On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins.

We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.

Effects of 1,25-dihydroxyvitamin D3 on growth of mouse neuroblastoma cells.

Epitopes of the 1,25-dihydroxyvitamin D(1,25(OH)2D3) receptor have been shown in developing dorsal root ganglia in fetal mice, as well as in cells maintained in culture [Johnson, J.A., Grande, J.P., Windebank, A.J. and Kumar, R., 1,25-Dihydroxyvitamin D3 receptors in developing dorsal root ganglia of fetal rats, Dev. Brain Res., 92 (1996) 120-124]. To investigate a possible role for 1,25(OH)2D3 in neural cell growth and development, a murine neuroblastoma cell line that expresses 1,25(OH)2D3 receptors, was treated with 1,25(OH)2D3. Treatment with 1,25(OH)2D3 resulted in a decrease in cell proliferation, a change in cell morphology, and the expression of protein markers of mature neuronal cells. The decrease in cell proliferation was accompanied by an increase in the expression of nerve growth factor (NGF). Anti-NGF monoclonal antibody added to the growth medium blocked the decrease in cell proliferation caused by 1,25(OH)2D3 treatment. Our results show that the sterol hormone 1,25(OH)2D3, causes a decrease in the proliferation of mouse neuroblastoma cells through alterations in the expression of NGF.

Inhibitors of renal epithelial phosphate transport in tumor-induced osteomalacia and uremia.

Tumors such as sclerosing hemangiomas are sometimes associated with hypophosphatemia and osteomalacia, both of which disappear on removal of the tumor. We identified a heat labile, 8,000-25,000 dalton, inhibitor of renal epithelial phosphate transport in supernatants of cultured sclerosing hemangioma cells obtained from a patient with oncogenic osteomalacia and hypophosphatemia. The inhibitor does not alter glucose or alanine transport in renal epithelial cells, and has a mechanism of cellular action distinct from that of parathyroid hormone (PTH) in that it inhibits phosphate transport in renal epithelia without increasing concentrations of cyclic 3',5' adenosine monophosphate (cAMP); it's activity is not blocked by a PTH receptor antagonist. Sclerosing hemangioma cells also produce a material that cross-reacts with antisera directed against PTH and tumor tissue sections immunostain with PTH antibodies. We have characterized a cDNA that encodes the PTH immunoreactive material. In its longest open reading frame the cDNA encodes a protein of 381 amino acids that does not resemble PTH in its primary structure. Opossum kidney cells transfected with the cDNA do not produce a product that inhibits phosphate transport. Dialysates from patients with end-stage renal disease also contain a substance(s) that inhibits phosphate and glucose transport in opossum kidney cells. The inhibitor(s) of phosphate uptake in dialysates is a heat labile, approximately 30,000 dalton substance that inhibits phosphate transport by a cAMP-independent mechanism. Determination of the structures and physiology of these phosphate transport inhibitors is likely to yield insights into the control of phosphate homeostasis.

The highly efficient production of full-length and mutant rat brain calcium-binding proteins (calbindins-D28K) in a bacterial expression system.

We expressed gram amounts of full-length and mutant rat brain calcium-binding proteins (calbindins-D28K) lacking one or two "EF-hand" motifs in a bacterial expression system. The cDNA for the full-length rat calcium-binding protein was cloned into the NdeI and BamHI sites of the pET3a vector. Additionally, constructs of the rat brain calcium-binding protein lacking EF-hand 2 (delta 2 mutant), EF-hand 6 (delta 6 mutant), and EF-hands 2 and 6 (delta 2, 6 mutant) were constructed using the same vector. These chimeric plasmids were used to transfect BL21 (DE3) pLysS Escherichia coli cells. Following transformation, the cells were grown in the presence of isopropylthiogalactoside in order to induce bacterial T7 polymerase, which resulted in the production of large amounts of the proteins of interest in the bacterial cytosol. Expressed full-length and delta 2 and delta 2,6 mutant proteins represented 50% or more of total bacterial protein. The delta 6 protein was not expressed. Cell lysis followed by purification of the proteins on DEAE-cellulose routinely resulted in gram yields of the proteins. The purified proteins displayed the appropriate amino acid composition and amino-terminal amino acid sequence. When analyzed by matrix-assisted laser desorption mass spectrometry the proteins were found to have the appropriate molecular weights (within the accuracy limits of the instrument). The expressed proteins bound to a polyclonal antiserum raised against chick intestinal calcium-binding protein. In addition, the full-length, delta 2, and delta 2,6 mutants bound calcium as assessed by a 45Ca blotting procedure. The production of large amounts of readily purified vitamin D-dependent calcium-binding proteins should be useful in biophysical studies of the proteins.

