Amaral, Jennifer M.; Abrams, Steve; Karaviti, Lefkothea; McKay, Siripoom V.
Background. Late transient neonatal hypocalcemia with hyperphosphatemia is potentially life-threatening. The use of dihydroxycholecalciferol in the management of neonatal hypocalcemia is unexplored. Objective. We hypothesized adding dihydroxycholecalciferol to intravenous continuous calcium infusion (CaI) will achieve accelerated correction of hypocalcemia. Design/Methods. A controlled double-blind randomized placebo group was organized to compare the addition of dihydroxycholecalciferol ...
Skliar, M I; Fernandez, M C; Faienza, H; Orsatti, M B; Puche, R C; Boland, R L; Skliar, M I
The erythrocytes of rats treated with 1, 25-dihydroxycholecalciferol or 1, 25-dihydroxycholecalciferol glycoside showed decreased levels of 2, 3-diphosphoglycerate. The same result has been obtained in vitro, indicating a direct effect of the sterol on the red cell. The glycoside is less active than the free sterol in vivo and more active in vitro. The decreased levels of diphosphoglycerate induced tissue hypoxia as shown by a higher plasma lactate/pyruvate ratio and a three fold increase in plasma erythropoietin concentration.
Full Text Available Background. Late transient neonatal hypocalcemia with hyperphosphatemia is potentially life-threatening. The use of dihydroxycholecalciferol in the management of neonatal hypocalcemia is unexplored. Objective. We hypothesized adding dihydroxycholecalciferol to intravenous continuous calcium infusion (CaI will achieve accelerated correction of hypocalcemia. Design/Methods. A controlled double-blind randomized placebo group was organized to compare the addition of dihydroxycholecalciferol to CaI in 3–14 day old neonates presenting with hypocalcemia, hyperphosphatemia and seizures. Ionized calcium and phosphorus were measured to adjust CaI and maintain eucalcemia. Time to resolution of hypocalcemia was defined as time from starting CaI to the first ionized calcium of mmol/L. CaI was discontinued when ionized calcium levels were mmol/L on two measurements and the infant tolerated feeds. Results. Fourteen neonates were studied without statistical difference between groups. Time to correction of hypocalcemia for 1,25 dihydroxycholecalciferol versus placebo was versus hours respectively (. The duration of CaI was versus hours respectively (. Conclusions. The addition of dihydroxycholecalciferol to standard CaI therapy reduced the duration of CaI, but did not reduce the time to correct hypocalcemia in neonates with late transient hypocalcemia.
Kanis, J.A.; Cundy, T.; Bartlett, M.; Smith, R.; Heynen, G.; Warner, G.T.; Russell, R.G.G.
Small doses (1 to 10 μg daily) of 24,25-dihydroxycholecalciferol (24,25-(OH) 2 D 3 ), a renal metabolite of vitamin D of uncertain function, increased intestinal absorption of calcium in normal people and in patients with various disorders of mineral metabolism, including anephric subjects. In five of six patients studied, calcium balance increased, but, unlike 1,25-dihydroxycholecalciferol, 24,25-(OH) 2 D 3 did not increase plasma or urinary calcium concentrations. These results suggest that 24,25-(OH) 2 D 3 may be an important regulator of skeletal metabolism in man with potential value as a therapeutic agent. (author)
Full Text Available Background. Late transient neonatal hypocalcemia with hyperphosphatemia is potentially life-threatening. The use of 1.25 dihydroxycholecalciferol in the management of neonatal hypocalcemia is unexplored. Objective. We hypothesized adding 1.25 dihydroxycholecalciferol to intravenous continuous calcium infusion (CaI will achieve accelerated correction of hypocalcemia. Design/Methods. A controlled double-blind randomized placebo group was organized to compare the addition of 1.25 dihydroxycholecalciferol to CaI in 3–14 day old neonates presenting with hypocalcemia, hyperphosphatemia and seizures. Ionized calcium and phosphorus were measured to adjust CaI and maintain eucalcemia. Time to resolution of hypocalcemia was defined as time from starting CaI to the first ionized calcium of ≥1.1 mmol/L. CaI was discontinued when ionized calcium levels were ≥1.1 mmol/L on two measurements and the infant tolerated feeds. Results. Fourteen neonates were studied without statistical difference between groups. Time to correction of hypocalcemia for 1,25 dihydroxycholecalciferol versus placebo was 7.2 ± 1.9 versus 11.5 ± 3.4 hours respectively (p=.26. The duration of CaI was 15.0 ± 1.5 versus 24.8 ± 4.4 hours respectively (p=.012. Conclusions. The addition of 1.25 dihydroxycholecalciferol to standard CaI therapy reduced the duration of CaI, but did not reduce the time to correct hypocalcemia in neonates with late transient hypocalcemia.
