Experiments that changed nutritional thinking. Proceedings of a minisymposium. Atlanta, Georgia, April 1995 and Washington, DC, April 1996.
Explore the source record for details and available documents.
Diet↗
Biomedical subjects
H F DeLuca
Publications and source records attributed to H F DeLuca.
Retinol is specifically required during midgestation for neonatal survival.
Previous work has demonstrated that vitamin A-deficient, retinoic acid-supplemented pregnant rats cannot complete gestation without the administration of retinol. As little as 2 micrograms administered on day 10 of gestation is sufficient to prevent the characteristic fetal resorption that begins at day 15 of gestation. This single dose of retinol supports continued development through day 20 of gestation. However, if gestation is allowed to proceed to parturition, the newborn pups die within a few minutes of being severed from the umbilical cord. The pups are born with a pink and healthy skin tone, but within seconds of umbilical separation, they begin to gasp for air, become cyanotic in appearance, and die within several minutes from an apparent inability to obtain oxygen. Histological examination of these neonates demonstrates delayed pulmonary development. Branching and scalloping of ducts and saccule and subsaccule formation are decreased. This phenotype is consistent with that observed in respiratory distress syndrome seen in some premature human infants.
Animals↗
Hypophosphatemia and the development of rickets in osteopetrotic (op/op) mice.
Our previous work has shown that op/op mice hyperabsorb dietary calcium in the vitamin D-deficient state and shunt that calcium into bone. Under these conditions, the op/op mice are hypocalcemic. The purpose of this study was to examine calcium metabolism and bone mineralization in vitamin D-deficient op/op mice. First, the op/op mice and their normal littermates were placed on a vitamin D-deficient, low phosphorus diet to limit bone mineralization. Under these circumstances, op/op mice survived, even when calcium was also removed from the diet. If the diet contained phosphate, op/op mice died from hypocalcemic tetany when calcium was also removed from the diet. Furthermore, serum calcium levels became similar to wild type in the op/op mice administered the vitamin D-deficient, low phosphorus diet, and op/op mice were able to increase serum calcium in response to 1,25-dihydroxyvitamin D3. The op/op mice developed rickets when their serum phosphorus level was too low to support bone mineralization. The op/op mice became hypophosphatemic on regimens in which normal mice were able to maintain normal serum phosphorus levels. It appears that the op/op mouse simply requires a higher dietary calcium and phosphorus level to prevent rickets and hypocalcemic tetany since the bone is not available as a source of these minerals. However, the ability of the op/op mouse to mineralize bone at low serum calcium and phosphorus levels remains unexplained.
Animals↗
Association between intestinal vitamin D receptor, calcium absorption, and serum 1,25 dihydroxyvitamin D in normal young and elderly women.
The exact mechanism for the decrease in intestinal calcium absorption with age is not yet understood. A decrease with age in serum 1,25-dihydroxyvitamin D (1,25(OH)2D) or a decrease in the intestinal vitamin D receptor (VDR) protein concentration are possible causes. The objective of this study was to examine the effect of age on these factors. Fifty-nine young women age 25-35 years were compared with 41 elderly women age 65-83 years who underwent measurements of VDR, calcium absorption using a 20 mg and 100 mg calcium carrier, and calciotropic hormones. Calcium absorption by both tests was lower in the elderly women compared with the young women (p < 0.05). Serum 1,25(OH)2D and duodenal VDR protein concentration were not significantly different between the two age groups. Serum 1,25(OH)2D correlated with the 20 mg calcium absorption test in both young (r = 0.35, p < 0.007) and elderly women (r = 0.58, p < 0.0001) and with the 100 mg calcium absorption in the elderly (r = 0.32; p < 0.05). VDR did not correlate with calcium absorption in young women or elderly women, nor did VDR correlate with serum 1,25(OH)2D and serum 25-hydroxyvitamin D. In summary, the decrease in calcium absorption cannot be explained by a decrease in intestinal VDR. The correlation between serum 1,25(OH)2D and both calcium absorption tests only accounts for 12-30% of the variance in the age-related change in the calcium absorption tests. Other factors, not yet understood, are responsible for the decline in calcium absorption with age.
