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.     

Ultrastructural changes in the lysosomes of rachitic intestinal absorptive cells

In chicks maintained on a rachitogenic (vitamin D deficient) diet, the number of intestinal absorptive cell calcium-lysosomes is markedly decreased in comparison to normal animals. In addition, the majority (better than 50%) of these rachitic calcium-lysosomes are atypical in their fine structure resembling the lamellar bodies seen in certain diseases (Tay-Sachs disease, Duchenne's muscular dystrophy). Such atypical organelles are characterized by their internal membranous swirls reminiscent of myelin figures. This information appears to be a further indication that lysosomes are normally involved in calcium homeostatic mechanisms and therefore sensitive to circulating vitamin D levels.     

Studies on the mode of action of calciferol. X. 24-Nor-25-hydroxyvitamin D3, an analog of 25-hydroxyvitamin D3 having "anti-vitamin" activity.

24-Nor-25-hydroxyvitamin D₃, an analog of 25-hydroxyvitamin D₃, has been chemically synthesized in six steps. This steroid was tested in chicks, $\text{in vivo}$, for its ability to generate the classic vitamin D mediated responses of stimulation of intestinal calcium transport and bone calcium mobilization. Although the 24-nor-25-OH-vitamin D₃ itself exhibited no biological activity in these assays, the analog was found to inhibit the normal responses produced by a physiological dose of vitamin D₃. These results suggest that 24-nor-25-OH-vitamin D₃ may satisfy certain requirements expected of a calciferol “anti-vitamin.”     

Lysosomal proliferation in rachitic avian intestinal absorptive cells following 1,25-Dihydroxycholecalciferol

Lysosomes in chick intestinal absorptive cells from rachitic (vitamin D-deficient) and vitamin D-replete animals were studied utilizing transmission electron microscopic histochemistry and ultrastructural morphometry. Absorptive cells from rachitic animals, serum calcium = 7.3±0.3 mg%, contained an average of 4.0±0.3 supranuclear lysosomes. In rachitic chicks sacrificed 9 hr post-injection of 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D, the values for both serum calcium, 9.8 ± 0.2 mg%, and the number of apical absorptive cell lysosomes, 12.9±0.6, were increased over non-injected or vehicle-only injected animals. Lysosomes in vitamin D-replete absorptive cells were characterized by their intense staining with pyroantimonate, indicative of their high calcium content. The same organelles also produced a positive reaction for acid phosphatase. Rachitic lysosomes, also acid phosphatase positive, were only lightly stained with pyroantimonate. The lysosomal proliferation apparently induced by 1,25-dihydroxycholecalciferol may be a further indication that these organelles play a role in intestinal calcium transport and/or intracellular calcium homeostasis within the absorptive cell.     

Evidence for aqueous soluble vitamin D-like substances in the calcinogenic plant, Tristetum flavescens

A crude aqueous extract of the leaves of $\text{T. flavescens}$ when administered orally to vitamin D-deficient chicks produced significant increases in plasma phosphate but had little effect on plasma calcium. When chicks, fed a high strontium diet to inhibit endogenous 1,25(OH)₂ vitamin D₃ production and intestinal calcium transport, were dosed with the extract or synthetic 1,25(OH)₂D₃⁴⁵Ca absorption from the duodenum $\text{in vivo}$ was stimulated, whereas vitamin D₃ was ineffective. Partial purification of the crude extract on a Sephadex GH25 column yielded two factors, one of which mimicked 1,25 (OH)₂D₃ activity in chicks fed the high strontium diet while the other produced a significant increase in plasma phosphate. The presence of these substances, together with previously demonstrated organic solvent soluble vitamin D-type activity, may account for the calcinogenic nature of the plant.     

