Effect of 26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 on the expression of vitamin-D-responsive genes in vitamin-D-deficient mice

26,26,26,27,27,27-Hexafluoro-1,25-dihydroxyvitamin D3 (ST-630) is a newly developed agent to maintain the levels of calcium and phosphorus in blood. Herein, we investigated the effect of this compound on the expression of vitamin-D-responsive genes in vitamin-D-deficient mice. ST-630 was more effective than 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3] with respect to the induction of Cyp24 and calbindin-D9k mRNAs in the kidney and in the small intestine. Moreover, the increase in mRNA levels of vitamin-D-responsive genes induced by ST-630 lasted longer than that induced by 1,25(OH)2D3. These results indicate that ST-630 was more effective in inducing Cyp24 and calbindin-D9k gene expression than 1, 25(OH)2D3 when both compounds were injected into vitamin-D-deficient mice.     

Effect of 1,25 (OH)2 vitamin D3 on glomerulosclerosis in subtotally nephrectomized rats

In the past, there has been considerable concern that treatment with active vitamin D might accelerate progression independent of hypercalcemia and hypercalcuria. Nevertheless, 1,25(OH)2D3 has known antiproliferative properties and has also been shown to inhibit renal growth. Since glomerular growth is a permissive factor for the development of glomerulosclerosis, we reasoned that 1,25(OH)2D3 might even attenuate progression. To test this working hypothesis we performed two experiments of 8 and 16 weeks duration, respectively, to compare subtotally nephrectomized (SNX) rats treated with ethanol and SNX treated with 1,25(OH)2D3. Control animals were sham operated and pair-fed with SNX animals. 1,25(OH)2D3 (3 ng/100 g body wt/day) was administered by osmotic minipump. 1,25(OH)2D3 had no significant effect on systolic blood pressure and only a transient effect on weight gain. SNX reduced the number of glomeruli (left kidney) from an average of 3.3 x 10(4) to 1.2 x 10(4) per kidney. Mean glomerular volume was 3.87 +/- 0.71 x 10(6) microns 3 in sham operated animals and significantly (P < 0.05) higher (10.1 +/- 1.75 x 10(6) microns 3) in untreated animals 16 weeks after SNX. Glomerular volume was significantly (P < 0.05) less in 1,25(OH)2D3 treated SNX [10.1 +/- 1.75 in ethanol vs. 7.04 +/- 1.78 in 1,25(OH)2D3 treated SNX]. In parallel, there was significantly (P < 0.01) less glomerulosclerosis [glomerulosclerosis index 1.16 +/- 0.14 in the ethanol treated SNX vs. 0.80 +/- 0.16 in SNX treated with 1,25(OH)2D3] in the eight week experiment. Albuminuria was significantly (P < 0.01) lower in 1,25(OH)2D3 treated than in ethanol treated SNX (mean 0.785 mg/24 hr, range 0.43 to 1.80, vs. 3.75 mg/24 hr, 1.29 to 14.2). The morphological data were directionally analogous in a second 16 week experiment. Only slight changes of the vascular sclerosis index and tubulointerstitial index were seen in SNX and were not affected by 1,25(OH)2D3 further. To prove that the effect of 1,25(OH)2D3 was independent of PTH, parathyreoidectomized SNX rats without or with 1,25(OH)2D3 treatment were examined seven days post-SNX. PCNA staining showed suppression of cell proliferation. Furthermore, in situ hybridization for transforming growth factor-B (TGF-beta) showed less vascular and tubular expression in 1,25(OH)2D3 treated rats. We conclude that 1,25(OH)2D3 has antiproliferative actions during the compensatory growth of nephrons in response to subtotal nephrectomy. These effects are independent of PTH. The data document that 1,25(OH)2D3 reduces renal cell proliferation and glomerular growth as well as glomerulosclerosis and albuminuria as indicators of progressive glomerular damage.     

1,25(OH)2 vitamin D3 binding sites in male sex organs of the Siberian hamster (Phodopus sungorus). An autoradiographic study

Using autoradiography, binding sites for 1,25(OH)2 vitamin D3 are found in certain genital organs of male Siberian hamsters (Phodopus sungorus), in particular in basal epithelial cells and fibroblasts of the lamina propria of prostate glands. Scattered labeled cells are also present in the epithelium of coagulation and urethral glands. In contrast to the findings in mice, under the conditions of the experiment, 1,25(OH)2 vitamin D3 binding sites are not recognizable in other accessory sex glands and gonads. The frequency of basal epithelial cells with [3H]1,25(OH)2 vitamin D3 nuclear binding is higher in regressed dorsal prostate glands of animals living in short photoperiods. The data suggest that 1,25(OH)2 vitamin D3 may promote proliferation and differentiation in basal epithelial cells, modulated by the seasonal and functional status of the animal.     