Adult↗
Vitamin D and multiple sclerosis.
Recently, it has been clearly demonstrated that exogenous 1,25-dihydroxyvitamin D3, the hormonal form of vitamin D3, can completely prevent experimental autoimmune encephalomyelitis (EAE), a widely accepted mouse model of human multiple sclerosis (MS). This finding has focused attention on the possible relationship of this disease to vitamin D. Although genetic traits certainly contribute to MS susceptibility, an environmental factor is also clearly involved. It is our hypothesis that one crucial environmental factor is the degree of sunlight exposure catalyzing the production of vitamin D3 in skin, and, further, that the hormonal form of vitamin D3 is a selective immune system regulator inhibiting this autoimmune disease. Thus, under low-sunlight conditions, insufficient vitamin D3 is produced, limiting production of 1,25-dihydroxyvitamin D3, providing a risk for MS. Although the evidence that vitamin D3 is a protective environmental factor against MS is circumstantial, it is compelling. This theory can explain the striking geographic distribution of MS, which is nearly zero in equatorial regions and increases dramatically with latitude in both hemispheres. It can also explain two peculiar geographic anomalies, one in Switzerland with high MS rates at low altitudes and low MS rates at high altitudes, and one in Norway with a high MS prevalence inland and a lower MS prevalence along the coast. Ultraviolet (UV) light intensity is higher at high altitudes, resulting in a greater vitamin D3 synthetic rate, thereby accounting for low MS rates at higher altitudes. On the Norwegian coast, fish is consumed at high rates and fish oils are rich in vitamin D3. Further, experimental work on EAE provides strong support for the importance of vitamin D3 in reducing the risk and susceptibility for MS. If this hypothesis is correct, then 1,25-dihydroxyvitamin D3 or its analogs may have great therapeutic potential in patients with MS. More importantly, current research together with data from migration studies opens the possibility that MS may be preventable in genetically susceptible individuals with early intervention strategies that provide adequate levels of hormonally active 1,25-dihydroxyvitamin D3 or its analogs.
Animals↗
A group of deltanoids (vitamin D analogs) regulate cell growth and proliferation in small cell carcinoma cell lines.
A group of deltanoids has been used for studying the inhibition of cell growth and proliferation in two small cell lung carcinoma (SCLC) in vitro. The biologically active deltanoid, 1,25 dihydroxyvitamin D3 (1,25 (OH)2D3), has functions beyond its classical roles of stimulating calcium transport and serum calcium. It also causes the differentiation of a variety of precursor cells and suppresses growth. Although 1,25(OH)2D3 has an inhibitory effect on growth of certain malignant cells, its hypercalcemic effect has prevented clinical applications. Several new deltanoids, which showed comparable or even greater abilities to induce differentiation and to inhibit proliferation, have been identified. Furthermore, these synthetic deltanoids have been shown to be less effective on calcium metabolism and less hypercalcemic. We have selected four synthetic deltanoids; MC-903, 1 alpha-OH-pregnacalciferol, 19-nor-24 homo, and 19-nor-22(E). When compared with 1,25 (OH)2D3, these deltanoids showed considerable potency on cell growth and proliferation in the NCI-H82 and the NCI-H209 SCLC lines. Cells were treated with various concentrations of deltanoids. They inhibited the growth and proliferation of both SCLC cells in vitro in a time-and dose-dependent manner, as determined by cell number and 3H-thymidine uptake. 19-nor-22(E) showed an antiproliferative effect significantly comparable to 1,25(OH)2D3 in the NCI-H82 cell line 1 alpha-OH-pregnacalciferol, 19-nor-24 homo, and 19-nor-22(E) inhibited the cell growth in the NCI-H209 cells within the same significance as 1,25 (OH)2D3. The degree of the suppressive effect of the deltanoids was cell line dependent.