1,25-Dihydroxyvitamin D3 Controls a Cohort of Vitamin D Receptor Target Genes in the Proximal Intestine That Is Enriched for Calcium-regulating Components

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) plays an integral role in calcium homeostasis in higher organisms through its actions in the intestine, kidney, and skeleton. Interestingly, although several intestinal genes are known to play a contributory role in calcium homeostasis, the entire caste of key components remains to be identified. To examine this issue, Cyp27b1 null mice on either a normal or a high calcium/phosphate-containing rescue diet were treated with vehicle or 1,25(OH)₂D₃ and evaluated 6 h later. RNA samples from the duodena were then subjected to RNA sequence analysis, and the data were analyzed bioinformatically. 1,25(OH)₂D₃ altered expression of large collections of genes in animals under either dietary condition. 45 genes were found common to both 1,25(OH)₂D₃-treated groups and were composed of genes previously linked to intestinal calcium uptake, including S100g, Trpv6, Atp2b1, and Cldn2 as well as others. An additional distinct network of 56 genes was regulated exclusively by diet. We then conducted a ChIP sequence analysis of binding sites for the vitamin D receptor (VDR) across the proximal intestine in vitamin D-sufficient normal mice treated with vehicle or 1,25(OH)₂D₃. The residual VDR cistrome was composed of 4617 sites, which was increased almost 4-fold following hormone treatment. Interestingly, the majority of the genes regulated by 1,25(OH)₂D₃ in each diet group as well as those found in common in both groups contained frequent VDR sites that likely regulated their expression. This study revealed a global network of genes in the intestine that both represent direct targets of vitamin D action in mice and are involved in calcium absorption.     

Intestinal Cell Calcium Uptake and the Targeted Knockout of the 1,25D3-MARRS (Membrane-associated,Rapid Response Steroid-binding) Receptor/PDIA3/Erp57

We have crossed ERp57^flx/flx mice with commercially available mice expressing villin-driven cre-recombinase. Lysates of intestinal epithelial cells were prepared from knock-out (KO) mice and littermates (LM) and used in Western blot analyses with Ab099 against the N terminus of the 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) receptor: LM mice exhibited one positive band, which was absent in preparations from KO mice. Saturation analyses of cell lysates with [³H]1,25D₃ revealed negligible binding in preparations from either female or male KOs. Lysates from female and male LM mice had similar affinities but different numbers of binding sites. Isolated enterocytes were tested for steroid-stimulated calcium uptake. Treatment of cells from female or male LM mice with 1,25D₃ elicited enhanced calcium uptake in females and males within 5 min. Intestinal cells from KO mice exhibited a severely blunted or completely absent response to hormone. Confocal microscopy of intestinal cells revealed the presence of cell surface vitamin D receptors. However, antibodies to the vitamin D receptor failed to block 1,25D₃-stimulated calcium uptake. In chick enterocytes we have found that the PKA pathway mediates calcium uptake. The time course for activation of PKA in mouse enterocytes paralleled that for enhanced calcium uptake and for LM females reached 250% of controls within 5 min, and 150% of controls in cells prepared from LM males. Enterocytes from female or male KO mice failed to exhibit steroid hormone-stimulated PKA activity, but did respond to forskolin with enhanced calcium uptake. We conclude that the 1,25D₃-MARRS receptor is of central importance to steroid hormone-stimulated calcium uptake in mammalian intestinal cells.     

In vitro metabolism of vitamin D3 by isolated liver cells

Summary Liver cells were prepared from rats fed a rachitogenic diet to investigate the hepatic metabolism of [ — 1,2 —³H₂] vitamin D₃. Rat hepatocytes suspended in Hanks medium rapidly took up labeled vitamin D₃ from the incubation medium and converted this sterol to various metabolites, including 25-hydroxy vitamin D₃ (25-OH-D₃). There was a steady increment in the cellular production of 25-OH-D₃ and of the more polar metabolites of vitamin D₃ over 3 hr of incubation as determined by thin layer chromatography. Neither the addition of cyclic nucleotides or dexamethasone to, nor the removal of calcium or phosphate from the medium resulted in changes in the rate of conversion of vitamin D₃ to its products. Rats pretreated with sodium diphenylhydantoin converted labeled vitamin D₃ to its metabolites at the same rate as control rats. These data indicate that isolated liver cells retain the capacity for vitamin D₃ hydroxylation, but suggest that the rate of this process does not undergo rapid changes in response to metabolic stimulation.Recipient of Research Career Development Award 1 K04 HL-00089.     