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.     

The E1A oncogene induces resistance to the effects of 1,25-dihydroxyvitamin D3 on inhibition of growth of mouse keratinocytes

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited DNA synthesis in transformed mouse keratinocytes (Pam212) in a time- and dose-dependent manner as measured by [3H]thymidine incorporation. To investigate the mechanism through which 1,25-(OH)2D3 acts, we examined its effects on Pam212 cells further transformed with the E1A oncogene. Here, we show that transformation of the cells with the E1A oncogene induced resistance to the effects of 1,25-(OH)2D3 on inhibition of growth of Pam212 cells. While 1,25-(OH)2D3 treatment increased the level of expression of vitamin D receptor mRNA 20-fold in parental cells, the E1A-transformed cells failed to express vitamin D receptor mRNA even after treatment with 1,25-(OH)2D3. Transfection of the E1A-transformed cell line with an expression construct encoding the vitamin D receptor restored receptor expression as well as the inhibition of growth by 1,25-(OH)2D3. These results suggest that one of the mechanisms for acquisition of 1,25-(OH)2D3 resistance induced by E1A may involve loss of vitamin D receptor inducibility by 1,25-(OH)2D3.     

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)2D3 and 1,25-(OH)2D3 regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)2D3 affecting primarily growth zone (GC) cells and 24,25-(OH)2D3 affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)2D3 has been shown to increase phospholipase A2 activity in GC, while 24,25-(OH)2D3 has been shown to decrease phospholipase A2 activity in RC. Stimulation of phospholipase A2 in GC caused an increase in PKC, whereas inhibition of phospholipase A2 activity in RC cultures increased both basal and 24,25-(OH)2D3-induced PKC activity, suggesting that phospholipase A2 may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A2 inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A2 activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A2 inhibitors decreased both basal and 1,25-(OH)2D3-induced PKC activity in GC. When phospholipase A2 activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)2D3-induced PKC activity in RC and increased 1,25-(OH)2D3-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A2 activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A2 activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A2. These effects are cell maturation- and time-dependent and metabolite-specific.     

In vivo treatment with calcitriol (1,25(OH)2D3) reverses age-dependent alterations of intestinal calcium uptake in rat enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D3; [1, 25(OH)2D3] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)2D3 stimulation of Ca2+ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20-24-month-old rats given 1,25(OH)2D3 for 3 days (30 ng/100 g bw/day), calcitriol (10(-10) M, 3-5 minutes) stimulated Ca2&plus uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)2D3. When the aged rats were injected with 25(OH)D3, similar Ca2+ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1, 25(OH)2D3-dependent changes in Ca2+ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)2D3 stimulation of duodenal cell Ca2+ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)2D3 or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system.     

Influence of metabolic acidosis on serum 1,25(OH)2D3 levels in chronic renal failure

Metabolic acidosis has been shown to alter vitamin D metabolism. There is also evidence that calcium may modulate 1,25(OH)2D3 by a parathyroid hormone (PTH)-independent mechanism. To investigate the effect of rapid correction of chronic metabolic acidosis on serum 1,25(OH)2D3 levels by free calcium clamp in chronic renal failure, 20 patients with mild to moderate metabolic acidosis (mean pH 7.31 +/- 0.04) and secondary hyperparathyroidism (mean intact PTH 156.47 +/- 84.20 ng/l) were enrolled in this study. None had yet received any dialysis therapy. Metabolic acidosis was corrected by continuous bicarbonate infusion for 3-4 h until plasma pH was around 7.4, while plasma ionized calcium was held at the preinfusion level by calcium solution infusion during the entire procedure. The plasma pH, bicarbonate, total CO2, sodium, and serum total calcium levels were significantly increased while serum concentrations of alkaline phosphatase and albumin were significantly decreased after bicarbonate infusion. The plasma ionized calcium, potassium, serum magnesium, inorganic phosphorus, and 25(OH)D levels showed no significant change before and after bicarbonate infusion. The serum 1,25(OH)2D3 levels were significantly increased (38.66 +/- 11.77 vs. 47.04 +/- 16.56 pmol/l, p < 0.05) after correction of metabolic acidosis. These results demonstrate that rapid correction of metabolic acidosis raises serum 1,25(OH)2D3 levels in vitamin D-deficient chronic renal failure patients, and may underline the importance of maintaining normal acid-base homeostasis in the presence of secondary hyperparathyroidism in chronic renal failure.     

