The effects of 24R,25-dihydroxycholecalciferol and of 1 alpha,25-dihydroxycholecalciferol on ornithine decarboxylase activity and on DNA synthesis in the epiphysis and diaphysis of rat bone and in the duodenum.

The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.     

Monoclonal antibody with high affinity for 1,25-dihydroxycholecalciferol

We have developed a monoclonal antibody capable of detecting 1 pg/ml of 1,25-dihydroxycholecalciferol. At a dilution of 1:80,000 of ascitic fluid this antibody has an apparent K_D of 3.3 × 10^?11ML^?1. The immunogen used was a vitamin D analogue, calcitroic acid [1α, 3 β-dihydroxy-9, 10 seco-24-nor 5,7,10 (19) cholatriene-23-oic acid], conjugated to bovine serum albumin. Although this antibody is extremely sensitive, it also recognizes other important vitamin D₃ metabolites.     

Maternal-perinatal interrelationships of vitamin D metabolism in rats.

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite the ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1. Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet. Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected. Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol. Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

Maternal-perinatal interrelationships of vitamins D metabolism in rats

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite that ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1.Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet.Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 5/25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected.Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol.Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.     

Stimulation of creatine kinase BB activity by 1 alpha,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol in rat tissues.

Vitamin D metabolites stimulate creatine kinase BB activity in organs of vitamin D-deficient rats. In epiphyses of long bones, creatine kinase BB activity increases 2.6-fold 24 h after injection of 24R,25-dihydroxycholecalciferol but not of 1 alpha,25-dihydroxycholecalciferol. Contrariwise, 1 alpha,25-dihydroxycholecalciferol, but not 24R,25-dihydroxycholecalciferol, increases creatine kinase BB activity in diaphyses and in kidney. Neither metabolite affects creatine kinase activity in duodenal mucosa.     

Ternary solvent mixtures for improved resolution of hydroxylated metabolites of vitamin D2 and vitamin D3 during high-performance liquid chromatography

This paper reports the development of three new ternary solvent mixtures for the liquid-chromatographic separation of metabolites of vitamin D on microparticulate silica. All solvent systems offer reduced peak tailing and improved resolution of vitamin D compounds, particularly of 24(R),25-(OH)₂D₃, when compared to the commonly used hexane—isopropanol mixture. The new mixtures can be substituted for hexane—isopropanol systems presently used for preparative liquid-chromatographic steps prior to radioimmunoassay or competitive protein-binding assay of 24,25-(OH)₂D and 1,25-(OH)₂D in human plasma. Hexane—isopropanol—methanol (87:10:3) mixtures are recommended where the lipid content of samples is high, whereas hexane—ethanol—chloroform (80:10:10) promises to be a useful mixture for differentiating vitamin D₃ metabolites from their vitamin D₂ analogs. A combination of the two solvent systems permits the separate assay of both 24(R),25-(OH)₂D₃ and 24(R),25-(OH)₂D₂ as well as 1,25-(OH)₂D₃ and 1,25-(OH)₂D₂.     

Regulation of Metabolism of 25-Hydroxycholecalciferol by Kidney Tissue <Emphasis Type="Italic">in vitro</Emphasis> by Dietary Calcium

IT is now recognized that hydroxylated metabolites of vitamin D (that is, cholecalciferol) function as effectors of the physiological actions originally attributed to the unaltered vitamin¹. The activation of vitamin D by specific hydroxylation reactions and sequestration of the resultant metabolites by target tissues represents a hormonal control loop which is feed-back sensitive. 25-Hydroxycholecalciferol (25-HCC) and 1,25-dihydroxycholecalciferol (1,25-DHCC) have been shown to be participants in the control loop, vitamin D being first metabolized in the liver to 25-HCC² which in turn is hydroxylated in the C-1 position to 1,25-DHCC in the kidney^3,4. The metabolically active form in the intestine appears to be 1,25-DHCC^5,6.     

Stimulation of (3H)uridine incorporation into nuclear RNA of rat kidney by vitamin D metabolites

Within 30–60 min after administration of 25-hydroxycholecalciferol or 30 min after 1,25-dihydroxycholecalciferol, the incorporation of [³H]uridine into the nuclear RNA of kidney is stimulated 1.6-fold or 3-fold, respectively. The results suggest that 1,25-dihydroxycholecalciferol is the active form responsible for the stimulation of RNA synthesis. It is suggested that specific RNA and protein synthesis may be involved in the renal reabsorption of ions initiated by vitamin D or its metabolites.     

