Synthesis, biologic activity, and protein binding characteristics of a new vitamin D analog, 22-hydroxyvitamin D3

We synthesized a novel vitamin D analog, 22-hydroxyvitamin D3 9 and tested its biologic activity (and antivitamin properties) in vivo in vitamin D-deficient rats, and in vitro in the chick embryonic duodenum. We examined its ability to bind to the sterol carrier protein, vitamin D binding protein and the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. The new vitamin 9 was synthesized from 3 beta-hydroxy-22,23-dinorcholenic acid 1 in 12 steps. The vitamin 9 displayed no vitamin D agonist activity in the intestine or in bone in vivo and did not block the activity of vitamin D3 or 25-hydroxyvitamin D3. It was a weak vitamin D3 agonist in the chick embryonal duodenum in vitro. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. Vitamin 9 bound to the chick intestinal cytosol receptor with low affinity. 22-Hydroxyvitamin D3 and various vitamin D sterols were bound to vitamin D binding protein in the following order: 25-hydroxyvitamin D3. (24R)-24,25-dihydroxyvitamin D3, and (25S)-25,26-dihydroxyvitamin D3 greater than 22-hydroxyvitamin D3 greater than 11 alpha-hydroxyvitamin D3 greater than 1,25-dihydroxyvitamin D3 greater than vitamin D3. We conclude that the introduction of a hydroxyl group at C-22 in the side chain of the vitamin D3 molecule decreases its biological activity.     

Characterization of a specific, high affinity binding macromolecule for 1 alpha, 25-dihydroxyvitamin D3 in cultured chick kidney cells

Cytosol prepared from vitamin D3-deficient kidney cells in culture contains a 3.7 S protein that specifically binds 1,25-dihydroxyvitamin D3 with high affinity and low capacity. Whole kidney homogenate cytosol preparations are shown to possess two 1,25-dihydroxyvitamin D3 binding macromolecules. One of the binding proteins sediments at 3.5 to 3.7 S while the second sediments at 6.0 S. The 6.0 S component has a greater affinity for 25-dihydroxyvitamin D3 than for 1,25-dihydroxyvitamin D3. Cultured cell cytosol was found to have little 6.0 S 25-hydroxyvitamin D3 binding protein. Scatchard analysis of the cultured cell cytosol reveals an equilibrium binding constant (KD) of 5.6 x 10 (-11) with 57 fmol of sites/mg of protein. The receptor-like protein has a Mr = 72,000 and as with other steroid receptors it aggregates in the presence of low potassium concentrations. Analog competition for receptor binding reveals the following potency order: 1,25-dihydroxyvitamin D3 > 25-hydroxyvitamin D3 > 1 alpha-hydroxyvitamin D3 > 24(R),25-dihydroxyvitamin D3; the receptor had no detectable affinity for vitamin D3. The kidney cells respond to 1,25-dihydroxyvitamin D3 by diminishing 25-hydroxyvitamin D3 1 alpha-hydroxylation and increasing 24R-hydroxylation. Cultured cells provide a preparation of cytosol which has allowed extensive characterization of the renal 1,25-dihydroxyvitamin D3 receptor and should facilitate investigations into the role this receptor plays in renal control of vitamin D3 metabolism.     

Characterization of 25-hydroxyvitamin D binding protein from intestinal cells

We have previously purified a cytosolic vitamin D metabolite binding protein (cDBP) from rat enterocytes, which has characteristics distinct from other vitamin D binding proteins. In these studies, we demonstrate that cDBP in a semi-purified fraction from human intestinal cells (Caco-2 cells) binds 25-hydroxyvitamin D (25OHD) with at least a 1000-fold greater affinity than 1, 25-dihydroxyvitamin D (1,25(OH)(2)D) or 24,25-dihydroxyvitamin D. Treatment of cells with 1,25(OH)(2)D reduced 25OHD binding to approximately one third that of the untreated cells (0.42 CPM/mg total protein vs 1.34 CPM/mg total protein, respectively). Finally, the cDBP is not immunoreactive to antibodies prepared against the C-terminus of the nuclear vitamin D receptor (VDR). In summary, cDBP bound 25OHD with greater affinity than either 1,25(OH)(2)D or 24,25 dihydroxyvitamin D, the cytosolic binding activity was down-regulated by 1,25(OH)(2)D and cBDP is distinct from the nuclear VDR.     