Anti-tumor effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in seborrheic keratosis

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a drug with potent antiproliferative action on keratinocytes that have nuclear receptors for 1,25(OH)2D3. We investigated the effects of 1,25(OH)2D3 on widespread seborrheic keratoses in 51 patients with these tumors. The data indicated that resolution of these tumors was dependent on both tumor size and dose of 1,25(OH)2D3. Among 15 patients treated with a high dose (0.5 microgram/d) of oral 1,25(OH)2D3, the lesions of widespread seborrheic keratoses changed from brown-black papules to brownish papules with erythema and/or crust as early as 2 wk after the start of treatment. The tumors finally developed into an atrophic scar or brownish pigmented macule. Histologically, vacuolation of the spinous cells, vesicle formation, and liquefaction degeneration of the basaloid cells were observed. Numerous lymphocytes had infiltrated in the papillary dermis. Among 36 patients treated with a low dose (0.25 microgram/d) of 1,25(OH)2D3, brownish papules became pale to normal in color and reduced in size, without erythematous change. Histologically, acanthosis of the epidermis was reduced, but degenerative change of the tumor cells was not observed. These data suggest that oral therapy of 1,25(OH)2D3 is an acceptable method well suited to the removal of seborrheic keratoses, especially those that are predominantly small tumors.     

Effect of vitamin D3 and 1,25(OH)2D3 on growth of neoplastically derived cell lines and their alkaline phosphatase activity

Inhibitory effects of 1,25(OH)2D3 and D3 on growth of four neoplastically derived cells were observed in human acute leukemia cell culture CEM-C-1 and CEM-C-7, human cervical carcinoma cell lines C-4-1 and human epithelioid carcinoma cells of cervix HeLa S3K. Concurrently, in dexamethasone-responsive cells C-4-1 and HeLa S3K there was a 1,25(OH)2D3 and D3 induced elevation of alkaline phosphatase with 1,25(OH)2D3 showing the greater effects. It is supposed that vitamins D3-induced alkaline phosphatase activity in malignant cells, which is proposed to be a possible marker of cell differentiation, can be associated with the membrane effects of these vitamins.     

Impaired vitamin D metabolism and response in spontaneously diabetic GK rats

Several studies of diabetes mellitus patients have demonstrated abnormalities in calcium, phosphate and vitamin D metabolism. In an earlier study, the authors reported impaired renal processing of phosphate in spontaneously diabetic GK rats, an animal model of type II diabetes mellitus. In the present study, which represents an extension of the earlier study, vitamin D metabolism and response are examined in 20-week-old GK rats. Serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] was found to be lower in GK rats than in Wistar rats. After intraperitoneal administration of 0.5 micrograms/kg 1,25-(OH)2D, serum calcium increased in GK rats, but not in Wistar rats, while serum phosphate remained unchanged in GK rats, but increased in Wistar rats. Although serum 1,25-(OH)2D rose abruptly in 3 h and decreased thereafter in both GK and Wistar rats, the decrease in serum 1,25-(OH)2D at 6 h was more marked in GK rats than in Wistar rats. Serum 24,25-dihydroxyvitamin D was consistently higher in GK rats than in Wistar rats. Northern blotting and dot blotting with use of a cDNA probe for the 24-hydroxylase gene showed an increased expression of the gene in the kidney of GK rats. These results demonstrate impaired vitamin D metabolism in GK rats. Increased activity of 24-hydroxylase, in addition to impaired phosphate metabolism, may play a role in impaired vitamin D metabolism in GK rats.     

Changes in 1,25-(OH)2D3 synthesis and its receptor expression in spleen cell subpopulations of mice infected with LPBM5 retrovirus

This study examines the influence of chronic retroviral infection of mice with a LPBM5 virus mixture on the paracrine system involving immune cells and 1,25-(OH)2D3 in the spleen. Plasma ionized calcium, 25-(OH)D and 1,25-(OH)2D of infected mice were unchanged. In contrast, the specific binding of 1,25-(OH)2D3 to spleen cytosol and the number of monocyte/macrophages expressing 1,25-(OH)2D3 receptors (VDR) were markedly increased. The retroviral infection also influenced the local production of 1,25-(OH)2D3 in the spleen. It did not alter this production in monocyte/macrophages but increased that in isolated T cells. Isolated B cells in control mice did not produce 1,25-(OH)2D3, but they increased the ability of isolated T cells to produce this metabolite during coculture incubations. Infection altered this cell interaction as 1,25-(OH)2D3 production in infected T cells decreased when these cells were cocultured with infected B cells. Thus, chronic retroviral infection alters both the local vitamin D metabolism and VDR expression by immune cells in mice. These findings suggest close local interactions between 1,25-(OH)2D3 and immune system activation during retroviral infection.     