Vitamin D metabolite-binding proteins in human tissue

Serum and post-microsomal supernatants of human lymphocyte, erythrocyte, skeletal muscle and parathyroid adenoma homogenates were examined for specific binding of 25-hydroxycholecalciferol (25-OHD₃) and 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃). Muscle, lymphocytes and parathyroid adenomata extracts contained a 6-S 25-OHD₃-binding protein which was not found in erythrocyte extracts, and which was distinct from the smaller serum transport α-globulin. A cathodal, 1,25-(OH)₂D₃-binding protein, which sedimented at 3–4 S was also detected in parathyroid tissue. These observations suggest the possibility of direct physiologic interaction between vitamin D metabolites and nucleated human tissues other than intestine and bone.     

In vitro cultures of Solanum malacoxylon Sendt.: nutritional requirements and sterol production

Callus cultures were established from hypocotyl, root and leaf explants of Solanum malacoxylon. The growth rate of calli was evaluated on Murashige and Skoog medium as well as Gamborg's B5 medium. Sterols were isolated from calli grown on both media however the B5 proved to be more effective in promoting the production of these metabolites. No calcitriol was found in the cultures. Feeding experiments with vitamin D₃ were scarcely effective in promoting the production of vitamin D₃ hydroxylated metabolites.Abbreviations GC/MS gas-liquid chromatographic-mass spectrometric analysis - CHCL₃ chloroform - GLC gas liquid chromatography - PCV packed cell volume - TLC thin layer chromatography     

Production of C-24- and C-23-oxidized metabolites of 1,25-dihydroxycholecalciferol by cultured kidney cells (LLC PK1) and their presence in kidney in vivo.

1,25-Dihydroxy[3H]cholecalciferol was converted into several more-polar metabolites by a cultured pig kidney cell line (LLC PK1). The production of metabolites was stimulated by pretreating the cells with unlabelled 1,25-dihydroxycholecalciferol. A similar profile of metabolites was observed on high-pressure-liquid-chromatographic analysis of an extract from the kidneys of rats dosed intravenously with 1,25-dihydroxy[3H]cholecalciferol. Among the metabolites detected were 1,24,25-trihydroxycholecalciferol, 1,25-dihydroxy-24-oxocholecalciferol, 1,23,25-trihydroxy-24-oxocholecalciferol and 1,25-dihydroxycholecalciferol-26,23-lactone. The results are in accord with data reported for intestinal 1,25-dihydroxycholecalciferol metabolism [Napoli, Pramanik, Royal, Reinhardt & Horst (1983) J. Biol. Chem. 258, 9100-9107]. These data indicate that C-23- and C-24-oxidation of 1,25-dihydroxycholecalciferol are phenomena common to calciferol target tissues, and that regulation of 1,25-dihydroxycholecalciferol homoeostasis is dependent on the rate of its metabolism in addition to the rate of its synthesis.     

Evidence for a 1,25-dihydroxycholecalciferol-dependent spermine-binding protein in chick duodenal mucosa

The spermine-binding activity of a cytosol protein fraction from chick duodenal mucosa changes in relation to the circulating level of 1,25-dihydroxycholecalciferol. The spermine-binding activity increases very rapidly within 1–2 hours after the rachitic chick was dosed intracardially with 1,25-dihydroxycholecalciferol. The clear and reproducible response is prevented by actinomycin D and cycloheximide. This increase is one of the earliest events induced by the active form of vitamin D₃ in the duodenal cell of rachitic chicks.     

Synthesis and composition of vitamin D-3 metabolites in Solanum malacoxylon

The synthesis of vitamin D-3 hydroxylated metabolites in Solanum malacoxylon was investigated. When crude leaf homogenates and subcellular fractions were incubated with [³H]vitamin D-3 and [³H]25-hydroxy-vitamin D-3 under conditions described for animal vitamin D-3-25-hydroxylase and 25-hydroxy-vitamin D-3-1α-hydroxylase, respectively, labelled metabolites identified on the basis of their chromatographic properties as 25-hydroxy-vitamin D-3 and 1,25-dyhydroxy-vitamin D-3 were formed. Other unidentified product metabolites were also detected. Vitamin D-3-25-hydroxylase activity was localized in microsomes and 25-hydroxy-vitamin D-3-1α-hydroxylase in mitochondria and microsomes. Chromatography of sterols isolated from leaf extracts preincubated with β-glucosidase on Sephadex LH-20 columns permitted the isolation of three biologically active fractions with elution properties similar to vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3, respectively. Ultraviolet spectra characteristic of vitamin D-3 and its metabolites were obtained after purification of the fractions by TLC. Co-chromatography of individual fractions with authentic metabolites on TLC provided further evidence that the plant contains vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3 as glucoside derivatives. These results suggest that a similar pathway of vitamin D-3 hydroxylation as in animals may be operative in S. malacoxylon.     

