Synthesis of 25-hydroxy[23,24-3H]vitamin D3

A synthesis of 25-hydroxy[23,24-³H]vitamin D₃ leading to a radiochemically pure product with a specific acitivity of 78 Ci/mmol is described. The structure of the product was confirmed by comparison with unlabeled material and its biological activity was established by in vitro conversion to 1α,25-dihydroxy[23,24-³H]vitamin D₃ using the chick kidney 1α-hydroxylase system.     

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.     

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.     

Studies on vitamin D3 metabolism. Discrete liver cytosolic binding proteins for vitamin D3 and 25-hydroxyvitamin D3

Two separate liver cytosolic proteins have been partially purified and identified by their selectivity for binding either [1α,2α(n)-³H]vitamin D₃ or 25-hydroxy [26(27)-methyl-³H]vitamin D₃. The chromatographic properties of the two proteins were not distinguishable by ion-exchange nor were they dependent upon the vitamin D₃ nutritional status of the birds. However, in molecular exclusion chromatography, the binding proteins can be successfully resolved into two discrete entities. Their binding properties suggest that they are not identical with plasma vitamin D₃ binding protein.     

Does estradiol stimulate in vivo production of 1,25-dihydroxyvitamin D3 in the rat?

Female rats were injected with estradiol benzoate (3 mg/kg daily) or corn oil for 8 days before receiving 325 picomoles of 25-[26,27³H]-hydroxyvitamin D₃ intravenously. Eighteen hours later lipids were extracted from plasma, gut mucosa and kidneys and vitamin D₃ metabolites were separated using Sephadex LH-20 and chloroform: hexanes (65:35) column chromatography. Estradiol treatment increased 1,25-[26,27-³H]-dihydroxyvitamin D₃ content in all three isssues.     

Parathyroid hormone is not involved in 1,25-dihydroxyvitamin D regulation of 25-hydroxyvitamin D metabolism in vivo

1,25-Dihydroxyvitamin D₃ administration to vitamin D-deficient rats suppresses accumulation of 1,25-dihydroxy-[3α-³H]vitamin D₃ and stimulates accumulation of 24,25-dihydroxy-[3α-³3H]vitamin D₃ from 25-hydroxy-[3α-³H]vitamin D₃ equally well in the presence and absence of parathyroid glands. These results demonstrate that this regulatory action is not mediated by the parathyroid glands and support conclusions from $\text{in}\text{vitro}$ studies that this represents a direct action of 1,25-dihydroxyvitamin D₃.     

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.     

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.     

Isolation,identification, and biological activity of 23,25,26-trihydroxyvitamin D3, an in vitro and in vivo metabolite of vitamin D3

Kidney homogenates from vitamin D₃-supplemented chicks incubated with 25-hydroxyvitamin D₃ [25(OH)D₃] produce significant quantities of a new, unknown vitamin D metabolite. This metabolite was isolated in pure form from such incubation mixtures by using Sephadex LH-20 column chromatography followed by high-pressure liquid chromatography. This metabolite has been identified as 23,25,26-trihydroxyvitamin D₃ [23,25,26(OH)₃D₃] by loss of radioactivity from 25-hydroxy[23,24-³H]vitamin D₃ and 25-hydroxy-[26,27-methyl-³H]vitamin D₃, ultraviolet absorption spectrophotometry, mass spectrometry, and periodate cleavage oxidation followed by mass spectrometry. This same metabolite was also isolated from the serum of rats given large doses of vitamin D₃, and structurally characterized as 23,25,26-trihydroxyvitamin D₃. As yet, the stereochemistry at the C-23 and C-25 positions of the natural product remains unknown. A comparison of responses to a single dose level (500 ng) of 23,25,26(OH)₃D₃ or 25(OH)D₃ over 96 h in vitamin D-deficient rats indicated that the new metabolite had no capability to mediate bone calcium mobilization and that it was only weakly active in stimulating intestinal calcium transport.     