Bacterial synthesis of truncated forms of the human vitamin D receptor and characterization of anti-receptor monoclonal antibodies.

We biosynthesized full-length (amino acids 1-427) and truncated human 1,25-dihydroxyvitamin D3 receptor proteins that encompassed only the putative DNA binding domain (amino acids 1-112) or the DNA binding domain and parts of the sterol binding domain (amino acids 1-193 and 1-328) in a bacterial expression system. We also prepared monoclonal antibodies against the full-length vitamin D receptor. The binding properties of the monoclonal antibodies were characterized by their ability to bind to full-length and truncated vitamin D receptor protein constructs. Seven of twelve monoclonal antibodies recognized the full-length receptor protein. These antibodies bound to truncated hVDR proteins with decreasing affinities as successive truncations were made from the carboxy-terminal end of the receptor protein. The five remaining monoclonal antibodies recognized the full-length and truncated receptor proteins with equally low affinities. Truncated forms of the vitamin D receptor and region-specific antibodies will be useful in assessing the properties of the receptor.

A vasopressin analog that binds but does not activate V1 or V2 vasopressin receptors is not internalized into cells that express V1 or V2 receptors.

To assess whether receptor binding is sufficient to initiate vasopressin receptor endocytosis in cells expressing the vasopressin V1 or V2 receptors, we synthesized a novel fluorescent-labeled vasopressin analog, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)-D-tyrosine, 4-valine, 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-CLVP), that binds to vasopressin receptors but does not activate intracellular events such as the mobilization of intracellular calcium or the activation of adenylate cyclase. We compared the manner in which this analog was endocytosed in cells expressing V1 (A-10, rat smooth muscle cells) or V2 (LLC-PK1, porcine kidney cells) receptors with that of a full agonist, [1-(beta-mercaptopropionic acid), 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) [Lutz et al. (1990) J. Biol. Chem. 265, 4657-4663; Lutz et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,6507-6511]. We showed that R-CLVP bound to both types of receptors with good affinity. It failed to increase cyclic AMP concentrations in LLC-PK1 cells and did not increase the mobilization of intracellular calcium in A-10 cells. It bound to the surface of both these cell types in a diffuse manner and it did not undergo receptor endocytosis in either cell type. In contrast, R-MLVP, an agonist that bound to both receptor subtypes and elicited changes in intracellular cyclic AMP and calcium, bound to the surface of these cells in a diffuse manner at early times after exposure, and rapidly underwent endocytosis. We conclude that binding of vasopressin to its receptors alone is insufficient to cause receptor endocytosis, and other events distal to the receptor are required to initiate endocytosis. R-CLVP should be a useful analog in determining the factors responsible for initiating receptor endocytosis.

Synthesis and biological activity of a biotinylated vasopressin analog.

To study vasopressin receptor-mediated endocytosis using electronmicroscopy methods and to develop avidin affinity columns for receptor purification, we synthesized and tested the biological properties of a biotinylated vasopressin (VP) analog [1-(2-mercapto) propionic acid] 8-[lysine-N6-biotin] VP (B-MLVP). B-MLVP was prepared by coupling biotin to the epsilon amine of the lysine residue in [1-(2-mercapto) propionic acid] 8-(lysine) VP (MLVP). The structure of HPLC purified B-MLVP was confirmed by fast atom bombardment mass spectrometry. B-MLVP effectively competed for arginine vasopressin (AVP) binding sites in canine renal plasma membranes on the surface of LLC-PK1 kidney cells. Dissociation constants of 15 nM and 202 nM were calculated from the results of competition binding assays conducted with membranes and cells, respectively. B-MLVP stimulated adenylate cyclase activity and elevated cellular 3',5',cyclic-AMP (cAMP) content in a manner similar to AVP, indicating it is an agonist of VP action in renal tissue. These observations indicate that B-MLVP is an agonist of VP action and may be used to study renal VP receptors by employing avidin coupled to various reporter groups.

The synthesis and biological activity of four novel fluorescent vasopressin analogs.