Vitamin D metabolites specific binding by rat intestinal cytosol

The intestine of ricketic rats, a recognized target tissue of vitamin D, contains s soluble macromolecule capable of specific in vitro binding of both 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. Its sedimentation behavior on linear 5–20% sucrose gradients suggests as a molecular weight of approximately 100 000. The binding is specific for sterols possessing both an open B ring and 25-hydroxyl group, and is destroyed by pre-incubation with trypsin. The binding affinity for 25-hydroxycholecalciferol (K_A = 2 · 10⁹ 1/mole) was 2.8 times that for 1,25-dihydroxycholecalciferol.     

Human serum binding protein for vitamin D and its metabolites (DBP): evidence that actin is the DBP binding component in human skeletal muscle

The tissue protein which tightly binds the human serum binding protein for vitamin D and its metabolites (HDBP) was studied in soluble extracts of human skeletal muscle. A tissue protein-HDBP complex was effected in vitro by the addition of human serum Cohn IV to high-speed supernatant from muscle, and the complex was partially purified by ion-exchange chromatography, gel filtration, and affinity chromatography. The faster-sedimenting complex was retained longer than HDBP on DEAE-Sephacel columns, and was estimated to have a size of 100,000 daltons by gel filtration. The complex displayed inhibitory activity to deoxyribonuclease I (DNase I), whereas HDBP alone did not. When the complex was applied to affinity chromatography columns, immunoassayable HDBP was retained by DNase I-agarose and two dominant proteins of ~58,000 and 45,000 M_r were retained by the IgG fraction of anti-HDBP serum covalently bonded to amino-agarose, as revealed by sodium dodecyl sulfate-polyacrylamide electrophoresis. Pure HDBP does not bind to nor inhibit DNase I, but an actin-HDBP complex does. These data suggested that the tissue component with high affinity for HDBP was actin. Incubation of equimolar amounts of polymerized actin and pure HDBP in its apo form resulted in the depolymerization of the actin. This depolymerizing activity was also observed with HDBP saturated with cholecalciferol, 25-hydroxycholecalciferol, 24R, 25-dihydroxycholecalciferol, or 1,25-dihydroxycholecalciferol.     

The synthesis and biological activity of 25,26-dihydroxy-cholecalciferol, a polar metabolite of vitamin D3

The synthesis of 25,26-dihydroxycholecalciferol, a biologically active metabolite of cholecalciferol (vitamin D₃) is described. 3β-Hydroxy-27-nor-5-cholesten-25-one was converted in three steps to 5,7-cholestadiene-3β,25 (RS), 26-triol. The latter compound was irradiated with ultraviolet light to give 25 (RS), 26-dihydroxyprecholecalciferol; this compound underwent thermal isomerisation to yield 25 (RS), 26-dihydroxycholecalciferol. The structure of the final product was confirmed by ultra-violet spectroscopy, mass spectroscopy and by periodate degradation to the known 25-oxo-27-nor-cholecalciferol. 25 (RS), 26-Dihydroxycholecalciferol was able to stimulate the intestinal absorption of calcium but had little or no effect on the healing of rickets.     

Studies on calciferol metabolism. VII. The effects of actinomycin D and cycloheximide on the metabolism, tissue and subcellular localization, and action of vitamin D3

It was originally postulated, primarily on the basis of experiments employing actinomycin D, that calciferol (vitamin D) mediated its characteristic physiological responses in the intestine via the activation of information stored in the intestinal genome. A more recent alternative hypothesis suggested that actinomycin D blocked the biological response to calciferol by inhibiting the mandatory metabolism of cholecalciferol to 1,25-dihydroxycholecalciferol. Presented in this paper are the results of recent experiments studying the effects of both actinomycin D and cycloheximide on the metabolism, subcellular localization, and action of cholecalciferol or its metabolites, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. Actinomycin D was found to inhibit calcium transport stimulated by cholecalciferol or its metabolites without inhibiting their metabolism or localization in the target tissue, the intestinal mucosa. However, actinomycin D had to be administered in four doses at 2-hr intervals to block the stimulation of calcium transport by 1,25-dihydroxycholecalciferol. Actinomycin D was also found not to lower the renal levels of 25-hydroxycholecalciferol-1-hydroxylase, which were measured in vitro. In contrast, cycloheximide was found to inhibit the localization of the sterols in the intestine. Also cycloheximide lowered the renal enzyme levels which were measured in vitro following administration of the antibiotic in vivo. From these data it can be calculated that the 25-hydroxycholecalciferol-1-hydroxylase appears to have a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approximately 3 hr. Thus, the inhibition of intestinal calcium transport by these two antibiotics may in fact occur at two different target organs; cycloheximide by a lowering of the kidney levels of 25-hydroxycholecalciferol-1-hydroxylase and actinomycin D by blocking the action of 1,25-dihydroxycholecalciferol in the intestine.     

