Evolving Role of Vitamin D in Immune-Mediated Disease and Its Implications in Autoimmune Hepatitis.

Vitamin D has immunomodulatory, anti-inflammatory, antioxidant, and anti-fibrotic actions that may impact on the occurrence and outcome of immune-mediated disease. The goals of this review are to describe the nature of these expanded roles, examine the implications of vitamin D deficiency in autoimmune hepatitis, and identify opportunities for future investigation. Abstracts were identified in PubMed by multiple search terms. Full-length articles were selected for review, and secondary and tertiary bibliographies were developed. Vitamin D receptors are expressed on the principal cell populations involved in the innate and adaptive immune responses. Macrophages and dendritic cells can produce 1,25-dihydroxyvitamin D within the microenvironment. This active form of vitamin D can inhibit immune cell proliferation, promote an anti-inflammatory cytokine profile, expand regulatory T cells, enhance glucocorticoid actions, increase glutathione production, and inhibit hepatic stellate cells. Vitamin D deficiency has been commonly present in patients with immune-mediated liver and non-liver diseases, and it has been associated with histological severity, advanced hepatic fibrosis, and non-response to conventional glucocorticoid therapy in autoimmune hepatitis. Vitamin D analogues with high potency, low calcemic effects, and independence from hepatic hydroxylation are possible interventions. In conclusion, vitamin D has properties that could ameliorate immune-mediated disease, and vitamin D deficiency has been a common finding in immune-mediated liver and non-liver diseases, including autoimmune hepatitis. Loss of vitamin D-dependent homeostatic mechanisms may promote disease progression. Vitamin D analogues that are independent of hepatic hydroxylation constitute an investigational opportunity to supplement current management of autoimmune hepatitis.

RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.

RORα and RORγ are expressed in human skin cells that produce the noncalcemic 20-hydroxyvitamin D3 [20(OH)D3] and 20,23-dihydroxyvitamin D3 [20,23(OH)2D3]. Chinese hamster ovary (CHO) cells stably expressing a Tet-on RORα or RORγ expression vector and a ROR-responsive element (RORE)-LUC reporter, and a mammalian 2-hybrid model examining the interaction between the ligand binding domain (LBD) of RORα or RORγ with an LBD-interacting LXXLL-peptide, were used to study ROR-antagonist activities. These assays revealed that 20(OH)D3 and 20,23(OH)2D3 function as antagonists of RORα and RORγ. Moreover, 20(OH)D3 inhibited the activation of the promoter of the Bmal1 and G6pase genes, targets of RORα, and 20(OH)D3 and 20,23(OH)2D3 inhibited Il17 promoter activity in Jurkat cells overexpressing RORα or RORγ. Molecular modeling using crystal structures of the LBDs of RORα and RORγ revealed docking scores for 20(OH)D3, 20,23(OH)2D3 and 1,25(OH)2D3 similar to those of the natural ligands, predicting good binding to the receptor. Notably, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3 inhibited RORE-mediated activation of a reporter in keratinocytes and melanoma cells and inhibited IL-17 production by immune cells. Our study identifies a novel signaling pathway, in which 20(OH)D3 and 20,23(OH)2D3 act as antagonists or inverse agonists of RORα and RORγ, that opens new possibilities for local (skin) or systemic regulation.-Slominski, A. T., Kim, T.-K., Takeda, Y., Janjetovic, Z., Broz˙yna, A. A., Skobowiat, C., Wang, J., Postlethwaite, A., Li, W., Tuckey, R. C., Jetten, A. M. RORα and ROR γ are expressed in human skin and serve as receptors for endogenously produced noncalcemic 20-hydroxy- and 20,23-dihydroxyvitamin D.

In vivo deletion of CAR resulted in high bone mass phenotypes in male mice.