We synthesized and tested the biological properties of four fluorescent vasopressin analogs: [1-(2-mercapto)propionic acid]-8-lysine-N6-5-dimethylamino-naphthalene-1-sulfonyl vasopressin (D-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxyfluorescein vasopressin (F-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-2-N-methylanthranilamide vasopressin (MA-MLVP), and [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxytetramethylrhodamine vasopressin (R-MLVP). All fluorescent analogs were prepared by coupling the appropriate fluorochrome to the 6-amino group of the lysine residue in [1-(2-mercapto)propionic acid]-8-lysine vasopressin (MLVP) which was synthesized by the Merrifield solid-phase method. The structures of high performance liquid chromatography-purified MLVP and the fluorescent analogs were confirmed by fast atom bombardment mass spectrometry. F-MLVP, MA-MLVP, and R-MLVP effectively competed for 8-arginine vasopressin (AVP)-binding sites in canine renal plasma membranes and on the surface of porcine kidney cells (LLC-PK1, ATCC CL101). Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 32, 8.8, and 26 nM, respectively, were calculated from the results of competition binding assays conducted with membranes. D-MLVP did not bind to plasma membranes. Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 390, 38, and 160 nM, respectively, were calculated from the results of competition binding assays conducted with cells. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased adenylate cyclase activity in canine renal plasma membranes to values 2.4, 2.9, and 2.6 times that of basal activity, respectively. A maximally active concentration of AVP (1 microM) increased adenylate cyclase activity in canine renal plasma membranes to a value 2.7 times that of basal activity. D-MLVP did not stimulate adenylate cyclase activity. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased the cAMP content of porcine kidney cells from a basal level of 43 to 267, 160, and 469 pmol/mg of cell protein, respectively. Specific binding of these fluorescent analogs to receptors on the surface of LLC-PK1 cells was observed by fluorescence microscopy. These observations indicate that F-MLVP, MA-MLVP, and R-MLVP are biologically active fluorescent vasopressin analogs which are well-suited to the study of renal vasopressin receptors by fluorescence microscopy.

Synthesis and biological activity of novel vitamin D analogues: 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 and 24,24-difluoro-1 alpha,25-dihydroxy-26,27-dimethylvitamin D3.

We synthesized 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 (16), and 24,24-difluoro-1 alpha, 25-dihydroxy-26,27-dimethylvitamin D3 (21), from 3 beta-hydroxy-22,23-dinorcholenic acid 3-acetate. Compound 16 was found to be a highly potent vitamin D analogue with bioactivity similar to that of 25-hydroxyvitamin D3 in vivo. Compound 16 was bound by vitamin D binding protein with an affinity slightly less than that of 25-hydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with approximately the same affinity as that of 25-hydroxyvitamin D3. In the organ-culture duodenum, 16 induced the synthesis of calcium binding protein with a potency approximately 1/20 that of 1,25-dihydroxyvitamin D3. Compound 21 was also noted to be a highly potent vitamin D analogue with bioactivity in vivo similar to that of 1,25-dihydroxyvitamin D3. It was bound to vitamin D binding protein with an affinity considerably less than that of 1,25-dihydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with an affinity slightly less than that of the native hormone. In the organ-culture duodenum, 21 was noted to be about 3 times more active than 1,25-dihydroxyvitamin D3 in the induction of calcium binding protein. The introduction of fluorines at C-24 and extension of the sterol side chain at C-26 and C-27 by methylene groups results in vitamin D analogues that have biological activity in vivo similar to those of the respective nonfluorinated natural sterols.

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, 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.

The synthesis and biological activity of 22-fluorovitamin D3: a new vitamin D analog.