Intranuclear localization and receptor proteins for 1,25-dihydroxycholecalciferol in chick intestine

1. The intranuclear distribution of cholecalciferol and its metabolites was studied in the intestine of rachitic chicks. 2. At high doses of cholecalciferol the nuclei contain the vitamin and its 25-hydroxy metabolite, but over 80% of this is localized on the nuclear membranes. The hormone, 1,25-dihydroxycholecalciferol, is found within the cell nuclei irrespective of the intake of cholecalciferol, but significant amounts could not be found with chromatin isolated free of nuclear membranes. 3. 1,25-Dihydroxycholecalciferol is associated in the nucleus with an acidic protein. Since one of the actions of 1,25-dihydroxycholecalciferol is to control the synthesis of mRNA for calcium-binding protein it was to be expected that the hormone would be bound to chromatin, as with the other steroid hormones. It is suggested that the hormone-receptor complex exists as part of an equilibrium mixture of the complex bound to the DNA and in a free form. 4. A protein extract of nuclei was obtained, which when incubated at 4 degrees C for 1h took up the 1,25-dihydroxycholecalciferol. The nature of this binding was studied. 5. There appear to be two nuclear proteins able to bind the hormone one of which is the intestinal nuclear receptor. The binding sites on this protein are saturable with the hormone, have an association constant of 2x10(9)m(-1) and show a high chemical specificity for the 1,25-dihydroxycholecalciferol. The number of nuclear binding sites for the hormone provided by this receptor is similar to the maximum intestinal hormone concentration so far observed. Its sedimentation coefficient is 3.5S, and is very close to that observed for the nuclear protein to which is attached the 1,25-dihydroxycholecalciferol formed in vivo from vitamin D. 6. The cytoplasmic protein has an association constant of 1x10(9)m(-1)and a sedimentation coefficient of 3.0S, but its relation with the nuclear receptor is not yet clear.     

Comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites

The comparison of equilibrium and disequilibrium assay conditions for ergocalciferol, cholecalciferol and their major metabolites were investigated to evaluate: (1) optimization of sensitivity (2) crossreactivity of these compounds in their respective assays and (3) side chain steric requirements of the vitamin D molecule for optimum binding to the calciferol binding protein or bovine thymus receptor. Disequilibrium assay conditions improved assay sensitivity 30-fold for the calciferol assay and approx 3-fold for metabolites in the 25-hydroxycalciferol and 1,25-dihydroxycalciferol assays. Ergocalciferol compounds were uniformly less efficient in their association with the proteins tested than were their cholecalciferol counterparts, with one exception. In the calciferol assay, cholecalciferol had greater affinity for the the calciferol binding protein than did ergocalciferol. In the 25-hydroxycalciferol assay affinity for the calciferol binding protein was 25-hydroxycholecalciferol = 24,25-dihydroxycholecalciferol greater than 25-hydroxyergocalciferol greater than 25S,26-dihydroxycholecalciferol greater than 24,25-dihydroxyergocalciferol greater than 25,26-dihydroxyergocalciferol. In the assay for 1,25-dihydroxycalciferol, bovine thymus receptor recognized 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol equally. From the forthcoming data it appears that hydroxyl and/or methyl groups on the calciferol side chain alter the ability of these physiological compounds to associate with the calciferol binding protein.     

Competitive protein binding assay for 24,25-dihydroxycholecalciferol

A competitive protein binding assay which measures 24,25-dihydroxycholecalciferol in human serum has been developed using the binding protein from vitamin D-deficient rat kidney. As 25-hydroxycholecalciferol and 25,26-dihydroxycholecalciferol also interact with the binding protein, possible interference by these compounds in the assay has been overcome by preparative chromatography of serum extracts on Sephadex LH 20 prior to assay. The mean serum level of 24,25-dihydroxycholecalciferol in seven normal volunteers was 1.68 ± 0.82 ng/ml whereas patients receiving large therapeutic doses of vitamin D were found to have higher levels. None was detectable in the serum of a vitamin D-deficient patient.