Constitutive androstane receptor (CAR) was originally identified as xenobiotic sensor that regulates the expression of cytochrome P450 genes. However, recent studies suggest that this nuclear receptor is also involved in the regulation of energy metabolism including glucose and lipid homeostasis. This study investigated the role of CAR in the regulation of bone mass in vivo using CAR(-/-) mice. Endogenous mRNA expression of CAR was observed in both primary osteoblasts and osteoclast precursors. CAR(-/-) mice have exhibited significant increase in whole body bone mineral density (BMD) by 9.5% (P < 0.01) and 5.5% (P < 0.05) at 10 and 15 weeks of age, respectively, compared with WT mice in males. Microcomputed tomography analysis of proximal tibia demonstrated a significant increase in trabecular bone volume (62.7%), trabecular number (54.1%) in male CAR(-/-) mice compared with WT mice. However, primary culture of calvarial cells exhibited no significant changes in osteogenic differentiation potential between CAR(-/-) and WT. In addition, the number of tartrate-resistant acid-phosphatase positive osteoclasts in the femur and serum level of CTx was not different between CAR(-/-) and WT mice. The higher BMD and microstructural parameters were not observed in female mice. Interestingly, serum level of testosterone in male CAR(-/-) mice was 2.5-fold higher compared with WT mice and the mRNA expressions of Cyp2b9 and 2b10 in the liver, which regulate testosterone metabolism, were significantly down-regulated in male CAR(-/-) mice. Furthermore, the difference in BMD between CAR(-/-) and WT mice disappeared at 8 weeks after performing orchiectomy. CAR(-/-) mice also exhibited significant increase in serum 1,25(OH)2 D3 levels but Cyp 27B1 which converts 25(OH)D3 to 1,25(OH)2 D3 was significantly down-regulated compared to WT mice. These results suggest that in vivo deletion of CAR resulted in higher bone mass, which appears to be a result from reduced metabolism of testosterone due to down-regulation of Cyp2b.

Production of 22-hydroxy metabolites of vitamin d3 by cytochrome p450scc (CYP11A1) and analysis of their biological activities on skin cells.

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D(3), producing 20S-hydroxyvitamin D(3) [20(OH)D(3)] and 20S,23-dihydroxyvitamin D(3) [20,23(OH)(2)D(3)] as the major metabolites. These are biologically active, acting as partial vitamin D receptor (VDR) agonists. Minor products include 17-hydroxyvitamin D(3), 17,20-dihydroxyvitamin D(3), and 17,20,23-trihydroxyvitamin D(3). In the current study, we have further analyzed the reaction products from cytochrome P450scc (P450scc) action on vitamin D(3) and have identified two 22-hydroxy derivatives as products, 22-hydroxyvitamin D(3) [22(OH)D(3)] and 20S,22-dihydroxyvitamin D(3) [20,22(OH)(2)D(3)]. The structures of both of these derivatives were determined by NMR. P450scc could convert purified 22(OH)D(3) to 20,22(OH)(2)D(3). The 20,22(OH)(2)D(3) could also be produced from 20(OH)D(3) and was metabolized to a trihydroxyvitamin D(3) product. We compared the biological activities of these new derivatives with those of 20(OH)D(3), 20,23(OH)(2)D(3), and 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. 1,25(OH)(2)D(3), 20(OH)D(3), 22(OH)D(3), 20,23(OH)(2)D(3), and 20,22(OH)(2)D(3) significantly inhibited keratinocyte proliferation in a dose-dependent manner. The strongest inducers of involucrin expression (a marker of keratinocyte differentiation) were 20,23(OH)(2)D(3), 20,22(OH)(2)D(3), 20(OH)D(3), and 1,25(OH)(2)D(3), with 22(OH)D(3) having a heterogeneous effect. Little or no stimulation of CYP24 mRNA expression was observed for all the analogs tested except for 1,25(OH)(2)D(3). All the compounds stimulated VDR translocation from the cytoplasm to the nucleus with 22(OH)D(3) and 20,22(OH)(2)D(3) having less effect than 1,25(OH)(2)D(3) and 20(OH)D(3). Thus, we have identified 22(OH)D(3) and 20,22(OH)(2)D(3) as products of CYP11A1 action on vitamin D(3) and shown that, like 20(OH)D(3) and 20,23(OH)(2)D(3), they are active on keratinocytes via the VDR, however, showing a degree of phenotypic heterogeneity in comparison with other P450scc-derived hydroxy metabolites of vitamin D(3).