Increased biological potency of hexafluorinated analogs of 1,25-dihydroxyvitamin D3 on bovine parathyroid cells

1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is known to be involved in regulating the proliferation of parathyroid cells and PTH synthesis through reactions involving its nuclear receptor. We evaluated the effects of 1,25-(OH)₂D₃ and its hexafluorinated analog, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D₃ (26,27-F₆-1,25-(OH)₂D₃), on parathyroid cells. The 1,25-(OH)₂D₃ and 26,27-F₆-1,25-(OH)₂D₃ each inhibited [³H]thymidine incorporation and ornithine decarboxylase (ODC) activity, which is important in cell proliferation, in primary cultured bovine parathyroid cells. The inhibitory effect of 26,27-F₆-1,25-(OH)₂D₃ on PTH secretion from parathyroid cells was significantly more potent than that of 1,25-(OH)₂D ₃ between 10⁻¹¹ M and 10⁻⁸ M. Study of 26,27-F₆-1,25-(OH)₂D₃ metabolism in parathyroid cells in vitro elucidated its slower degradation than that of 1,25-(OH)₂D₃. After 48 h of incubation with [1β-³H]26,27-F₆-1,25-(OH)₂D₃, two HPLC peaks, one for [1β-³H]26,27-F₆-1,25-(OH)₂D₃, and a second larger peak for [1β-³H]26,27-F₆-1,23(S),25-(OH)₃D₃, were detected. No metabolites were detected after the same period of incubation with 1,25-(OH)₂[26,27-³H]D₃. We observed that 26,27-F₆-1,23(S),25-(OH)₃D₃ was as potent as 1,25-(OH)₂D₃ in inhibiting the proliferation of parathyroid cells.Data suggest that the greater biological activity of 26,27-F₆-1,25-(OH)₂D₃ is explained by its slower metabolisms and by the retention of the biological potency of 26,27-F₆-1,25-(OH)₂D₃ even after 23(S)-hydroxylation.     

Characteristics of [3H]1 alpha, 25-(OH)2D3 binding to nuclear fractions from rat pituitary adenoma GH3 cells

Specific high affinity binding sites for [³H]1α, 25-dihydroxy-vitamin D₃ were observed in nuclear fractions of rat pituitary adenoma GH₃ cells. Crude nuclear (P1) sites demonstrated a pharmacological specificity for vitamin D₃ metabolites and analogues that was in accord with the characteristics of 1α, 25-dihydroxyvitamin D₃ receptors in recognized target organs. GH₃ cells grown in serum-containing medium contained significant amounts of 1α, 25-dihydroxy-vitamin D₃ in a P1 extract, whereas no 1α, 25-dihydroxyvitamin D₃ was detectable in P1 extracts from cells cultured in the absence of serum. Binding of [³H]1α, 25-dihydroxyvitamin D₃ to the P1 fraction was unaffected by prior depletion of intracellular 1α, 25-dihydroxyvitamin D₃, suggesting that association of [³H]1α, 25-dihydroxyvitamin D₃ to nuclear sites is not attributable to translocation of a cytosolic hormone-receptor complex and molecular exchange. The results support the concept that 1α, 25-dihydroxyvitamin D₃ has a physiological role in mediating pituitary hormone secretion.     

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.     

Synthesis of 1α-[2-H]Hydroxyvitamin D3

1α-[2-³H]Hydroxyvitamin D₃ was synthesized chemically. The preparation was radiochemically pure and had a high enough specific activity (4.2 Ci/mmol) to permit experiments using 62.5 pmol/rat. This preparation had as much effect as synthetic 1α-hydroxyvitamin D₃ in increasing the serum Ca level of vitamin D-deficient rats.     

Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples

C-3-substituted 25-hydroxyvitamin D₃ analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3β-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue–DBP complex. When compared to 25-hydroxyvitamin D₃-DBP, the binding of some antibodies to the analogue–DBP complex of the 25-hydroxyvitamin D₃ derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-³H]-25-hydroxyvitamin D₃ from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.     