24R,25-dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 are both indispensable for calcium and phosphorus homeostasis

The essential role of vitamin D throughout the life of most mammals and birds as a mediator of calcium homeostasis is well established. In view of the complex endocrine system existent for the regulated metabolism of vitamin D₃ to both 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 24R,25-dihydroxyvitamin D₃ [24R,25-(OH)₂D₃] (both produced by the kidney), an intriguing problem is to elucidate whether only one or both of these dihydroxyvitamin D₃ metabolites is required for the generation of all the biological responses mediated by the parent vitamin D₃. In contrast to the accumulated knowledge concerning the short term actions of 1,25(OH)₂-D₃ on stimulating intestinal calcium absorption and bone calcium reabsorption, relatively little is known of the biological function of 24,25(OH)₂D₃. We report now the results of a nine month study in which chicks were raised on a vitamin D-deficient diet from hatching to sexual maturity and received as their sole source of “vitamin D” either 24,25(OH)₂D₃ or 1,25(OH)₂D₃ singly or in combination. Specifically we are describing the integrated operation of the vitamin D endocrine system as quantitated by the individual measurement in all birds of 22 variables related to “vitamin D status” and as evaluated by the statistical procedure of multivariate discriminant analysis. Twelve of these variables involved detailed analysis of the bone including quantitative histology and the other 10 variables reflect various manifestations of vitamin D action, e.g. serum Ca²⁺ and Pi levels, vitamin D-dependent calcium binding protein (CaBP) in the intestine and kidney, egg productivity etc. As evaluated by the multivariate analysis, it is clear that 24,25(OH)₂D₃ and 1,25(OH)₂D₃ are simultaneously required for normalization of calcium homeostasis.     

Intestinal Na/Ca exchanger protein and gene expression are regulated by 1,25(OH)2D3 in vitamin D-deficient chicks

The role of 1,25(OH)₂D₃ on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)₂D₃ were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)₂D₃ simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1 μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)₂D₃. NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)₂D₃. Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)₂D₃ treatment. Rapid effects of 1,25(OH)₂D₃ on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)₂D₃ on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)₂D₃ enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

Chemical synthesis and biological activity of 3-deoxy-1alpha-hydroxyvitamin D3 an analog of 1alpha,25-(OH)2-D3, the active form of vitamin D3

The chemical synthesis of 3-deoxy-1α-hydroxyvitamin D₃ from cholesterol is described. This steroid is a highly important analog of the hormonally active form of vitamin D, 1α, 25-dihydroxyvitamin D₃; it is the only analog presently available for structure-function studies which lacks the 3β-hydroxyl but retains the key 1α-hydroxyl of 1α, 25-dihydroxyvitamin D₃. The new steroid is highly biologically active; it stimulated intestinal calcium absorption significantly more rapidly than vitamin D₃ and as rapidly as 1α, 25-dihydroxyvitamin D₃.     

Adaptation of intestinal calcium absorption: parathyroid hormone and vitamin D metabolism.

It has already been demonstrated that the adaptation of intestinal calcium absorption of rats on a low calcium diet can be eliminated by thyroparathyroidectomy plus parathyroid hormone administration. This treatment elevates intestinal and plasma levels of 1,25-dihydroxyvitamin D₃ in rats on a high calcium diet while producing no change in rats on a low calcium diet. It therefore appears likely that the modulation of intestinal calcium absorption by dietary calcium is mediated by the parathyroid glands and the renal biogenesis of 1,25-dihydroxyvitamin D₃. Changes in the other unknown vitamin D metabolite levels as a result of dietary calcium are also modified by thyroparathyroidectomy and parathyroid hormone administration, but the effect of these metabolites on intestinal calcium transport is unknown.     