Support of embryonic chick survival by vitamin D metabolites

The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.     

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.     

23-Keto-25-hydroxyvitamin D3: a vitamin D3 metabolite with high affinity for the 1,25-dihydroxyvitamin D specific cytosol receptor

A new metabolite of 23,25-dihydroxyvitamin D3 has been generated with kidney homogenates prepared from vitamin D treated chicks. The metabolite was purified with three high-performance liquid chromatographic steps and was identified as 23-keto-25-hydroxyvitamin D3 by ultraviolet absorption spectroscopy, mass spectrometry, and chemical reactivity. The R stereoisomer of 23,25-dihydroxyvitamin D3 was 10-fold more effective as an in vitro precursor to 23-keto-25-hydroxyvitamin D3 than was the naturally occurring S stereoisomer. Approximately 500 ng of 23-keto-25-hydroxyvitamin D3 was necessary to produce the same degree of intestinal-calcium transport as 25 ng of vitamin D3--a difference of about 20-fold. 23-Keto-25-hydroxyvitamin D3 was not active at stimulating bone calcium resorption at the doses and times tested. This new vitamin D3 metabolite, however, had greater affinity than 25-hydroxyvitamin D3 to both the rat plasma vitamin D binding protein and the 1,25-dihydroxyvitamin D specific cytosol receptor. Heretofore, only 1 alpha-hydroxylated metabolites of 25-hydroxyvitamin D3 or analogues possessing a pseudo 1 alpha-hydroxy group were known to bind to the 1,25-dihydroxyvitamin D receptor with higher affinity than 25-hydroxyvitamin D3. Ketone formation at the 23 position, therefore, is the first side-chain modification of 25-hydroxyvitamin D3 that results in enhanced binding to the 1,25-dihydroxyvitamin D receptor binding protein.     

End product inhibition of hepatic 25-hydroxyvitamin D production in the rat: specificity and kinetics

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.     

1 alpha-hydroxylation of 24-hydroxyvitamin D2 represents a minor physiological pathway for the activation of vitamin D2 in mammals

C24-Hydroxylation was evaluated as a possible activation pathway for vitamin D2 and vitamin D3. Routine assays showed that 24-hydroxyvitamin D2 and 1,24-dihydroxyvitamin D2 could be detected in rats receiving physiological doses (100 IU/day) of vitamin D2; however, 24-hydroxyvitamin D3 could not be detected in rats receiving similar doses of vitamin D3. In rats, 24-hydroxyvitamin D2 was very similar to 25-hydroxyvitamin D2 at stimulating intestinal calcium transport and bone calcium resorption. The biological activity of 24-hydroxyvitamin D2 was eliminated by nephrectomy, suggesting that 24-hydroxyvitamin D2 must undergo 1 alpha-hydroxylation to be active at physiological doses. In vivo experiments suggested that when given individually to vitamin D deficient rats, 24-hydroxyvitamin D2, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 were 1 alpha-hydroxylated with the same efficiency. However, when presented simultaneously, 24-hydroxyvitamin D2 was less efficiently 1 alpha-hydroxylated than either 25-hydroxyvitamin D3 or 25-hydroxyvitamin D2. 1,24-Dihydroxyvitamin D2 was also approximately 2-fold less competitive than either 1,25-dihydroxyvitamin D2 or 1,25-dihydroxyvitamin D3 for binding sites on the bovine thymus 1,25-dihydroxyvitamin D receptor. These results demonstrate that 24-hydroxylation followed by 1 alpha-hydroxylation of vitamin D2 represents a minor activation pathway for vitamin D2 but not vitamin D3.     

The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitamin D3 and 1,25-dihydroxy-26,27-dimethylvitamin D3: highly potent novel analogs of vitamin D3