Plachot, J J; Du Bois, M B; Halpern, S; Cournot-Witmer, G; Garabedian, M; Balsan, S
Growth plates of 18-day-old rabbits were incubated in a protein-free synthetic medium, either without any additive, with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] (10(-10) M), with 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] (10(-10) M and 10(-9) M), with both metabolites, or with the ethanol solvent alone. Cartilages, before and after 5 days of incubation, were studied by light and electron microscopy. The intracellular calcium distribution was analyzed by the potassium pyroantimonate method, and the calcium content was verified by x-ray microprobe analysis. When compared to nonincubated samples the cartilages incubated for 5 days without any additive as well as the cartilages incubated with the solvent alone showed excessive hydratation and hypertrophy of the chondrocytes, which had lost their columnar arrangement. The matrix and the cells were devoid of mineral. The ultrastructure of the cells was well preserved. These changes were largely prevented by the presence of both vitamin D3 metabolites. With regard to calcium distribution, 1,25-(OH)2D3 maintained calcium in mitochondria and crystals in matrix vesicles, whereas 24,25-(OH)2D3 only partly maintained mitochondrial mineral. In the chondrocytes incubated with this latter metabolite, small calcium granules were seen in the cytoplasm; most vesicles were devoid of crystals, and amorphous precipitates were seen in the matrix. These data demonstrate the in vitro influence of vitamin D3 metabolites on the organization and mineralization of the cartilage matrix and on the distribution of intracellular calcium in chondrocytes. Furthermore, they support the hypothesis that the in vitro action of 1,25-(OH)2D3 is different from that of 24,25-(OH)2D3 in that 1,25-(OH)2D3 may influence calcium storage in mitochondria and matrix vesicles, whereas 24,25-(OH)2D3 is likely to be involved in calcium transport and release.
O'Riordan, J.L.H.; Hendy, G.N.; Fraher, L.J.; Papapoulos, S.E.; Sandler, L.M.; Clemens, T.L.
Recent interest is centred on the development of improved assays for 1,25-dihydroxycholecalciferol (1,25-(OH) 2 D 3 ) the hormonal form of vitamin D. Because radioimmunoassays have been successful when applied to the measurement of steroid hormones it seemed reasonable to use this approach to assay 1,25-(OH) 2 D 3 . These techniques have now been developed and applied to clinical studies. Clearly, the radioimmunoassay using tritium as label, is proving to be a versatile tool in the clinical study of the production and disposition of the hydroxylated vitamin D metabolites. This technique has a distinct practical advantage over existing radioreceptor assays in that antibodies provide a continuous supply of stable binding reagent whereas radioreceptor assays require preparation of intestinal binding protein from vitamin D deficient chickens. Moreover, with the radioimmunoasay approach there is considerable potential for further development. For example, by immunizing more animals with the same or different immunogens it may be possible to produce antisera with even higher affinity and with greatest structural specificity. These possibilities are now being investigated. (Auth.)
Koo, S.I.; Fullmer, C.S.; Wasserman, R.H.
The effect of cholecalciferol on the intestinal absorption of 65 Zn was assessed in zinc-deficient and zinc-replete rachitic chicks, using the in situ ligated loop techniques. Cholecalciferol did not significantly affect 65 Zn absorption in either group, although the synthesis of the intestinal calcium-binding protein (CaBP) in both groups was similar. In an analogous study, 1,25-dihydroxycholecalciferol increased 47 Ca absorption and induced the synthesis of CaBP but exerted no effect on 65 Zn absorption in zinc-deficient rachitic chicks. When fed a diet adequate in cholecalciferol, more CaBP was present in the intestine of the zinc-adequate group than in the zinc-deficient group, possibly due to the greater rate of growth and therefore the greater need for calcium by the former group. These results suggest that cholecalciferol and its most active metabolite do not directly affect zinc absorption and, by inference, that the vitamin D-dependent transport mechanism is not involved in zinc homeostasis, or in the interaction between calcium and zinc
Goulding, A.; McIntosh, J.; Campbell, D.