Calcitriol↗
Synthesis and biological activity of 1 alpha, 25-dihydroxy-18-norvitamin D3 and 1 alpha, 25-dihydroxy-18,19-dinorvitamin D3.
1 alpha, 25-dihydroxy-18-norvitamin D3 and 1 alpha, 25-dihydroxy-18,19-dinorvitamin D3 were prepared via Wittig-Horner coupling of 25-hydroxy-18-nor Grundmann type ketone with the corresponding A-ring phosphine oxides. Configuration at C-13 in the 18-nor Grundmann type alcohol (C,D-ring synthon), obtained by oxidative degradation of vitamin D3, was determined by 1H NMR spectroscopy and molecular mechanics calculations. Additional proof of the assigned trans-C/D-junction of the key intermediate 18-nor Grundmann type ketone follows from its chiroptical properties (circular dichroism data) and further chemical transformations. 1 alpha, 25-Dihydroxy-18-norvitamin D3 was found more potent than 1 alpha, 25-dihydroxyvitamin D3 in binding to the porcine intestinal vitamin D receptor (5-10x), in differentiation of HL-60 cells (5-10x), and in inhibition of HL-60 proliferation. 1 alpha, 25-Dihydroxy-18, 19-dinorvitamin D3 appeared equally active as 1 alpha, 25-dihydroxyvitamin D3 in these activities. In vivo, 1 alpha, 25-dihydroxy-18-norvitamin D3 was only slightly less active than 1 alpha, 25-dihydroxyvitamin D3 in intestinal calcium transport and bone calcium mobilization, while 1 alpha, 25-dihydroxy-18,19-dinorvitamin D3 showed activities 10 times lower. These studies imply that deletion of C-18 does not impair activity of analogs of 1 alpha, 25-dihydroxyvitamin D3.
Animals↗
Analysis of binding of the 1,25-dihydroxyvitamin D3 receptor to positive and negative vitamin D response elements.
The binding of the 1,25-dihydroxyvitamin D3 receptor to the vitamin D response elements (VDREs) in the rat osteocalcin (OSC-DRE), mouse osteopontin (MOP-DRE), rat calbindin D-9k (CaBP-DRE), and human parathyroid hormone genes (PTH-DRE) was studied. Binding of VDR to the three positive VDREs is cooperative. The degree of cooperativity is highest with the calbindin VDRE compared with either the OSC-DRE or the MOP-DRE. This cooperativity is largely absent in the case of the negative element, the PTH-DRE. The VDR binds in order of decreasing affinity to PTH-DRE > OSC-DRE = MOP-DRE > CaBP-DRE. Thus, the greatest affinity is associated with the lowest degree of cooperativity. Further study has revealed that the PTH-VDRE actually consists of two repeat elements like all other VDREs and is not a single six-base sequence. A nuclear factor has also been found that binds downstream from the GGTTCA element in the PTH promoter. The binding site of this factor overlaps the PTH-DRE nucleotide sequence.
Animals↗
1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis.
Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease believed to be a model for the human disease multiple sclerosis (MS). Induced by immunizing B10.PL mice with myelin basic protein (MBP), EAE was completely prevented by the administration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. 1,25-(OH)2D3 could also prevent the progression of EAE when administered at the appearance of the first disability symptoms. Withdrawal of 1,25-(OH)2D3 resulted in a resumption of the progression of EAE. Thus, the block by 1,25-(OH)2D3 is reversible. A deficiency of vitamin D resulted in an increased susceptibility to EAE. Thus, 1,25-(OH)2D3 or its analogs are potentially important for treatment of MS.
Animals↗
Conformationally restricted analogs of 1 alpha, 25-dihydroxyvitamin D3 and its 20-epimer: compounds for study of the three-dimensional structure of vitamin D responsible for binding to the receptor.