Antisense oligonucleotides targeted against protein kinase Cbeta and CbetaII block 1,25-(OH)2D3-induced differentiation

It is now recognized that protein kinase C (PKC) plays a critical role in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) promotion of HL-60 cell differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKCalpha, PKCbeta, PKCbetaI, and PKCbetaII on HL-60 promyelocyte cell differentiation and proliferation were examined. Cellular differentiation was determined by nonspecific esterase activity, nitro blue tetrazolium reduction, and CD14 surface antigen expression. Differentiation promoted by 1,25-(OH)2D3 (20 nM for 48 h) was inhibited similarly in cells treated with PKCbeta antisense (30 microM) 24 h prior to or at the same time as hormone treatment (86 +/- 9% inhibition; n = 4 versus 82 +/- 8% inhibition; n = 4 (mean +/- S.E.), respectively). In contrast, cells treated with PKCbeta antisense 24 h after 1, 25-(OH)2D3 were unaffected and fully differentiated. PKCalpha antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCbetaI- and PKCbetaII-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCbetaII antisense (30 microM) completely blocked CD14 expression induced by 1, 25-(OH)2D3, whereas PKCbetaI antisense had little effect. Interestingly, PKCbetaII antisense blocked differentiation by 87 +/- 7% (n = 2, mean +/- S.D.) but had no effect on 1,25-(OH)2D3 inhibition of cellular proliferation. These results indicate that the effects of 1,25-(OH)2D3 on HL-60 cell differentiation and proliferation can be dissociated by blocking PKCbetaII expression.     

A novel nonsense mutation in the first zinc finger of the vitamin D receptor causing hereditary 1,25-dihydroxyvitamin D3-resistant rickets

Hereditary 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-resistant rickets (HVDRR) is a rare autosomal recessive disorder resulting in target organ resistance to the active form of vitamin D [1,25-(OH)2D3]. Point mutations in the vitamin D receptor (VDR) gene have been identified in HVDRR. We investigated the molecular basis of HVDRR in a Brazilian family with two affected siblings. The propositus is a 12-yr-old boy born to first cousin parents who exhibited the classical pattern of the HVDRR, including early-onset rickets, total alopecia, convulsions, hypocalcemia, secondary hyperparathyroidism, and elevated 1,25-(OH)2D3 serum levels. His younger sister also developed clinical and biochemical features of HVDRR at 1 month of age and died at 4 yr of age. Genomic DNA was isolated from peripheral blood of the boy and from dried umbilical cord tissue of his affected sister. We amplified exons 2 and 3 of the VDR gene, which encode the zinc finger DNA-binding domain by PCR. Direct sequencing of the PCR products revealed a homozygous substitution of cytosine for thymine at nucleotide position 88 in exon 2 of the VDR gene in both affected siblings. This point mutation determined the substitution of a stop codon (TGA) for arginine (CGA) at amino acid position 30 at the first zinc finger of the DNA-binding domain of the VDR. This substitution generated a truncated receptor missing 397 residues. The parents and a normal sister were heterozygous for this mutation. In conclusion, we describe a novel nonsense mutation in the first zinc finger of the VDR that generated a severely truncated form of this receptor.     

Stimulation of osteoclast formation by 1,25-dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic cells: studies using VDR knockout mice

Previous studies have shown that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays important roles in the formation of osteoclasts through its actions on osteoblastic cells. We have generated mice lacking vitamin D receptor (VDR) by gene targeting (VDR-/-). These mice had tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and exhibited similar levels of parameters for bone resorption to those in wild type mice. The present studies were undertaken to clarify whether effects of 1,25(OH)2D on osteoclast formation require VDR in osteoblasts, and to examine mechanisms of the formation of osteoclasts without VDR-mediated actions using VDR-/- mice. When wild-type calvarial osteoblasts and spleen cells were co-cultured with 1,25(OH)2D, TRAP-positive osteoclasts were formed regardless of the genotypes of spleen cells. In contrast, when osteoblasts from VDR-/- mice were co-cultured, no osteoclasts could be formed even with wild-type spleen cells. Parathyroid hormone and interleukin-1alpha stimulated osteoclast formation by co-cultures from VDR-/- mice, and the generated osteoclasts showed resorbing activity. These results demonstrate that VDR-mediated actions of 1,25(OH)2D in osteoblasts are essential for osteoclast formation by 1,25(OH)2D, and that functionally intact osteoclasts can be formed without 1,25(OH)2D actions under stimulations by other agents. It is suggested that osteoclastic bone resorption can be maintained without 1,25(OH)2D actions by other stimulatory agents.     

The influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol, 25-hydroxycholecalciferol, and 1,25 dihydroxycholecalciferol in young broiler chickens

Three experiments were conducted to determine the influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol (vitamin D3), 25-hydroxycholecalciferol [25-(OH)D3], and 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in young broiler chicks. Two levels of vitamin A (1,500 and 45,000 IU/kg or 450 and 13,500 microg) were fed in all experiments. In Experiment 1, chicks were fed six levels of vitamin D3 (0, 5, 10, 20, 40, and 80 microg/kg). High dietary vitamin A decreased bone ash (P < 0.001), and increased the incidence of rickets (P < or = 0.02). Linear and quadratic responses to vitamin D3 levels were significant (P < 0.01) for body weight, bone ash, incidence and severity of rickets, and plasma calcium. In Experiment 2, six levels of 25-(OH)D3 (0, 5, 10, 20, 40, and 80 microg/kg) were added to the basal diet. Adding 25-(OH)D3 increased (P < 0.001) body weight, bone ash, and plasma calcium, and decreased rickets and plasma vitamin A. Adding 25-(OH)D3 overcame the reduction in bone ash produced by high dietary vitamin A showing a significant (P < 0.02) interaction. In Experiment 3, six levels of 1,25-(OH)2D3 (0, 2, 4, 8, 16, and 32 microg/kg) were added to the basal diet. High dietary vitamin A increased (P < 0.01) the incidence and severity of rickets. Adding 1,25-(OH)2D3 increased (P < 0.01) body weight, bone ash, plasma calcium, and reduced rickets and plasma and liver vitamin A. Adding 1,25-(OH)2D3 overcame the reduction in bone ash, and the increase in rickets produced by high vitamin A was significant (P < or = 0.05). These results indicate that high dietary vitamin A (45,000 IU/kg) interferes with the utilization of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3, increasing the requirement for each of them. Moreover, 45,000 IU/kg of dietary vitamin A ameliorated the potential toxic effects of feeding high levels of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3 to young broiler chickens. Further work is necessary to find the minimum levels of these vitamins needed to cause these effects.     

Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis

The serum level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D], the biologically most potent metabolite of vitamin D, is tightly regulated within narrow limits in human healthy adults. 1,25-(OH)2D deficiency is rare and is associated with disturbances in calcium and bone metabolism. We have previously reported a marked decrease in serum levels of 1,25-(OH)2D in human immunodeficiency virus (HIV)-infected patients. The present study was designed to further examine the causes and consequences of severe 1,25-(OH)2D deficiency in these patients. The design was a prospective cohort study. Fifty-four HIV-infected patients clinically classified according to the revised criteria from Centers for Disease Control and Prevention and healthy controls were studied. Parameters related to vitamin D and calcium metabolism as well as immunological and nutritional status were determined. Twenty-nine of the patients (54%) had serum levels of 1,25-(OH)2D below the lower reference limit, and 18 of these had undetectable levels. In contrast, HIV-infected patients had normal serum levels of 25-hydroxyvitamin D and vitamin D-binding protein. HIV-infected patients as a group had modestly depressed serum calcium and PTH levels. There were, however, no correlations between these parameters and serum levels of 1,25-(OH)2D. There were no differences in serum calcium or PTH levels or nutritional status when patients with severe 1,25-(OH)2D deficiency were compared to other patients, but patients with undetectable 1,25-(OH)2D had significantly elevated serum phosphate levels. Furthermore, patients with undetectable 1,25-(OH)2D levels were characterized by advanced clinical HIV infection, low CD4+ lymphocyte counts, and high serum levels of tumor necrosis factor-alpha (TNFalpha). We conclude that inadequate 1alpha-hydroxylation of 25-hydroxyvitamin D seems to be the most likely cause of 1,25-(OH)2D deficiency in HIV-infected patients, possibly induced by an inhibitory effect of TNFalpha. The low 1,25-(OH)2D and high TNFalpha levels observed may impair the immune response in HIV-infected patients both independently and in combination and may represent an important feature of the pathogenesis of HIV-related immunodeficiency. Markedly depressed 1,25-(OH)2D serum levels are also present in certain other disorders characterized by immunological hyperactivity. Thus, the findings in the present study may not only represent a previously unrecognized immune-mediated mechanism for induction of 1,25-(OH)2D deficiency in human disease, but may also reflect the importance of adequate serum levels of 1,25-(OH)2D for satisfactory performance of the immune system in man.