Metabolic balance studies were undertaken to determine whether sodium bicarbonate (NaHCO 3 ) supplements (4.5 mmol/day) altered 7-day cumulative calcium (Ca) phosphorus (P) balances in growing rats consuming either a basal diet providing 0.6% Ca and 0.3% P, or this diet plus 1,25-dihydroxycholecalciferol [40 ng 1,25(OH) 2 D 3 /day]. Feeding bicarbonate lowered urinary Ca but raised fecal Ca so that Ca balance became less positive. However, 1,25(OH) 2 D 3 increased net absorption of Ca and P to the same degree when given to control rats and rats consuming bicarbonate. Nevertheless, bicarbonate-fed rats had lower net Ca absorption than controls, even when treated with high doses of 1,25(OH) 2 D 3 . Changes in net Ca absorption induced by bicarbonate may occur at a point in the gut distal to the duodenum since duodenal 45 Ca absorption was decreased by bicarbonate feeding. The present results show that bicarbonate consumption depressed net Ca absorption in the rat. The effect appears to be independent of changes in 1,25(OH) 2 D 3 metabolism because it is manifest in animals receiving high doses of 1,25(OH) 2 D 3 , which stimulate alimentary Ca absorption maximally, and because bicarbonate-fed rats are able to respond normally to exogenous 1,25(OH) 2 D 3 by increasing their net absorption of Ca and P. In view of this demonstration that NaHCO 3 supplements elevate fecal Ca loss in the rat, it is suggested that studies should be undertaken to determine whether bicarbonate exerts similar adverse effects on Ca balance in humans
Dudeja, P.K.; Wali, R.K.; Klitzke, A.; Sitrin, M.D.; Brasitus, T.A.
Diabetes was induced in rats by administration of a single i.p. injection of streptozotocin (50 mg/kg body wt). After 7 d, diabetic rats were further treated with insulin or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] for an additional 5-7 d. Control, diabetic, diabetic + insulin, and diabetic + 1,25(OH)2D3 rats were then killed, their proximal small intestines were removed, and villus-tip epithelial cells were isolated and used to prepare brush-border membrane vesicles. Preparations from each of these groups were then analyzed and compared with respect to their amiloride-sensitive, electroneutral Na(+)-H+ exchange activity, using 22 Na uptake as well as acridine orange techniques. The results of these experiments demonstrated that (a) H+ gradient-dependent 22 Na uptake as well as Na+ gradient-dependent transmembrane H+ fluxes were significantly increased in diabetic vesicles compared to their control counterparts, (b) kinetic studies demonstrated that this enhanced 22 Na uptake in diabetes was a result of increased maximal velocity (Vmax) of this exchanger with no change in apparent affinity (Km) for Na+, (c) serum levels of 1,25(OH)2D3 were significantly lower in diabetic animals compared with their control counterparts; and (d) insulin or 1,25(OH)2D3 treatment restored the Vmax alterations to control values, without any significant changes in Km, concomitant with significantly increasing the serum levels of 1,25(OH)2D3 in diabetic animals. These results indicate that Na(+)-H+ activity is significantly increased in proximal small intestinal luminal membranes of streptozotocin-induced diabetic rats. Moreover, alterations in the serum levels of 1,25(OH)2D3 may, at least in part, explain this enhanced antiporter activity and its correction by insulin
Caniggia, A; Vattimo, A
Measurement of 47Calcium absorption was performed on eleven women with post-menopausal osteoporosis. The study was repeated after 10 days treatment with 1 microgram daily of 1,25(OH)2D3. These patients showed a statistically significant improvement of fractional calcium absorption that was inversely correlated to the basal values. The prompt improvement of the intestinal calcium transport in post-menopausal osteoporotic women, a few days after the administration of physiological doses of 1,25(OH)2D3, suggests that these patients synthesize inappropriately small amounts of 1,25(OH)2D3 because of their oestrogen deficiency. This could be an important pathogenetic factor in post-menopausal osteoporosis, as the efficiency of the adaptation of calcium absorption to low calcium intakes is dependent on 1,25(OH)2D3.
The effects of vitamin D3 follow its metabolisation in the liver and then in the kidney. Its most active metabolite is 1,25 (OH)2D3, produced by the liver precursor 25(OH)D3. In chronic renal insufficiency, demineralising osteopathy can be corrected by administering 1,25 (OH)2D3 to make up for its under-production by the kidneys. An assessment if is made of 47Ca intestinal transport in patients with chronic renal insufficiency before and after such treatment. It was found that the effects of the metabolite on calcium transport were dose-dependent.
Wasserman, R H; Bar, A; Corradino, R A; Taylor, A N; Peterlik, M
Some properties of the vitamin D dependent CaBP have been briefly summarized. In addition to providing possible insight into the molecular basis of vitamin D action, the measurement of intestinal CaBP in animals subjected to different conditions and treatments has proven useful in assessing the effective vitamin D status of that animal. Using measurements of both the degree of intestinal /sup 47/Ca absorption in situ and duodenal CaBP levels, some aspects of the vitamin D-like factor in the South American plant Solanum malacoxylon were investigated. A vitamin D assay based on CaBP as end point indicated that the plant contains about 1.3 x 10/sup 5/ IU vitamin D/sub 3/ equivalents per kg. The Solanum factor, together with an adequate calcium intake, are necessary conditions for the product of gross toxic symptoms in the chick. Using experimental conditions that inhibit the conversion of 25-(OH)D/sub 3/ to 1,25-(OH)/sub 2/D/sub 3/ by the kidney enzyme system (i.e., a high stable strontium diet), it was shown that the Solanum factor can cause a reversal of this inhibition. This suggested that the Solanum factor mimics the action of 1,25-(OH)/sub 2/D/sub 3/, and this was confirmed by Walling and Kimberg (personal communication) since, in their hands, the administration of S. malacoxylon extract to nephrectomized rats was able to stimulate intestinal calcium transport in vitro. Similar results were brought forth at this meeting by Dr. Mautalen of Argentina. The Solanum factor was effective in an intestinal organ culture system, indicating that the factor acts directly on the gut and, if modification of the factor is needed for biological activity, the necessary enzymes are present in the intestinal tissue.