Two proteins play important roles in the expression of vitamin D function: the specific nuclear receptor protein (vitamin D receptor, VDR) and the transport protein (vitamin D binding protein, DBP). This study was conducted to clarify the conformation of vitamin D responsible for binding to those proteins. For the purpose, the side chain mobility of 1,25(OH)2D3 (1) and its 20-epimer, 20-epi-1,25(OH)2D3 (2), was analyzed by a systematic conformational search. The results were depicted as a three-dimensional dot map, which indicates that the side chains of the two vitamins (1 and 2) occupy different spatial regions that are separated in two areas. We denoted these areas as A and G for 1 and EA and EG for 2. Four analogs, the diastereomers at C(20) and C(22) (3-6) of 22-methylated 1,25(OH)2D3 whose side chains were confined to occupy G, A, EA, and EG, respectively, were designed. These analogs (3-6) were synthesized efficiently by a stereoselective conjugate addition of organocuprate to steroidal E- and Z-22-en-24-ones as the key step. In binding to the VDR the affinities of the analogs (3-6) relative to 1,25-(OH)2D3 (1) were 1/60, 1/3, 20, and 1/100, respectively. These results indicate that the A region is responsible for binding of 1 to VDR and the EA region for binding of 2. Only isomer 4 showed significant affinity for DBP, indicating only the A region is responsible for binding to DBP. Thus, 5 showed clear separation of binding affinities for two proteins, VDR and DBP. Having the highest known VDR affinity, (22R)-22-methyl-20-epi-1,25(OH)2D3 (5) has potential both as a therapeutic agent and as a tool to study the molecular mechanism of vitamin D-mediated gene transcription.
Animals↗
Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme.
Human 25-hydroxyvitamin D-24-hydroxylase has been expressed in Spodoptera frugiperda (Sf21) insect cells using the previously cloned cDNA in baculovirus (AcNPV-P450cc24). The activity of recombinant h-P450cc24 required adrenodoxin, adrenodoxin reductase, and NADPH. Incubation of this reconstituted system with 25-OH-[26,27-(3)H]D3 substrate produced several metabolites that were resolved on a normal-phase cyano HPLC system. These products exactly comigrated with authentic standards for 24-oxo-25-OH-D3, 23(S),25-(OH)2D3, 24(R),25-(OH)2D3, and 24-oxo-23(S),25-(OH)2D3. The soluble proteins from Sf21 cells infected with wild-type baculovirus produced neither 24,25-(OH)2D3 nor any of the other 25-OH-D3 metabolites. The products were isolated and subjected to a normal-phase amino HPLC for further separation, purification, and characterization. Comigration on two HPLC systems, periodate cleavage reactions, and NaBH4 reduction established clearly the identity of these metabolites. Incubation of recombinant h-P450cc24 with 25-OH-[3 alpha-3H]D3 led to the isolation of an additional product that comigrated with 24,25,26,27-tetranor-23-OH-D3. Treatment of putative 24,25,26,27-tetranor-23-OH-[3 alpha-3H]D3 with acetic anhydride changed its migration on amino HPLC to a less polar position, indicating acetylation of a hydroxyl group(s). These data demonstrate conclusively that h-P450cc24 is a multicatalytic enzyme catalyzing most, if not all, of the reactions in the C-24/C-23 pathway of 25-OH-D3 metabolism. It is likely that this enzyme by itself converts 25-OH-D3 and 1,25-(OH)2D3 to one of its final excretion products.
Animals↗
Metabolites of all-trans-retinol in day 10 conceptuses of vitamin A-deficient rats.