20,23-dihydroxyvitamin D3, novel P450scc product, stimulates differentiation and inhibits proliferation and NF-kappaB activity in human keratinocytes.

We have examined effects of the 20,23-dihydroxyvitamin D3 (20,23(OH)2D3), on differentiation and proliferation of human keratinocytes and the anti-inflammatory potential of 20,23(OH)2D3 from its action on nuclear factor-kappaB (NF-kappaB). 20,23(OH)2D3 inhibited growth of keratinocytes with a potency comparable to that for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell cycle analysis showed that this inhibition was associated with G1/G0 and G2/M arrests. 20,23(OH)2D3 stimulated production of involucrin mRNA and inhibited production of cytokeratin 14 mRNA in a manner similar to that seen for 1,25(OH)2D3. Flow cytometry showed that these effects were accompanied by increased involucrin protein expression, and an increase in the cell size and granularity. Silencing of the vitamin D receptor (VDR) by corresponding siRNA abolished the stimulatory effect on involucrin gene expression demonstrating an involvement of VDR in 20,23(OH)2D3 action. This mode of action was further substantiated by stimulation of CYP24 gene expression and stimulation of the CYP24 promoter-driven reporter gene activity. 20,23(OH)2D3 displayed several fold lower potency for induction of CYP24 gene expression than 1,25(OH)2D3. Finally, 20,23(OH)2D3 inhibited the transcriptional activity of NF-kappaB in keratinocytes as demonstrated by EMSA, NF-kappaB-driven reporter gene activity assays and measurements of translocation of p65 from the cytoplasm to the nucleus. These inhibitory effects were connected with stimulation of the expression of IkappaBalpha with subsequent sequestration of NF-kappaB in the cytoplasm and consequent attenuation of transcriptional activity. In summary, we have characterized 20,23(OH)2D3 as a novel secosteroidal regulator of keratinocytes proliferation and differentiation and a modifier of their immune activity.

Resurrection of vitamin D deficiency and rickets.

The epidemic scourge of rickets in the 19th century was caused by vitamin D deficiency due to inadequate sun exposure and resulted in growth retardation, muscle weakness, skeletal deformities, hypocalcemia, tetany, and seizures. The encouragement of sensible sun exposure and the fortification of milk with vitamin D resulted in almost complete eradication of the disease. Vitamin D (where D represents D2 or D3) is biologically inert and metabolized in the liver to 25-hydroxyvitamin D [25(OH)D], the major circulating form of vitamin D that is used to determine vitamin D status. 25(OH)D is activated in the kidneys to 1,25-dihydroxyvitamin D [1,25(OH)2D], which regulates calcium, phosphorus, and bone metabolism. Vitamin D deficiency has again become an epidemic in children, and rickets has become a global health issue. In addition to vitamin D deficiency, calcium deficiency and acquired and inherited disorders of vitamin D, calcium, and phosphorus metabolism cause rickets. This review summarizes the role of vitamin D in the prevention of rickets and its importance in the overall health and welfare of infants and children.

A specific high-affinity binding macromolecule for 1,25-dihydroxyvitamin D3 in fetal bone.

Cytosol fractions were prepared from fetal rat or embryonic chick calvaria and analyzed for binding of vitamin D3 metabolites on sucrose density gradients. Both cytosol fractions contain a 3.5S macromolecule which specifically binds 1,25-dihydroxy-[3H]vitamin D3 and in addition, a 5 to 6S macromolecule which binds 25-hydroxy-[3H]vitamin D3. In rat calvaria cytosol, 1,25-dihydroxy-[3H]vitamin D3 also binds to the 5 to 6S macromolecule but appears to have greater affinity for the 3.5S component.

The essentiality of vitamin D metabolites for embryonic chick development.