A sensitive radioimmunoassay for 1,25-dihydroxycholecalciferol, the most potent derivative of vitamin D 3 is described and has been used to measure its concentration in human serum from normal subjects, patients with clinical osteomalacia,, anephric patients and those with chronic renal failure. The absorption and clearance of 1,25-dihydroxycholecalciferol have been studied in healthy and uremic patients by following the changes of its circulating concentration after administration of a single (2ug) dose of synthetic 1,25-dihydroxycholecalciferol orally. The radioimmunoassay was also used to investigate the underlying cause of the hypercalcaemia of sarcoidosis. (author)
Fairney, A.; Baggiolini, E.; Uskokovic, M.R.
The invention relates to the determination of 1 α, 25-dihydroxycholecalciferol and of optical enantiomers and racemates thereof. More particularly, the invention is concerned with a radioimmunoassay and a reagent for the determination of 1 α, 25-dihydroxycholecalciferol and of optical enantiomers and racemates thereof, with novel antigens and antibodies useful in the said process and with a process for the preparation of the said antigens and antibodies. The invention is also concerned with novel haptens useful in the preparation of said antigens and with a process for the preparation of said haptens. (author)
nuclear pellet was resuspended in Buffer A, Cytometer (Coulter Corp., Miami, FL) equipped with an argon laser . sonicated 2 x 10 s, and stored at -80 ’C...rickets type II with alopecia . Proc Natl Acad Sci  Mezzetti G, Barbiroli B, Oka T. 1,25-Dihydroxycholecalciferol re- USA 1997;94:9831-5. ceptor
Mykkanen, H.M.; Wasserman, R.H.
The uptake of selenite by purified brush border membrane vesicles isolated from duodena of rachitic or vitamin D-treated chicks was studied by using radioactive selenite and a rapid filtration technique. Cholecalciferol treatment (500 IU at 72 h) significantly enhanced selenite uptake, a response that decreased when the vesicles were stored at room temperature for 2.5 h prior to the uptake measurement. Preincubation of the vesicles in 1.0 mmol/L H2O2 reduced [75Se]selenite uptake, indicating the involvement of oxidizable groups in the uptake reaction. Iodoacetic acid (IAA), a sulfhydryl-blocking reagent, at 1-2 mmol/L concentration eliminated the difference in selenite uptake due to cholecalciferol and had no effect on vesicles from rachitic animals. A higher concentration of IAA (10 mmol/L) enhanced selenite uptake manyfold and increased the absolute difference due to cholecalciferol treatment. Single intravenous doses of 100 IU cholecalciferol, 100 IU ergocalciferol, or 0.1 micrograms 1,25-dihydroxycholecalciferol also stimulated selenite uptake, suggesting a general response to vitamin D compounds. Normal animals given a single dose of 1,25-dihydroxycholecalciferol 12 h prior to killing also responded. Treatments that enhanced the uptake of [75Se]selenite also increased the amount of membrane-bound sulfhydryl groups, suggesting the involvement of membrane-bound sulfhydryl groups in the vitamin D response. A significant increase in selenite uptake by intravenous 1,25-dihydroxycholecalciferol occurred within 10 min. This rapid effect provides a new tool to probe early biochemical effects of vitamin D on intestinal epithelium
Eilam, Y; Szydel, N; Harell, A
Both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) exerted direct effects on Ca2+ transport and accumulation in primary cultures of bone cells. The following changes were recorded. (1) A significant decrease in the amount of intracellular exchangeable Ca2+. (2) A marked increase in the rate constants of efflux from the 'slow'-turnover intracellular Ca pool. (3) A marked increase in the 'initial rate' of Ca influx into the cells. Thus, vitamin D metabolites caused an increase in the turnover of Ca2+ in bone cells and altered the steady-stae level of intracellular exchangeable Ca2+. Whereas the changes in the rate of efflux were abolished in the presence of inhibitors of protein synthesis, the increase in the rate of influx was not sensitive to these inhibitors. It is suggested that the changes in the two fluxes were mediated by different mechanisms and that the changes in influx were due to a direct effect of vitamin D metabolites on the cellular membranes.