It has been previously demonstrated that vitamin A-deficient rats supplemented with retinoic acid cannot complete gestation. Resorption of fetuses invariably occurs in these animals beginning Day 15 of gestation. Retinol must be administered on or before Day 10 of gestation in order to prevent this phenomenon. The administration of as little as 2 micrograms on Day 10 is sufficient to allow continuation of gestation through parturition. This study examines the metabolites of all-trans-retinol in the conceptuses of vitamin A-deficient, retinoic acid-supported pregnant animals at Day 10 of gestation. The retinoids recovered in this study could be identified as all-trans-retinol, retinyl palmitate, and other retinyl esters. Virtually no radiolabeled retinoic acid was found in the conceptuses of these animals over the 12-h period examined. This may suggest that retinoic acid that may be required by the fetus can arise directly from maternal sources. Additionally, the isolation of an early appearing, very polar metabolite of retinol is reported. This aqueous-soluble compound accounts for more than 20% of the radioactivity recovered at 1 h post-dose. The amount of this compound increases through 6 h post-dose. This metabolite is not found in urine from the same animals and is not likely to be an excretion product.
Animals↗
Effect of 1,25-dihydroxyvitamin D3 on mouse thymus: role of extracellular calcium.
We recently reported that mice treated with 1,25-dihydroxyvitamin D3 ( 1,25-(OH)2D3) or 19-nor-1,25-(OH)2D2 experienced a severe loss of their thymocytes and decreased proliferation in response to concanavalin A mitogen. The present study investigated the effect of short-term treatment with 1,25-(OH)2D3 on the thymic architecture and thymocyte subsets. Daily treatment with 1,25-dihydroxyvitamin D3 at 20 ng per mouse for 4 days induced significant involution of thymic tissue. The atrophy was predominantly observed in the cortical component. Flow cytometric analysis of thymocyte subsets showed that the CD4 + CD8 + population was the primary target. Since the treated mice experienced profound hypercalcemia, we studied the effect of 1,25-(OH)2D3 on animals fed a vitamin D-deficient, low calcium diet or the same diet containing vitamin D for 25 days prior to treatment. The low calcium fed mice showed severe hypocalcemia and slight thinning of thymic cortex. Treatment with 1,25-(OH)2D3 moderately improved the hypocalcemia but had no further effect on the thymus of these animals. On the other hand, hypercalcemia and thymic atrophy were found in the animals fed the diet containing vitamin D. Overall, the atrophy effect on the thymus caused by 1,25-(OH)2D3 treatment was prevented by eliminating the hypercalcemia observed in + D + Ca treated animals. Thus, thymic atrophy probably resulted from hypercalcemia and not from 1,25-(OH)2D3 itself.
Animals↗
An enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein.
In this paper, we detail an enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein. The receptor protein of cell and tissue homogenates is bound between two monoclonal antibodies specific for different epitopes on the receptor protein. The first antibody is bound to the well of an ELISA plate and the second is biotinylated. The receptor-antibody complex is detected with avidin-alkaline phosphatase and rho-nitrophenyl phosphate. The amount of receptor in each sample is determined by comparison with a standard curve made from purified receptor protein. This assay is highly sensitive, measuring as little as 2 fmol of receptor, and has an intra-assay coefficient of variation of 6.6% and an interassay coefficient of variation of 13.8%. The assay can be used to measure the receptor from mammalian and avian species and is independent of the presence of hormone. By eliminating the need for a radio-iodinated monoclonal antibody and incorporating the ease of a plate assay, we have a significantly improved method for measuring the vitamin D receptor protein. This paper also presents Western analysis of the antibodies used to demonstrate that they do not recognize other steroid hormone receptors.
Animals↗
Recent advances in the molecular biology of vitamin D action.