Laying hens maintained on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D produce eggs which appear normal but which produce embryos having a defective upper mandible and which die at 18 to 19 days of embryonic life. Hens maintained on 25-hydroxyvitamin D3, on the other hand, produce normal embryos. Hens fed a vitamin D deficient diet produce eggs which develop the same embryonic defect. Injection of the affected eggs from the 1,25-dihydroxyvitamin D3 fed hens with vitamin D3, 25-hydroxyvitamin D3, or 1,25-dihydroxyvitamin D3 greatly increases the percentage of normal embryos. It therefore appears that 1,25-dihydroxyvitamin D3 is not transferred from hen to egg in sufficient amounts to support embryonic development and that vitamin D or its metabolites, or both, are necessary for normal chick embryo development.

Side chain metabolism of 25-hydroxy-[26,27-14C] vitamin D3 and 1,25-dihydroxy-[26,27-14C] vitamin D3 in vivo.

Radioactive CO2 was detected in expired air after the administration of 25-hydroxy-[26,27-14C] vitamin d3 to vitamin D-deficient hypocalcemic rats; 14co2 was also detected after the administration of 1,25-dihydroxy-[26,27-14C] vitamin D3 to rats raised on the same diet. Nephretcomy totally abolished 14CO2 formation after administration of 25-hydroxy-[26,27-14C] vitamin D3 but not after the administration of 1,25-dihydroxy-[26,27-14C] vitamin D3. The production of 14CO2 commenced within 4 hours after injection of 1,25-dihydroxy-[26,27-14C] vitamin D3, suggesting a possible relevance of this reaction to the function of 1,25-dihydroxyvitamin D3. These results at least demonstrate a new metabolic pathway of vitamin D3 metabolism involving the oxidation of a portion of the side chain of 1,25-dihydroxyvitamin D3 to CO2.

Stimulation of 24,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3.

Vitamin D-deficient rats produce [(3)H]1,25-dihydroxyvitamin D(3) from [(3)H]25-hydroxyvitamin D(3) regardless of dietary content of calcium or phosphate. A daily dose of 130 picomoles of 1,25-dihydroxyvitamin D(3) for a period of 5 days reduces production of [(3)H]1,25-dihydroxyvitamin D(3) to essentially zero and stimulates production of [(3)H]24,25-dihydroxyvitamin D(3). A daily dose of 325 picomoles of 25-hydroxyvitamin D(3) has a similar but less dramatic effect. On the other hand, 650 picomoles daily of 24,25-dihydroxyvitamin D(3) given to vitamin D-deficient rats had no effect. Thus it appears that 1,25-dihydroxyvitamin D(3) is an important factor in the regulation of kidney metabolism of 25-hydroxyvitamin D(3).

Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid.

After mature rats that had been fed on a vitamin D3-deficient diet were injected with tritium-labeled 1,25-dihydroxyvitamin D3, radioactivity became concentrated in nuclei of luminal and cryptal epithelium of the duodenum, jejunum, ileum, and colon; in nuclei of the epithelium of kidney distal tubules including the macula densa, and in podocytes of glomeruli; in nuclei of the epidermis including outer hairshafts and sebaceous glands; and in nuclei of certain cells of the stomach, anterior and posterior pituitary, and parathyroid. These results reveal cell types that contain receptors for 1,25-dihydroxyvitamin D3 or metabolites of this compound both in known or hypothesized target tissues and in tissues that were previously unknown to participate in vitamin D3 metabolism.

1 alpha, 25-Dihydroxyvitamin D3 nuclear receptors in pituitary.

Specific binding of 1 alpha,25-dihydroxyvitamin D(3) was found in nuclear and cytosol fractions of the bovine pituitary. For nuclear binding. the dissociation constant was 0.1 namomole per liter, and maximum binding was 104 femtomoles per milligram of protein. In competition studies, 25-hydroxyvitamin D(3) was 300 times weaker than 1 alpha,25-dihydroxyvitamin D(3). The existence of high-affinity sites supports a physiologic role for 1 alpha,25-dihydroxyvitamin D(3) in the pituitary.