We synthesized 22-fluorovitamin D3 from (22S) cholest-5-ene-3 beta, 22-diol-3 beta-acetate 2. Compound 2 was treated with diethylaminosulfur trifluoride to give 22-fluorocholest-5-en-3 beta-acetate 3 and (E) 22-dehydrocholest-5-en-3 beta-acetate. Compound 3 was treated with N-bromosuccinimide to give a mixture of the respective 5,7- and 4,6-dienes. The 5,7-diene of 3 was separated from the 4,6-diene using the dienophile 4-phenyl-1,2,4-triazoline-3, 5-dione. 22-Fluoro-5 alpha,8 alpha-(3,5-dioxo-4-phenyl-1, 2,4-triazolino)-cholest-6-en-3 beta-acetate 4 was purified by flash chromatography and treated with lithium aluminum hydride to generate 22-fluorocholesta-5,7-dien-3 beta-ol 5. Photolysis of the diene 5, followed by thermal equilibration, resulted in the synthesis of 22-fluorovitamin D3 7. The vitamin 7 increased active intestinal calcium transport only at a dose of 50,000 pmol/rat, whereas vitamin D3 increased intestinal calcium transport at a dose of between 50 and 500 pmol/rat. 22-Fluorovitamin D3 7 did not mobilize bone and soft tissue calcium at a dose as high as 50,000 pmol/rat, whereas vitamin D3 was successful in doing so at a dose of 500 pmol/rat. When tested in the duodenal organ culture system, 22-fluorovitamin D3 7 had approximately 1/25th the potency of vitamin D3. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. 22-Fluorovitamin D3 7 bound to the rat plasma vitamin D binding protein less avidly than vitamin D3. 22-Fluorovitamin D3 was bound very poorly to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. We conclude that the introduction of fluorine at the C-22 position results in a vitamin D sterol with decreased biologic activity when compared to vitamin D3. The presence of a fluorine group at C-22 position inhibits the binding of the vitamin to rat vitamin D binding protein when compared to the binding of its hydrogen analog, vitamin D3.

Biological activity of the C-1, C-3, C-25, beta-D-glucopyranosides of 1,25-dihydroxyvitamin D3.

We have shown previously that the 3-beta-D-glucopyranosides of vitamin D3, 1 alpha-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 are biologically active in vivo. In order to determine whether the presence of the beta-D-glucopyranoside moiety linked to either the C-3, the C-25 or the C-1 hydroxyl function of the molecule affected the biological activity of the conjugates 1, 2, or 3 derived from 1,25-dihydroxyvitamin D3, we administered increasing amounts of compounds to vitamin D-deficient rats maintained on a low calcium diet. The aglycone, 1,25-dihydroxyvitamin D3 (4), also was administered to another group of such animals. When administered i.v., all three beta-D-glucopyranosides increased intestinal calcium transport in doses as low as 50 pmol/rat. A dose of approximately 500 pmol/rat was required to increase bone calcium mobilization in these same animals. The three glucosides were found to be equally active in both biological responses. A dose of only 5 pmol of 1,25-dihydroxyvitamin D3 (4) increased both intestinal calcium transport and bone calcium mobilization. We also performed similar experiments after the p.o. administration of these compounds to vitamin D-deficient rats maintained on a low calcium diet. The glucopyranosides 1, 2 and 3 were able to increase calcium transport in the intestine, as well as mobilize bone calcium at doses of between 500 and 5000 pmol/rat. Once again, the compounds were equipotent, but were less active than 1,25-dihydroxyvitamin D3 (4). After the i.v. or p.o. doses of the glucosides, plasma concentrations of 1,25-dihydroxyvitamin D3 increased in a dose-dependent manner. We conclude that: The C-3, C-25 and C-1 beta-D-glucopyranosides of 1,25-dihydroxyvitamin D3 are biologically active and equipotent in vivo, most likely as a result of hydrolysis to the free aglycone and they are less active than the aglycone in this respect.

Biologic activity of 3beta-D-glucopyranosides of vitamin D compounds.

Polar glycosidic conjugates of vitamin D compounds occur in the vegetable and possibly in the animal kingdom. The biologic activity of these conjugates has not been examined systematically. To obtain more information on the biological role of such sterol conjugates, we examined the biological activity of the 3beta-D-glucopyranosyl conjugates of vitamin D3 5, 25-hydroxyvitamin D3 6, 1alpha-hydroxyvitamin D3 7 and 1alpha,25-dihydroxyvitamin D3 8. When these compounds were administered i.v. we found that a dose of between 50 and 500 pmol/rat of the four glucopyranosides tested increased active intestinal calcium transport and increased bone calcium mobilization in vitamin D-deficient rats fed a low calcium diet. Under the same conditions, corresponding doses of the parent vitamin D3 compounds elicited comparable increases in both intestinal calcium transport and bone calcium mobilization. When these compounds were administered p.o. 3beta-D-glucopyranosyl vitamin D3 5 exhibited no biological activity at doses of up to 5000 pmole/rat, whereas the corresponding glycosides of 25-hydroxyvitamin D3 6, 1alpha-hydroxyvitamin D3 7 and 1,25-dihydroxyvitamin D3 8 were active at doses of 500 to 1000 pmol/rat in the intestinal calcium transport system. When the glucopyranosyl conjugates were administered i.v. to vitamin D-deficient rats, 25-hydroxyvitamin D3 and 1alpha,-25-dihydroxyvitamin D3 were detected in the serum at levels less than or equal to those noted in animals dosed with the respective free sterols.