1,25-Dihydroxyvitamin D3 metabolism. The effect of dietary calcium and phosphorus

Rats maintained on tritiated 1,25-dihydroxyvitamin D₃ as their sole source of vitamin D and placed on diets differing in calcium content had similar intestinal levels of tritiated 1,25-dihydroxyvitamin D₃. Since 1,25-dihydroxyvitamin D₃ administration eliminated adaptation of intestinal calcium transport, it appears that increased production of 1,25-dihydroxyritamin D₃ is responsible for the stimulation of calcium transport by low dietary calcium. When maintained on tritiated 1,25-dihydroxyvitamin D₃, rats fed a low-phosphorus diet had somewhat higher levels of tritiated 1,25-dihydroxyvitamin D₃ in the duodenum and plasma than rats on a normal-phosphorus diet. In addition to stimulating 1,25-dihydroxyvitamin D₃ synthesis, low dietary phosphorus may increase the accumulation of 1,25-dihydroxyvitamin D₃ in both intestine and plasma.     

Interrelationships of vitamin D,bone metabolism and blood calcium concentration in the chick

It is not known if the effects of vitamin D deficiency on chick bone are due to direct actions of the vitamin or if they are secondary to other changes, such as hypocalcemia. Day-old cockerels were fed either a rachitogenic diet containing no Ca (−D-Ca), 1.4% Ca (−D), or 3% Ca (−DHiCa) and given corn oil (−D groups) or vitamin D₃ in corn oil (+D and + D-Ca) p.o. for up to 21 days. Radii were harvested and incubated for 6–8 h in a defined medium. Medium samples were taken every 2 h and analysed for Ca, P₁ and lactate. Some bones were incubated in a respirometer to measure O₂ consumption. Compared to +D, −D birds showed evidence of D deficiency by decreased plasma Ca concentration (33%), bone and body weight (43%) and Ca release from bone (70%) and by histological changes in bone characteristic of rickets. Increases were seen in total and bone alkaline phosphatase activity in plasma (270 and 706%, respectively), P_i release (23%), O₂ consumption (23%) and lactate production (52%) by the −D radii. The marked hypocalcemia seen in the −D chicks did not occur in −DHiCa birds. Nevertheless, bone and body weights were decreased in this group and bone lactate production, O₂ consumption and total and bone alkaline phosphatase in plasma were increased. Rachitic bone lesions were only partially corrected by the high-Ca diet. Release of Ca and P_i from the −DHiCa bone? was not different than from +D radii. Comparing +D-Ca and −D-Ca groups with +D chicks, both were hypocalcemic with decreased bone weight, body weight and bone Ca release, while showing elevated lactate production and P_i release. The only difference between the +D-Ca and −D-Ca groups was a 50% decrease in Ca release by −D-Ca bone. The results suggest that in chicks: (1) some, but not all, of the effects of vitamin D deficiency on bone can be corrected by normalizing plasma Ca and (2) evaluation of the effects of vitamin D deficiency on bone may require hypocalcemia, since some responses are masked by normocalcemia.     

Effect of alcohol on renal vitamin D metabolism in chickens.

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

Effect of dietary calcium and phosphorus on intestinal calcium absorption and vitamin D metabolism.

To understand better dietary regulation of intestinal calcium absorption, a quantitative assessment of the metabolites in plasma and duodenum of rats given daily doses of radioactive vitamin D₃ and diets differing in calcium and phosphorus content was made. All known vitamin D metabolites were ultimately identified by high-pressure liquid chromatography. In addition to the known metabolites (25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, 25,26-dihydroxyvitamin D₃, and 1,24,25-trihydroxyvitamin D₃), several new and unidentified metabolites were found. In addition to 1,25-dihydroxyvitamin D₃ and 1,24,25-trihydroxyvitamin D₃, the levels of some of the unknown metabolites could be correlated with intestinal calcium transport. However, whether or not any of these metabolites plays a role in the stimulation of intestinal calcium absorption by low dietary calcium or low dietary phosphorus remains unknown.     