Calcium absorption and calcium-binding protein synthesis: solanum malacoxylon reverses strontium inhibition.

The ingestion of diets containing high concentrations of stable strontium inhibits calcium absorption and intestinal calcium-binding protein synthesis and, as shown by others, does so by inhibiting the conversion of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol, the active form of vitamin D. The addition of the South American plant Solanum malacoxylon to strontium-containing diets counteracts the inhibitory action of dietary strontium, thereby indicating that the plant contains a factor which can mimic the action of 1,25-dihydroxycholecalciferol and representing the first such factor identified in a botanical source.

Brain target sites for 1,25-dihydroxyvitamin D3.

Autoradiographic studies with 3H-labeled 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] demonstrate, in certain neurons of rat forebrain, hindbrain, and spinal cord, a nuclear retention and concentration of radioactivity, which can be prevented by treatment with 1,25(OH)2D3, but not with 25-hydroxyvitamin D3. These results indicate the presence of brain receptors in addition to pituitary receptors for 1,25(OH)2D3 and suggest a central modulation of calcium homeostasis and other central effects for this hormone. The existence of a brain-pituitary axis for certain 1,25(OH)2D3-mediated endocrine-autonomic effects is postulated.

Vitamin D release across abdominal adipose tissue in lean and obese men: The effect of ß-adrenergic stimulation.

Obesity is characterized by a blunted lipolytic response in abdominal subcutaneous adipose tissue (SAT) and low circulating vitamin D levels. Here we investigated whether an impaired SAT lipolytic response coincides with an impaired SAT vitamin D release in eight lean and six obese men. 25-hydroxyvitamin D3 [25(OH)D3 ] and 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] fluxes across SAT were measured using arterio-venous blood sampling in combination with AT blood flow measurements after an overnight fast and during 1-hr intravenous infusion of the non-selective ß-adrenergic agonist isoprenaline (20 ng·kg FFM-1 ·min-1 ). 1,25(OH)2 D3 was released across abdominal SAT during isoprenaline infusion in lean [-0.01 (-0.04 to 0.00) pmol*100 g tissue-1 *min-1 , p = .017 vs. zero flux], but not in obese men [0.01 (0.00 to 0.02) pmol*100 g tissue-1 *min-1 , p = .116 vs. zero flux], and accompanied by an impaired isoprenaline-induced lipolytic response in abdominal SAT of obese versus lean men. Isoprenaline had no significant effects on net 25(OH)D3 release across abdominal SAT and plasma vitamin D metabolites in lean and obese men. To conclude, a blunted isoprenaline-mediated lipolysis is accompanied by reduced release of 1,25(OH)2 D3 vitamin D across abdominal SAT in obesity.

Vitamin D and pregnancy: the maternal-fetal metabolism of vitamin D.

A model of the maternal-fetal metabolism of vitamin D3 is depicted in Fig. 2. 25-OHD3 of maternal origin is metabolized by the maternal kidneys to the potent metabolite, 1,25-(OH)2D3, which acts on the maternal intestine, kidneys, and skeleton. The maternal kidneys and other organs can produce 24,25-(OH)2D3, although this pathway may be suppressed near the end of gestation. The placenta has selective permeability to the vitamin D3 metabolites, with 25-OHD3 crossing from the mother to the fetus more readily than the dihydroxylated metabolites. The onset of the placental synthesis of 1,25-(OH)2D3 during gestation is unknown. Likewise the regulation of the placental 25-OHD3-1 alpha-hydroxylase is unknown. 1,25-(OH)2D3 of placental origin may enter the maternal or the fetal circulation or act locally on the placenta by inducing the synthesis of proteins involved in the cellular transport of Ca. Perhaps one placenta cell type synthesizes 1,25-(OH)2D3 and another cell type possessing a cytoplasmic receptor for 1,25-(OH)2D3 responds to this metabolite. The function of the 24,25-(OH)2D3 produced by the placenta is unknown. The concentration of free 25-OHD3 and free 1,25-(OH)2D3 in the fetal circulation exceeds the maternal levels due to the differences in the DBP concentrations of the two bloodstreams. The 1,25-(OH)2D3 in the fetal bloodstream may originate from either the placenta or the fetal kidneys. The latter site may not be active in utero due to the hypercalcemia and hyperphosphatemia relative to the maternal levels of these ions. 1,25-(OH)2D3 in the fetal bloodstream acts on those fetal tissues containing cytoplasmic receptors for this metabolite. The intestinal mucosa apparently lacks these receptors until sometime during neonatal life. In contrast, fetal bone cells possess receptors for the 1,25-(OH)2D3. The 24,25-(OH)2D3 in the fetal bloodstream may also be involved in the growth and differentiation of the fetal skeleton. However, the precise role of both metabolites in the fetus remains conjectural.