Ferrari, S; Drusiani, E; Battini, R; Fregni, M
Calbindin D28K (formerly Vitamin D-Dependent Calcium Binding Protein) is a protein induced by 1,25-dihydroxycholecalciferol in several chicken tissues. A chicken genomic DNA library was screened with a synthetic oligonucleotide representing the sequence of Calbindin D18K cDNA from nt 146 to nt 176. The positive clone CBAl extends the 5'-end of the first exon by 451 bp. The sequence of a BamHI-SacII restriction fragment with coordinates -451 + 50 is shown. The BamHI-SacII fragment was subcloned 5' to the CAT gene of pUCCAT. The result is shown of a CAT assay on mouse fibroblasts 3T6 transiently transfected with pUCCAT, pUCCAT containing the BamHI-SacII fragment in the correct or opposite orientation or the SV40 promoter. /sup 14/C-chloramphenicol and its acetyl derivatives generated by purified CAT are also shown. The expression of CAT appears to be constitutive since the enzyme activity is not influenced by the presence (+) or absence (-) of 1,25-dihydroxycholecalciferol in the culture medium.
Shi Yuyuan; Zhang Peiyin; Li Yajuan
A modifed radioreceptor assay for 1,25-(OH) 2 -D 3 (1,25-Dihydroxy vitamin D 3 , Dihydroxycholecalciferol, DHCC) in serum was established, which was proved to have high sensitivity, stability and accuracy, and was applied in patients with rachitis. The results showed that DHCC level in serum in rachictic patients was lower than that of the control. The detection rate of DHCC was obviously higher than that through symptoms, laboratory tests and X-ray examination in patients with rachitis. Therefore, this assay technique can be used as a sensitive indicator for diagnosis of rachitis. It was also found that DHCC level in mothers was cor-relative with their infants, so that assay for DHCC level in pregnant mothers has significance in prevention and treatment of rachitis in their infants
Crissey, Susan D; Ange, Kimberly D; Jacobsen, Krista L; Slifka, Kerri A; Bowen, Phyllis E; Stacewicz-Sapuntzakis, Maria; Langman, Craig B; Sadler, William; Kahn, Stephen; Ward, Ann
Serum concentrations of several nutrients were measured in 12 captive wild felid species including caracal (Felis caracal), cheetah (Acinonyx jubatus), cougar (Felis concolor), fishing cat (Felis viverrinus), leopard (Panthera pardus), lion (Panthera leo), ocelot (Felis pardalis), pallas cat (Felis manul), sand cat (Felis margarita), serval (Felis serval), snow leopard (Panthera uncia) and tiger (Panthera tigris). Diet information was collected for these animals from each participating zoo (Brookfield Zoo, Fort Worth Zoo, Lincoln Park Zoological Gardens and North Carolina Zoological Park). The nutritional composition of the diets at each institution met the probable dietary requirements for each species except for the pallas cat. Blood samples were collected from each animal (n = 69) and analyzed for lipids (total cholesterol, triacylglycerides, HDL cholesterol and LDL cholesterol), vitamin D metabolites [25-hydroxycholecalciferol (25(OH)D) and 1,25-dihydroxycholecalciferol (1,25(OH)(2)D)], vitamin A (retinol, retinyl stearate and retinyl palmitate), vitamin E (alpha- and gamma-tocopherol) and selected carotenoids. Species differences were found for all except triacylglycerides and 1,25(OH)(2)D. Genus differences were found for retinol, retinyl palmitate, retinyl stearate, gamma-tocopherol and beta-carotene. Circulating nutrient concentrations for many of the species in this study have not been reported previously and most have not been compared with the animals' dietary intakes. The large number of animals analyzed provides a substantial base for comparing the serum nutrient concentrations of healthy animals, for both wild and captive exotic species.
Jespersen, B; Jensen, J D; Nielsen, H K
Bone mineral content, estimated by single-photon absorptiometry of the forearm, serum values of intact parathyroid hormone (PTH(1-84], osteocalcin, alkaline phosphatase, 1,25-dihydroxycholecalciferol (1,25(OH)2D3), and aluminium were determined during treatment with calcium carbonate (CaCO3......) or aluminium hydroxide (Al(OH)3) in 11 dialysis patients participating in a randomised cross-over study. Each treatment period lasted 6 months. Serum phosphorus was maintained in the range 1.5-2.0 mmol/l. During Al(OH)3 treatment bone mineral content (BMC) decreased by 11% per half-year (mean), but only by 3...... 0.05), osteocalcin decreased (89% versus 117%, P less than 0.01), alkaline phosphatase decreased (92% versus 116%, P less than 0.05), and aluminium decreased (56% versus 189%, P less than 0.05). 1,25(OH)2D3 remained unchanged in both periods. No increase in soft-tissue calcification was demonstrated...