We synthesized 25-hydroxy-26,27-dimethylvitamin D3, 9, and 1,25-dihydroxy-26,27-dimethylvitamin D3, 14, from chol-5-enic acid-3 beta-ol and tested their biological activity in vivo and in vitro. 9 was found to be highly potent vitamin D analog with bioactivity similar to that of 25-hydroxyvitamin D3. 9 bound to rat plasma vitamin D binding protein with approximately one-third the affinity of 25-hydroxyvitamin D3. In a duodenal organ culture system and in a competitive binding assay with chick intestinal 1,25-dihydroxyvitamin D receptor, 9 was significantly more potent than 25-hydroxyvitamin D3. 1,25-Dihydroxy-26,27-dimethylvitamin D3, 14 was also highly active in vivo. At doses of 1000-5000 pmol/rat, its action was more sustained than that of 1,25-dihydroxyvitamin D3. 14 bound to vitamin D binding protein about 18 times less effectively than 1,25-dihydroxyvitamin D3. 14 bound to the chick intestinal cytosol receptor with an affinity one-half that of 1,25-dihydroxyvitamin D3. In a duodenal organ culture system, 14 was about half as active as 1,25-dihydroxyvitamin D3. Extension of the sterol side chain, at C-26 and C-27, by methylene groups, prolongs the bioactivity of a vitamin D sterol hydroxylated at C-1 and C-25; the corresponding sterol, hydroxylated only at C-25, does not show any alteration of its bioactivity in vivo. These newly synthesized analogs may potentially be of therapeutic use in various mineral disorders.     

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 vitamin D3 in mouse kidney mitochondria: effect of the Hyp mutation and 1,25-dihydroxyvitamin D3 treatment

Side-chain oxidation of vitamin D is an important degradative pathway. In the present study we compared the enzymes involved in side-chain oxidation in normal and Hyp mouse kidney. Homogenates of normal mouse kidney catalyze the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3. After subcellular fractionation, total side-chain oxidative activity, estimated by the sum of the three products synthesized per milligram protein under initial rate conditions, coincided with the mitochondrial enzyme marker succinate-cytochrome-c reductase. Treatment of normal mice with 1,25-dihydroxyvitamin D3 (1.5 ng/g) resulted in an eightfold increase in mitochondrial enzyme activity, with no change in apparent Km but a significant rise in Vmax. With 24,25-dihydroxyvitamin D3 as the substrate, normal renal mitochondria produced 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and the synthesis of these metabolites could be increased sixfold by pretreatment with 1,25-dihydroxyvitamin D3. In the Hyp mouse, the side-chain oxidation pathway showed similar subcellular distribution of enzyme activity. However, product formation from 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 was twofold greater in mutant than in normal mitochondria. Furthermore, 1,25-dihydroxyvitamin D3 pretreatment of Hyp mice resulted in a 3.4-fold increase over basal metabolism of both 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. These results demonstrate that (i) kidneys from normal and Hyp mice possess basal and 1,25-dihydroxyvitamin D3 inducible enzyme system(s) in the mitochondrial fraction, which catalyze the side-chain oxidation of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, and (ii) the Hyp mutation appears to perturb the renal metabolism of both substrates only in the basal state.     

Differential effects of Vitamin D analogs on calcium transport

It is well known that the efficiency of intestinal active calcium transport is regulated by the Vitamin D receptor pathway and Vitamin D analogs seem to exhibit differential effects on intestinal active calcium transport. To investigate the molecular basis for the difference among Vitamin D analogs, we tested three Vitamin D analogs: 1,25-dihydroxyvitamin D(3), 19-nor-1,25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) ex vivo and in vitro. In 5/6 nephrectomized rat intestinal active calcium transport, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effects on intestinal active calcium transport at all the concentrations tested, while 1alpha-hydroxyvitamin D(2) at 0.33 and 0.67 microg/kg and 1,25-dihydroxyvitamin D(3) at 1microg/kg significantly stimulated calcium transport. In Caco-2 cells, 19-nor-1,25-dihydroxyvitamin D(2) did not show a significant effect on calcium transport, while 1,25-dihydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(2) (the active form of 1alpha-hydroxyvitamin D(2)) stimulated calcium transport by 934 and 501% at 0.1microM, respectively. 1,25-Dihydroxyvitamin D(2) potently induced the expression of CALB3 and TRPV6 mRNA with an EC(50) of 0.3 and 1.0nM, whereas 19-nor-1,25-dihydroxyvitamin D(2) was 10-fold less potent than 1,25-dihydroxyvitamin D(2) in inducing CALB3 and TRPV6 mRNA. The three Vitamin D analogs had no significant effect on the expression of PMCA1 mRNA. These Vitamin D analogs did not change the expression of Vitamin D receptor (VDR) up to 10nM, but stimulated CYP24A1 expression in a dose-dependent manner with the potency in the order of 1,25-dihydroxyvitamin D(3)>1,25-dihydroxyvitamin D(2)=19-nor-1,25-dihydroxyvitamin D(2). These results suggest that the differential effect of Vitamin D analogs on stimulating intestinal and Caco-2 calcium transport may be in part due to its different effect on stimulating CALB3 and TRPV6 mRNA expression.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