Production, degradation, and circulating levels of 1,25-dihydroxyvitamin D in health and in chronic glucocorticoid excess.

The decreased intestinal absorption of calcium and accelerated bone loss associated with chronic glucocorticoid excess may be mediated by changes in vitamin D metabolism, leading to decreased availability of circulating 1,25-dihydroxyvitamin D. This hypothesis was examined in 14 patients with either endogenous or exogenous glucocorticoid excess. Analysis of paired serum samples (mean +/- SE) in 13 patients during euglucocorticoidism and during hyperglucocorticoidism showed that glucocorticoid excess resulted in small decreases of plasma 25-hydroxy-vitamin D concentrations (22 +/- 2- 18 +/- 2 ng/ml; P < 0.05) but no significant changes in plasma 1,25-dihydroxyvitamin D (32 +/- 8- 23 +/- 6 pg/ml) or serum immunoreactive parathyroid hormone (21 +/- 2- 18 +/- 2 muleq/ml). Additionally, we studied plasma kinetics of [3H]1,25-dihydroxyvitamin D3 after intravenous bolus administration in 10 hyperglucocorticoid patients and in 14 normal controls. Assessment with a three-compartment model showed no significant abnormalities in production rates (hyperglucocorticoid patients 1.2 +/- 0.3 micrograms/d, controls 1.5 +/- 0.2 micrograms/d) or metabolic clearance rates (hyperglucocorticoid patients, 18 +/- 2%; controls, 14 +/- 2%) or feces (hyperglucocorticoid patients, 60 +/- 9%, controls, 54 +/- 6%). We conclude that glucocorticoid excess does not effect plasma levels, production, or degradation of 1,25(OH)2D in humans. Thus, other mechanisms must be postulated to explain satisfactorily the abnormalities of bone structure and intestinal calcium absorption that may occur after chronic glucocorticoid therapy.

Extraction of vitamin D metabolites by bones of normal adult dogs.

Using the isolated perfused canine tibia we examined the extraction of [(3)H]25(OH)D(3), [(3)H]1,25(OH)(2)D(3) and [(3)H]24,25(OH)(2)D(3) by bone of normal adult dogs. The studies were performed with and without vitamin D binding protein (DBP) in the perfusate to examine the effect of protein binding on the extraction of the vitamin D metabolites. An average of 48+/-2% of [(3)H]25(OH)D(3) was extracted by bone, when no DBP was present. However, addition of only a small amount of DBP ( approximately 720 ng/ml of perfusate) nearly completely abolished the extraction of [(3)H]25(OH)D(3) by bone. No degradation and/or transformation of the labeled 25(OH)D(3) could be demonstrated during passage through the isolated perfused bone. The extraction of [(3)H]24,25(OH)(2)D(3) in a DBP-free medium averaged 33+/-5%. Addition of 720 ng of DBP/ml of perfusate completely inhibited the extraction of this metabolite. The extraction of [(3)H]1,25(OH)(2)D(3) averaged 30+/-3% in a DBP free medium and no inhibition of the extraction was demonstrated after addition of DBP (720 ng/ml of perfusate). However, addition of DBP in a concentration of 14.4 mug/ml of perfusate reduced the extraction of 1,25(OH)(2)D(3) to 8+/-2%, a value still significantly higher than that seen after addition of 20 times less DBP to perfusions with 25(OH)D(3) and 24,25(OH)(2)D(3). It is concluded that the isolated perfused bone of normal dogs can extract significant amounts of 25(OH)D(3), 1,25(OH)(2)D(3), and 24,25(OH)(2)D(3). Small concentrations of DBP (720 ng/ml) in the perfusate significantly inhibited the extraction of 25(OH)D(3) and 24,25(OH)(2)D(3). A carrier role for DBP is suggested and it is proposed that the levels of free vitamin D are important for extraction of the metabolites by bone. Therefore, due to the different affinities of DBP for the various metabolites of vitamin D, only 1,25(OH)(2)D(3) is extracted in vitro in significant amounts by bone of normal adult dogs, in the presence of DBP.