Shireman, R.B.; Williams, D.; Remsen, J.F.
The plasma distribution and cellular uptake of [ 3 H]vitamin D 3 was studied in vitro using cultured human fibroblasts. Incubation of [ 3 H]vitamin D 3 (cholecalciferol) with plasma followed by sequential ultracentrifugal fractionation of the lipoproteins indicated that 2-4% of the radioactivity associated with the very low density lipoprotein (VLDL), 12% with low density lipoprotein (LDL), and approximately 60% with the high density lipoprotein (HDL). The remaining radioactivity, 25%, was associated with the sedimented plasma fractions. By comparison, an average of 86% of the radioactivity from [ 3 H] 1,25-dihydroxycholecalciferol associated with the sedimented plasma fractions. The uptake of [ 3 H]vitamin D 3 from plasma, LDL, or HDL was studied in cultured human cells; uptake by normal fibroblasts was greatest from LDL and least from plasma. The cellular association of vitamin D 3 was time, concentration, and temperature dependent. At a concentration of 50 μg LDL/ml of medium, the uptake of [ 3 H]vitamin D 3 from LDL at 37 0 C was rapid and reached a maximum at approximately 4 hr; it was slower from HDL but continued to increase slowly up to 24 hr. The significance of these in vitro findings is uncertain since much of the vitamin D 3 absorbed from the intestine reportedly associates with chylomicrons and is rapidly taken up by the liver
Inoue, D; Santiago, P; Horne, W C; Baron, R
Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.
Hsu, Chen Hsing; Yang, Chweishiun; Patel, S.R.; Stevens, M.G.
The authors have studied the effect of dietary vitamin D restriction on serum levels of vitamin D metabolites, measured by radioreceptor assay and radioimmunoassay in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Both WKY and SHR were fed a vitamin D-deficient or a vitamin D-supplemented diet beginning at 4 wk of age. In vitamin D-supplemented animals, the serum 1,25-dihydroxycholecalciferol [1,25(OH) 2 D 3 ] concentration of WKY was similar to the level of SHR. Plasma calcium concentration was not different between WKY and SHR. In animals fed a vitamin D-deficient diet, the serum concentration of 1,25-(OH) 2 D 3 of SHR was significantly lower than that of WKY. Plasma 25-hydroxycholecalciferol level was markedly decreased in both WKY and SHR. The SHR, but not the WKY, developed hypocalcemia. Despite hypocalcemia, fasting urinary Ca 2+ excretion of SHR exceeded that of WKY. They conclude that the lower 1,25(OH) 2 D 3 level in SHR fed a vitamin D-deficient diet may be due to a defect in the synthesis of 1,25(OH) 2 D 3 . The low level of 1,25(OH) 2 D 3 is associated with renal wasting of calcium and hypocalcemia in SHR
Han, Jinyou; He, Hong
The aim of the study was to investigate the effects of piezosurgery in accelerating the movement of orthodontic alveolar bone tooth of rats and the expression mechanism of bone morphogenetic protein-2 (BMP-2). Adult male Wistar rats (n=30), with an age range of 14-15 weeks, and an average weight of 250±16 g were used. The animals were randomly divided into the control and observation groups. The rats in the control group were injected with 25-dihydroxyvitamin (1,25-dihydroxycholecalciferol) into their dental ligament. The rats in the observation group were placed with an orthodontic device between the first molar and central incisor in the maxillary. On the first day after animal treatment, piezosurgery stimulation was performed on the first molar in maxillary. The changes of the movement distance of the first molar and gum surface temperature on days 1, 3, 5, 7 and 14 were then compared. Immunohistochemical staining was performed to detect the expression of BMP-2 of periodontal tissue in the tension side of the first molar. Tooth movement distance in the observation group on days 5, 7 and 14 was significantly longer than that in the control group (ppiezosurgery may significantly accelerate the movement of orthodontic alveolar bone tooth of rats and be associated with an increasing BMP-2 expression.
Woloszynska-Read, Anna; Johnson, Candace S.; Trump, Donald L.