1,25-Dihydroxyvitamin D3 binds specifically to rat vascular smooth muscle cells and stimulates their proliferation in vitro

Cultured vascular smooth muscle cells (VSMC) derived from rat aorta were found to contain a specific receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Its Kd (5.0 x 10(-11) M) and capacity (22.9 fmol/mg of cytosol protein) for 1,25-(OH)2D3, its sedimentation coefficient on a sucrose density gradient (3.2 S), its relative affinities for various vitamin D3 metabolites [1,25-(OH)2D3 greater than 25-hydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3 greater than vitamin D3] and its affinity for DNA-cellulose were similar to those reported for the 1,25-(OH)2D3 receptor in other tissues. 1,25-(OH)2D3 at concentrations of more than 10(-10) M caused dose-dependent enhancement of the proliferation of VSMC in DMEM with 10% FCS. 25-Hydroxyvitamin D3 stimulated the proliferation of VSMC only at its highest concentration tested (10(-6) M). These data show that 1,25-(OH)2D3 stimulates the proliferation of VSMC after its binding to a cytoplasmic receptor of the cells in vitro, and support the possibility that VSMC are target cells of the hormone.     

C-6 functionalized analogs of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3: synthesis and binding analysis with vitamin D-binding protein and vitamin D receptor.

In this article, we describe the development of a general synthetic strategy to functionalize the C-6 position of vitamin D3 and its biologically important metabolites, i.e. 25-hydroxyvitamin D3 (25-OH-D3) and 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We employed Mazur's cyclovitamin D method to synthesize vitamin D3 analogs with several functionalities at the C-6 position. In addition, we synthesized 6-(3-hydroxypropyl) and 6-[(2-bromoacetoxy)propyl] derivatives of 25-OH-D3 15 and 16, respectively, and 6-(3-hydroxypropyl) derivative of 1,25(OH)2D3 17. Competitive binding assays of 15-17 with human serum vitamin D-binding protein showed that all these analogs specifically bound to this protein, although with significantly lower affinity than the 25-OH-D3, the strongest natural binder, but with comparable affinity with 1,25(OH)2D3, the hormone. On the other hand, 6-[3-hydroxypropyl], 1alpha,25-dihydroxyvitamin D3 17 did not show any specific binding for recombinant nuclear vitamin D receptor. These results indicated that the region containing the C-6 position of the parent seco-steroid [1,25(OH)2D3] may be an important recognition marker towards vitamin D receptor binding. Information, delineated in this article, will be important for evaluating structure-activity relationship in synthetic analogs of vitamin D and its metabolites.     

Intestinal 1,25-dihydroxyvitamin D3 binding protein: Specificity of binding

The binding of metabolites of vitamin D and their analogs to the 3.7S chick intestinal cytosol receptor protein has been specifically studied by competitive binding techniques and polyethylene glycol precipitation of the complex. The structural requirements for the interaction between the vitamin D molecule and the receptor could be assessed without the nuclear chromatin binding step. These measurements have shown that 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ are equally competitive and are the most active. Of the structural features of the compounds, the 1α-hydroxyl is most important followed by the 25-hydroxyl and the 3β-hydroxyl. The addition of a second hydroxyl near carbon 25 markedly reduces binding whether on the 26 carbon or the 24 carbon. A hydroxyl on C-24 could substitute to some degree for the 25-hydroxyl inasmuch as 24-hydroxyvitamin D₃ was much more effective than vitamin D₃ but less effective than 25-hydroxyvitamin D₃. In general the patterns of binding affinities correlated well with the biological activity of the various analogs strongly supporting a physiological role for the 1,25-dihydroxyvitamin D₃ binding protein. It also suggests that of the two-step receptor mechanism, the structural specificity is located in the initial interaction of the 1,25-dihydroxyvitamin D₃ and the cytosol receptor.     