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.     

Vitamin D deficiency diminishes the severity and delays onset of experimental autoimmune encephalomyelitis

Multiple sclerosis incidence is clearly inversely related to sun exposure. This observation led to the idea that vitamin D might be responsible for this relationship. Providing super-physiologic doses of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃, suppresses an animal model of multiple sclerosis, i.e. experimental autoimmune encephalomyelitis (EAE) but causes unwanted hypercalcemia. Further, dietary calcium is needed for this activity of 1α,25-dihydroxyvitamin D₃. B10PL mice were maintained on a vitamin D-deficient diet for two generations to produce frank vitamin D deficiency. These animals showed delayed onset and reduced severity of EAE compared to control animals on the same diet and given vitamin D₃ or provided a vitamin D-containing chow diet. Thus, vitamin D deficiency interferes with the development of this autoimmune disease rather than increasing susceptibility.     

Biochemical properties of A-homovitamin D derivatives: 1,4-dihydroxy-3-deoxy-A-homo-19-nor-9, 10-secocholesta-5,7-dienes

A study has been made in the chick of the stereostructural requirements of A-ring-functionalized vitamin D analogs which elicit vitamin D₃ and 1,25-(OH)₂D₃-dependent biological responses of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Ring expansion of vitamin D₃ to produce (1S,4S), (1S,4R), or (1R,4S)-(7E)-1,4-dihydroxy-3-deoxy-A-homo-19-nor-9,10-secocholesta-5,7-dienes resulted in the loss of both ICA and BCM biological activity at dose levels of steroid of up to 650 nmol/0.1 kg birds. Accordingly the three A-homo analogs of vitamin D₃ were assessed for their ability to inhibit or increase the ICA or BCM responses of D₃ and 1,25-(OH)₂D₃. Only (1R,4S)-(7E)-diol-C, maintaining a cis-β,β-hydroxyl orientation showed antagonistic biological activity. Intraperitoneal doses (65–325 nmol) of diol-C administered in conjunction with D₃ (0.8–3.25 nmol) inhibited the BCM responses selectively and had no effect on the ICA response. Doses of analog-C (16.3-3.25 nmol) injected before and after the active hormone 1,25-(OH)₂D₃ (0.13–01.30 nmol) stimulated the ICA response of the latter above its normal levels (a synergistic response) when administered alone.     

Effect of 1,25-dihydroxyvitamin D3 on intestinal calcium absorption in strontium-fed rats.

These studies investigated the initial stimulation of intestinal calcium absorption in the rat by 1,25-dihydroxyvitamin D₃. To produce a functional vitamin D₃-deficiency, rats were fed a diet containing 2.4% strontium. After 10 days on the diet, intestinal calcium uptake, as measured by everted gut sacs, was significantly depressed. Strontium-fed rats were dosed orally with 1,25-dihydroxyvitamin D₃, and changes in intestinal calcium uptake, intestinal alkaline phosphatase activity, and intestinal calcium-binding protein were measured as a function of time after dose. Calcium uptake was significantly increased in the proximal 2.5 cm of the duodenum at 4 h and along the whole duodenum by 7 h. Intestinal alkaline phosphatase activity, measured in a Triton extract of the mucosal homogenate and in isolated brush border complexes, was also increased by 7 h. Using both gel electrophoresis and immunodiffusion against a specific antiserum, an increase in intestinal calcium-binding protein was detected in intestinal supernate at 4 h after dosing. Almost no calcium-binding protein was detectable in strontium-fed rats dosed with propylene glycol only. These time studies are consistent with a role for both alkaline phosphatase and calcium-binding protein in the 1,25-dihydroxyvitamin D₃-stimulated uptake of calcium by the intestine. In addition, the usefulness of strontium feeding for producing a functional vitamin D₃ deficiency in rats is demonstrated.