There are substantial preclinical and epidemiologic data that suggest that vitamin D plays a role in the prevention and treatment of cancer. Numerous observational studies have shown that low blood levels of 25(OH) vitamin D (cholecalciferol), estimated by geographical location, diet and activity assessment or measured serum levels are associated with a higher risk of cancer and worse cancer-specific survival as well as numerous morbidities to e.g. cardiovascular disease, stroke, infection, autoimmune disease, and neuromuscular dysfunction among large populations. A considerable number of in vitro and in vivo studies indicate that the most active metabolite of vitamin D – 1,25-dihydroxycholecalciferol or calcitriol – has anti-proliferative, pro-apoptotic, pro-differentiating, and anti-angiogenic properties. Combined treatment of calcitriol and many types of cytotoxic agents has synergistic or at least additive effects. However, clinical trials testing these hypotheses have been less encouraging, though a number of methodological, pharmacological, and pharmaceutical issues confound all trials ever conducted. In order to properly assess the clinical value of vitamin D, its metabolites and analogs in cancer prevention and treatment, more studies are needed. PMID:21872802
Yingyu Ma, Donald L. Trump, Candace S. Johnson
Full Text Available As a steroid hormone that regulates mineral homeostasis and bone metabolism, 1α, 25-dihydroxycholecalciferol (calcitriol also has broad spectrum anti-tumor activities as supported by numerous epidemiological and experimental studies. Calcitriol potentiates the anti-tumor activities of multiple chemotherapeutics agents including DNA-damaging agents cisplatin, carboplatin and doxorubicin; antimetabolites 5-fluorouracil, cytarabine, hydroxyurea, cytarabine and gemcitabine; and microtubule-disturbing agents paclitaxel and docetaxel. Calcitriol elicits anti-tumor effects mainly through the induction of cancer cell apoptosis, cell cycle arrest, differentiation, angiogenesis and the inhibition of cell invasiveness by a number of mechanisms. Calcitriol enhances the cytotoxic effects of gamma irradiation and certain antioxidants and naturally derived compounds. Inhibition of calcitriol metabolism by 24-hydroxylase promotes growth inhibition effect of calcitriol. Calcitriol has been used in a number of clinical trials and it is important to note that sufficient dose and exposure to calcitriol is critical to achieve anti-tumor effect. Several trials have demonstrated that safe and feasible to administer high doses of calcitriol through intermittent regimen. Further well designed clinical trials should be conducted to better understand the role of calcitriol in cancer therapy.
Johnson, Candace S.; Chung, Ivy; Trump, Donald L.
Calcitriol (1,25 dihydroxycholecalciferol) has significant antitumor activity in vitro and in vivo in a number of tumor model systems. We developed a system for isolation of fresh endothelial cells from tumors and Matrigel environments which demonstrate that CYP24, the catabolic enzyme involved in vitamin D signaling, is epigenetically silenced selectively in tumor-derived endothelial cells (TDEC). TDEC maintain phenotypic characteristics which are distinct from endothelial cells isolated from normal tissues and from Matrigel plugs (MDEC). In TDEC, calcitriol induces G0/G1 arrest, modulates p27 and p21, and induces apoptotic cell death and decreases P-Erk and P-Akt. In contrast, endothelial cells isolated from normal tissues and MDEC are unresponsive to calcitriol-mediated anti-proliferative effects despite intact signaling through the vitamin D receptor (VDR). In TDEC, which is sensitive to calcitriol, the CYP24 promoter is hypermethylated in two CpG island regions located at the 5′end; this hypermethylation may contribute to gene silencing of CYP24. The extent of methylation in these two regions is significantly less in MDEC. Lastly, treatment of TDEC with a DNA methyltransferase inhibitor restores calcitriol-mediated induction of CYP24 and resistance to calcitriol. These data suggest that epigenetic silencing of CYP24 modulates cellular responses to calcitriol. PMID:20304059
Full Text Available Vitamin D is a fat soluble vitamin and generated in human skin by ultraviolet (UV light. Today, vitamin D is considered to be a steroidal hormone and plays a central role in bone mineralization and calcium homeostasis. The active form of the vitamin D is 1, 25-dihydroxyvitamin D [1, 25-dihydroxycholecalciferol (DHCC] which mediatesproliferation, differentiation and various functions at the cellular level through Vitamin D receptors (VDR.Therefore, compromised vitamin D status is likely to be involved in progression or pathogenesis of various disorders. This assumption is consistent with findings from epidemiological studies that a compromised vitamin D status in humans increases the risk of autoimmune diseases, such as inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus (SLE, multiple sclerosis and type I diabetes mellitus. However, diseases like cancer, cardiovascular disorders and bone disorders are yet not focused. Thus the role of vitamin D in pathogenesis of various diseases is complex and controversial. This review briefly summarizes the role of vitamin D in development and progression of different human disorders.