Plasma levels of vitamin D metabolites in fetal and pregnant ewes

The plasma concentrations of calcium; inorganic phosphorus; 25-hydroxyvitamin D; 24,25-dihydroxyvitamin D; and 1,25-dihydroxyvitamin D were determined in sheep maternal and fetal arterial circulations. In addition, plasma concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were determined simultaneously across the uterine and umbilical circulations. Fetal arterial levels of calcium (r = 0.560); inorganic phosphorus (r = -0.095); and 1,25-dihydroxyvitamin D (r = 0.040) were significantly higher than and did not correlate with maternal arterial levels. Maternal levels of 25-hydroxyvitamin D were significantly higher than and correlated (r = 0.693) with fetal 25-hydroxyvitamin D levels. No significant difference existed between maternal and fetal arterial levels of 24,25-dihydroxyvitamin D. No significant difference was detected in the concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D across the uterine or umbilical circulations.     

6-Fluoro-vitamin D3: a new antagonist of the biological actions of vitamin D3 and its metabolites which interacts with the intestinal receptor for 1 alpha,25(OH)2-vitamin D3

The biological activity and the binding affinity for the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] intestinal receptor of a new fluorine-containing vitamin D compound, namely 6-fluoro-vitamin D3 (6-F-D3), is reported. A significant interaction of 6-F-D3 with the 1,25(OH)2D3 receptor was found, with a relative competitive index (RCI) of 0.26 +/- 0.04, which is intermediate between 25-hydroxyvitamin D3 (0.14 +/- 0.01) and 1 alpha-hydroxyvitamin D3 (0.46 +/- 0.08), where the RCI of 1,25(OH)2D3 is defined to be 100. In contrast, vitamin D3 was unable to interact with the 1,25(OH)2D3 receptor. Also, the biological activity of 6-F-D3 was assessed in vivo in the vitamin D-deficient chick. 6-F-D3 at doses up to 130 nmol displayed no biological action on either intestinal calcium absorption (ICA) or bone calcium mobilization (BCM) over the time interval of 14-48 h after dosing. However, when 130 nmol 6-F-D3 was given 2 h before and 6 h after vitamin D3 (1.62 nmol), a significant inhibition of vitamin D-mediated ICA was noted. Also, a dose of 130 nmol 6-F-D3 given 2 h before and 6 h after 1,25(OH)2D3 (0.26 nmol) significantly inhibited ICA, as measured at 12 h. 6-F-D3 is the first vitamin D analog found which has an ability to both bind to the 1,25(OH)2D3 receptor and to antagonize the production of biological responses by 1,25(OH)2D3.     

Up-regulation of the intestinal 1,25-dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D2 and vitamin D3

Concentrations of intestinal 1,25-dihydroxyvitamin D receptor were measured in rats receiving pharmacological amounts (25,000 IU/rat daily for 6 days) of either vitamin D2 or vitamin D3. The data showed that both hypervitaminosis D2 and hypervitaminosis D3 resulted in significant up-regulation of intestinal 1,25-dihydroxyvitamin D receptor (fmol/mg protein) relative to controls (409 +/- 24, vitamin D2-treated; 525 +/- 41, vitamin D3-treated; and 249 +/- 19, control). The 1,25-dihydroxyvitamin D receptor enhancement also was accompanied by elevated plasma 25-hydroxyvitamin D and hypercalcemia. These data suggest that increased target-tissue 1,25-dihydroxyvitamin D receptor may play a role in enhancing target-tissue responsiveness and, thus, have a significant role in mediating the toxic effects of hypervitaminosis D.     

The plasma binding protein for vitamin D is a site of discrimination against vitamin D-2 compounds by the chick

The binding of 25-hydroxy-[26,27-3H]vitamin D-3 and 25-hydroxy-[26,27-3H]vitamin D-2 to the vitamin D binding protein in the plasma of both rats and chicks has been studied. In the case of rats, sucrose density gradient analysis, competitive displacement, and Scatchard analysis demonstrate that 25-hydroxyvitamin D-3 and 25-hydroxyvitamin D-2 are bound equally well to the vitamin D binding protein. In contrast, 25-hydroxyvitamin D-2 is poorly bound, while 25-hydroxyvitamin D-3 is tightly bound to the vitamin D binding protein in chick plasma. On the other hand, the chick intestinal receptor binds 1,25-dihydroxyvitamin D-2 and 1,25-dihydroxyvitamin D-3 equally well with a KD of 7.10(-11) M for both compounds. These results strongly suggest that the failure of the plasma transport protein in chicks to bind the vitamin D-2 compounds may be responsible for their relative ineffectiveness in these animals.