Metabolic consequences of oral administration of 24,25-dihydroxycholecalciferol to uremic dogs.

24,25-dihydroxycholecalciferol [24,25-(OH)(2)D(3)], once considered a relatively inert metabolite of vitamin D(3), has been recently recognized as a metabolically active product in some species. In previous studies, we have shown that infusion of 24,25(OH)(2)D(3) into the thyroid artery of normal dogs results in prompt and complete suppression of parathyroid hormone (PTH) secretion. In this study, we have examined the metabolic consequences of oral administration of this metabolite in dogs with experimentally induced renal hyperparathyroidism. Dogs with comparable degrees of renal insufficiency (glomerular filtration rate, 10-15 ml/min) were treated for 3 wk with daily doses of either 2 mug of 24,25(OH)(2)D(3) or 50% ethanol, the vehicle in which the metabolite was suspended. After a 6-wk recovery period, treatments were reversed: dogs who had previously served as controls received the metabolite while dogs previously treated with metabolite received the vehicle. Administration of 24,25(OH)(2)D(3) resulted in a 40-60% decrease of immunoreactive PTH. This was associated with a small (0.1-0.2 mg/dl) but unequivocal decrease of serum ionized calcium. Calcium balance, which was slightly negative under control conditions, became slightly but definitively positive on treatment with 24,25(OH)(2)D(3). All other parameters measured, including total serum calcium, magnesium, phosphorus, creatinine, electrolytes, phosphorus excretion, and phosphorus balance, remained unchanged. The data support the hypothesis that 24,25(OH)(2)D(3) not only decreases PTH secretion but also functions as an anabolic hormone in bone under the conditions of this experiment.

Enterohepatic physiology of 1,25-dihydroxyvitamin D3.

After intravenous administration of radiolabeled 1,25-dihydroxyvitamin D3 to rats, approximately 25% of the administered radioactivity appeared in the bile within 24 h. Instillation of the biliary radioactivity into the duodena of other rats was followed by recovery of 15% of the radioactivity in newly secreted bile within 24 h. The process by which products of 1,25-dihydroxyvitamin D3 were excreted in bile was not saturable in the dose range tested (0.275-650 ng). The metabolites of 1,25-dihydroxyvitamin D3 present in bile were found to be much more polar than 1,25-dihydroxyvitamin D3 and were resolved into three fractions on high performance liquid chromatography. 60% of the radioactivity present in bile was retained selectively by DEAE-cellulose; the radioactive material could be eluted from the gel at a low pH or at high salt concentrations. When bile containing the radiolabeled metabolites was incubated at 37 degrees C and pH 5 with beta-glucuronidase, there was an increase in the amount of radioactivity comigrating with 1,25-dihydroxyvitamin D3. Treatment of the products of radiolabeled 1,25-dihydroxyvitamin D3 in bile with diazomethane, an agent which converts acids into methyl esters, transformed one of the metabolites into a less polar compound. These results demonstrate that there is a quantitatively important enterophepatic circulation of the products of 1,25-dihydroxyvitamin D3 in the rat.