Synthesis and biologic activity of 3 beta-thiovitamin D3.

We synthesized 3 beta-thiovitamin D3 from 7-dehydrocholesterol and tested its biological activity and protein binding properties. The thiovitamin was found to be a weak vitamin D agonist at high doses in vivo. It was poorly bound by both vitamin D-binding protein as well as by the intestinal cytosol receptor for 1,25-dihydroxyvitamin D. It did not increase the synthesis of calcium binding protein in the chick embryonic duodenum and did not block the activity of 1,25-dihydroxyvitamin D3 in this system. We conclude that 3 beta-thiovitamin D3 is a weak vitamin D agonist in vivo with no agonist activity or antagonist activity to 1,25-dihydroxyvitamin D3 in the chick embryonic duodenum.

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3.

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.

Synthesis and biological activity of 3 beta-hydroxy-9,10-secopregna-5,7,10[19]-triene-20-one: a side chain analogue of vitamin D3.

The synthesis, biological and antagonistic activity of 3 beta-hydroxy-9,10-secopregna-5,7,10[19]-triene-20-one (20-oxopregnacalciferol, 7) a shortened side chain analogue of vitamin D3, are described. At the highest dose tested the analogue was found to have small though significant bone and soft tissue mobilization activity; no significant increase in intestinal calcium transport was noted. The compound was found to possess no antagonistic activity against vitamin D3.

Synthesis and biological activity of vitamin D2 3 beta-glucosiduronate and vitamin D2 3 beta-sulfate: role of vitamin D2 conjugates in calcium homeostasis.

To ascertain the physiologic function of vitamin D2 conjugates in calcium homeostasis, we synthesized vitamin D2 3 beta-glucosiduronate and vitamin D2 3 beta-sulfate in pure form and tested their biological activity in vitamin D deficient rats fed a low calcium diet. Vitamin D2 3 beta-glucosiduronate was active in promoting calcium transport in the intestine at a dose of 100 pmol per rat. It increased calcium mobilization from bone and soft tissue at a dose of 1000 pmol per rat. This conjugate was less active than equimolar doses of vitamin D2. These results demonstrate that vitamin D2 3 beta-glucosiduronate can be utilized by the rat as a source of vitamin D. In contrast, vitamin D2 3 beta-sulfate was biologically inert. It failed to increase calcium transport in the duodenum of vitamin D deficient rats except at the highest doses tested (greater than 100,000 pmol/rat). It was similarly ineffective in increasing calcium mobilization from bone and soft tissue. Our results lead us to conclude that vitamin D2 3 beta-glucosiduronate is probably utilized by the rat after hydrolysis to the free sterol; on the contrary, the sulfate is not biologically active except at the highest doses tested.

Synthesis and biological activity of vitamin D3 3 beta-sulfate. Role of vitamin D3 sulfates in calcium homeostasis.

To determine the biological activity of vitamin D sulfates, we synthesized vitamin D3 3 beta-sulfate and tested its biological activity in vitamin D-deficient hypocalcemic rats. When vitamin D3 sulfate was administered as a single oral dose of 208,000 or 416,000 pmol (100 micrograms or 200 micrograms), it increased active calcium transport in the duodenum and was also able to mobilize calcium from bone and soft tissue. Dose levels below this failed to elicit a response. Vitamin D3 itself was active at doses as low as 260 pmol when administered in this manner. In order to test the biological activity of vitamin D3 sulfate in various doses when administered chronically, we tested the biological activity of vitamin D3 sulfate after 5 days or oral dosing: vitamin D3 sulfate was active at doses of 52,000 pmol/day (25 microgram), whereas vitamin D3 was active at doses of 65 to 260 pmol/day over a period of 5 days. When administered as a single intravenous dose, vitamin D3 sulfate exhibited no biological activity in doses as high as 52,000 pmol. Vitamin D3, however, was active at a dose of as low as 65 pmol. We conclude that vitamin D3 sulfate, a metabolite of vitamin D3 of heretofore unknown biological activity, is considerably less active than vitamin d3 itself.