Following the cloning and deletion analysis of the vitamin D receptor, most recent advances have been in the isolation and characterization of the DNA response elements found in the promoter region of target genes of vitamin D. Vitamin D, like the thyroid and retinoid hormones, binds to repeat sequences, but the repeats are separated by three nonspecified bases. The action of the VDR requires the presence of the RXR proteins and evidently other proteins that are involved in regulating transcriptions. A possible role of phosphorylation of the ligand binding domain of the VDR in transcription has also appeared. Very likely, the molecular events involved in vitamin D stimulation or suppression of a target gene will include its interaction with a number of transcription factors, both in the regulation of transcription and in the actual machinery involved in the transcription process through polymerase II. Although likely, it is not entirely clear whether the genomic action of vitamin D can account for all of its biological activities. Nongenomic actions of the vitamin D hormone have been reported, but convincing evidence that this is of biological importance in vivo is lacking. Advances in our understanding of the vitamin D mechanism of action can clearly be expected from physical studies of cloned and expressed vitamin D receptor and its subdomains, elucidation of the transcription factors in vitamin D-modulated transcription of target genes, elucidation of the role of phosphorylation in the transcription process, and the identification of important genes that are regulated in the specific target tissues responsive to vitamin D. This will definitely remain as a very active field of investigation well into the future.
Animals↗
Osteopetrotic (op/op) mice are unable to maintain serum calcium levels despite hyperabsorption of calcium.
The effect of vitamin D on serum calcium, vitamin D receptor (VDR) protein level, and fate of radioactive calcium ingested were compared in vitamin D-deficient osteopetrotic (op/op) mice and their wild-type vitamin D-deficient littermates. Vitamin D deficiency was achieved in mice after feeding them a vitamin D-deficient diet for 5-6 weeks. Serum calcium did not increase in op/op mice in response to 1,25-dihydroxyvitamin D3. Furthermore, op/op mice were not capable of increasing serum calcium levels by passively absorbing calcium from the diet. These defects in calcium homeostasis were neither the result of the inability of the VDR to bind its ligand, as determined by the hydroxylapatite assay, nor the result of abnormal regulation of intestinal VDR, as determined by the enzyme-linked immunosorbent assay. Upon administration of radioactive calcium by oral gavage, it was found that vitamin D-deficient, op/op mice had an extremely efficient mechanism to absorb dietary calcium, but the calcium absorbed was readily shunted into bone; thus serum calcium did not increase. In addition, op/op mice also possessed vitamin D-stimulated intestinal calcium absorption, but this process was muted by the overwhelming vitamin D-independent mechanism of dietary calcium absorption.
Administration, Oral↗
Vitamin D3 as a possible chemopreventive and therapeutic agent of cancers.
Explore the source record for details and available documents.
Cholecalciferol↗
Transcriptional control of the osteocalcin gene by 1,25-dihydroxyvitamin D-2 and its 24-epimer in rat osteosarcoma cells.
The effects of two vitamin D analogs, 1,25-dihydroxyvitamin D-2 and 24-epi-1,25-dihydroxyvitamin D-2, were examined on osteocalcin gene expression in the rat osteosarcoma cell line ROS 17/28. Our results indicate that these analogs are more transcriptionally active than 1,25-dihydroxyvitamin D-3, particularly the 24-epimer. Assessment of reporter gene chloramphenicol acetyltransferase (CAT) activity, using the vitamin D responsive element (VDRE) derived from the human osteocalcin gene promoter. revealed that both analogs stimulated CAT activity 5- to 10-fold. 1,25-Dihydroxyvitamin D-2 was slightly more active than 1,25-dihydroxyvitamin D-3, while the 24-epimer was twice as effective. 1,25-Dihydroxyvitamin D-3 also stimulated osteocalcin mRNA accumulation by 2-fold over vehicle-treated cells, 1,25-dihydroxyvitamin D-2 by 2.5-fold, and 24-epi-1,25-dihydroxyvitamin D-2 by 4-fold. Electrophoretic mobility shift assays using the osteocalcin vitamin D responsive element revealed no increase in DNA binding with either analog when compared to 1,25-(OH)2D3. Examination of CAT activity using the rat 24-hydroxylase VDRE indicated no significant difference in transcription with these compounds, suggesting that the vitamin D-2 analogs preferentially activate osteocalcin gene expression.
Animals↗