Part of strontium's action in suppressing 1α,25-(OH)₂D₃ and stimulating 24,25-(OH)₂D₃ synthesis in strontium rickets appears to involve a decrease in parathyroid hormone (PTH) secretion and/or action. Calcitonin (CT) was not implicated in the cation's action. Thyroparathyroidectomized rats showed a low level of 1α,25-(OH)₂D₃ production which increased four- to eightfold following chronic PTE treatment. PTH appears to be the major calcium regulatory hormone involved in modulation of renal 25-OHD₃ metabolism.