Chemical synthesis of [1β-3H]1α,25-dihydroxyvitamin D3 and [1α-3H]1β,25-dihydroxyvitamin D3: Biological activity of 1β,25-dihydroxyvitamin D3

The simple three-step preparation of [1β-³H]1α,25-dihydroxyvitamin D₃ and [1α-³H]1β,25-dihydroxyvitamin D₃ from 1α,25-dihydroxyvitamin D₃ is described. In the rat, 1β,25-dihydroxyvitamin D₃, when compared with its α-epimer, did not stimulate intestinal calcium transport or bone calcium mobilization at doses 1000-fold higher than the doses of the natural hormone, 1α,25-dihydroxyvitamin D₃.     

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.     

Synthesis of 25-hydroxy-[26,27-3H]vitamin D2, 1,25-dihydroxy-[26,27-3H]vitamin D2 and their (24R)-epimers

Synthesis of a C-24-epimeric mixture of 25-hydroxy-[26,27-3H]vitamin D2 and a C-24-epimeric mixture of 1,25-dihydroxy-[26,27-3H]vitamin D2 by the Grignard reaction of the corresponding 25-keto-27-nor-vitamin D2 and 1 alpha-acetoxy-25-keto-27-nor-vitamin D3 with tritiated methyl magnesium bromide is described. Separation of epimers by high-performance liquid chromatography afforded pure radiolabeled vitamins of high specific activity (80 Ci/mmol). The identities and radiochemical purities of 25-hydroxy-[26,27-3H[vitamin D2 and 1,25-dihydroxy-[26,27-3H]vitamin D2 D2 were established by cochromatography with synthetic 25-hydroxyvitamin D2 or 1,25-dihydroxyvitamin D2. Biological activity of 25-hydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the rat plasma binding protein for vitamin D compounds, and by its in vitro conversion to 1,25-dihydroxy-[26,27-3H]vitamin D2 by kidney homogenate prepared from vitamin D-deficient chickens. The biological activity of 1,25-dihydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3.     

Side chain oxidation of 1,25-dihydroxyvitamin D3 in the rat: Effect of removal of the intestine

Removal of the jejunum, ileum and colon in the rat reduces the amount of ¹⁴CO₂ formed after an intravenous injection of 325 pmoles of 1,25-dihydroxy-[26,27-¹⁴C]vitamin D₃, by 65.2 ± 13.2% at 4 hours and 67.1 ± 9.12% at 8 hours. This suggests that the intestine may be one of the sites where side chain oxidation occurs. It is possible that the liver may also be involved in this process as removal of a large portion of the gut may disturb hepatic metabolism secondary to a reduction in portal blood flow. The process is not bacterial inasmuch as “germ free” animals produce at least as much ¹⁴CO₂ after the administration of 1,25-dihydroxy-[26,27-¹⁴C]vitamin D₃ as do non-germ free controls.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

The synthesis of [26,27-C]dihydroxyvitamin D3, a tracer for positron emission tomography (PET)

1α,25-Dihydroxyvitamin D₃, an endogenous ligand with the highest affinity for the vitamin D receptor (VDR), was labeled with ¹¹C for use in biological experiments. The radionuclide was incorporated via the reaction of [¹¹C]methyllithium on a methyl ketone precursor in tetrahydrofuran at −10 °C. Deprotection of the labeled intermediate yielded 2.5–3 GBq [26,27-¹¹C]1α,25-dihydroxyvitamin D₃ [¹¹C-1,25(OH)₂ D₃] with specific radioactivity averaging 100 GBq/μmol at the end of synthesis and HPLC purification. The entire process took 48 min from the end of radionuclide production. In vitro binding experiments in rachitic chick purified VDR demonstrated the high affinity binding of this novel tracer. Thus; ¹¹C-1,25(OH)₂ D₃ is available for in vivo distribution studies and may be suitable for the positron emission tomography (PET) determination of VDR levels and occupancy in animals and humans.