Ko, Seon Yil; Kim, Min Sung; Han, Won Jeong; Kim, Se Won; Kim, Jung Keun
We performed the present study to investigate whether osteotropic hormones play roles on the nitric oxide (NO) production in culture of ROS17/2.8 osteoblastic cells. The osteoblastic cell line ROS17/2.8 cells were cultured in F12 medium supplemented with 5% fetal bovine serum (FBS) at 37.deg. C in a humidified atmosphere of 5% CO 2 in air. ROS17/2.8 cells were plated in 96-well plants at a density of 2-3 x 10 3 cells/well and grown to confluence. Then the cells were pretreated with osteotropic hormones (parathyroid hormone (PTH) 20-500 ng/mL, 1, 25-dihydroxycholecalciferol (1, 25[OH] 2 D 3 ) 1-100nM ; prostaglandin E 2 (PGE 2 ) 20-500 ng/mL) in the medium supplemented with 0.4% FBS for (72 hours and the cells were treated with cytokines (TNFα and IFNγ) in phenol red-free F12 medium for an additional 48 hours. NO synthesis was assessed by measuring the nitrite anion concentration, the reation product of NO, in the cell culture medium using Griess reagent. PTH and 1, 25[OH] 2 D 4 pretreatment induced a significant increase in NO production in the presence of TNFα and IFNγ. PGE 2 slightly induced NO production compared to the control group. But, PGE 2 pretreatment did not affect in NO production in the presence of TNFα and IFNγ. These results suggest that the actions of osteotropic hormones in bone metabolism may be partially mediated by NO in the presence of cytokines
BACKGROUND: 1,25-Dihydroxycholecalciferol (1,25(OH)(2)D(3)) has been shown to mitigate epithelial inflammatory responses after antigen exposure. Patients with cystic fibrosis (CF) are at particular risk for vitamin D deficiency. This may contribute to the exaggerated inflammatory response to pulmonary infection in CF. METHODS: CF respiratory epithelial cell lines were exposed to Pseudomonas aeruginosa lipopolysaccharide (LPS) and Pseudomonas conditioned medium (PCM) in the presence or absence of 1,25(OH)(2)D(3) or a range of vitamin D receptor (VDR) agonists. Levels of IL-6 and IL-8 were measured in cell supernatants, and cellular total and phosphorylated IκBα were determined. Levels of human cathelicidin antimicrobial peptide (hCAP18) mRNA and protein were measured in cells after treatment with 1,25(OH)(2)D(3). RESULTS: Pretreatment with 1,25(OH)(2)D(3) was associated with significant reductions in IL-6 and IL-8 protein secretion after antigen exposure, a finding reproduced with a range of low calcaemic VDR agonists. 1,25(OH)(2)D(3) treatment led to a decrease in IκBα phosphorylation and increased total cellular IκBα. Treatment with 1,25(OH)(2)D(3) was associated with an increase in hCAP18\\/LL-37 mRNA and protein levels. CONCLUSIONS: Both 1,25(OH)(2)D(3) and other VDR agonists significantly reduce the pro-inflammatory response to antigen challenge in CF airway epithelial cells. VDR agonists have significant therapeutic potential in CF.
Kristensen, Mette; Jensen, Marlene; Kudsk, Jane; Henriksen, Marianne; Mølgaard, Christian
In the Western world, increased consumption of carbonated soft drinks combined with a decreasing intake of milk may increase the risk of osteoporosis. This study was designed to reflect the trend of replacing milk with carbonated beverages in a group of young men on a low-calcium diet and studies the effects of this replacement on calcium homeostasis and bone turnover. This controlled crossover intervention study included 11 healthy men (22-29 years) who were given a low-calcium basic diet in two 10-day intervention periods with an intervening 10-day washout. During one period, they drank 2.5 l of Coca Cola per day and during the other period 2.5 l of semi-skimmed milk. Serum concentrations of calcium, phosphate, 25-hydroxycholecalciferol, 1,25-dihydroxycholecalciferol (1,25(OH)2D), osteocalcin, bone-specific alkaline phosphatase (B-ALP) and cross-linked C-telopeptides (CTX), plasma intact parathyroid hormone (PTH) and urinary cross-linked N-telopeptides (NTX) were determined at baseline and endpoint of each intervention period. An increase in serum phosphate (Pcola period compared to the milk period. Also, bone resorption was significantly increased following the cola period, seen as increased serum CTX (Pcola with a low-calcium diet induces increased bone turnover compared to a high intake of milk with a low-calcium diet. Thus, the trend towards a replacement of milk with cola and other soft drinks, which results in a low calcium intake, may negatively affect bone health as indicated by this short-term study.
Grant R Campbell
Full Text Available Low vitamin D levels in human immunodeficiency virus type-1 (HIV infected persons are associated with more rapid disease progression and increased risk for Mycobacterium tuberculosis infection. We have previously shown that 1α,25-dihydroxycholecalciferol (1,25D3, the active form of vitamin D, inhibits HIV replication in human macrophages through the induction of autophagy. In this study, we report that physiological concentrations of 1,25D3 induce the production of the human cathelicidin microbial peptide (CAMP and autophagic flux in HIV and M. tuberculosis co-infected human macrophages which inhibits mycobacterial growth and the replication of HIV. Using RNA interference for Beclin-1 and the autophagy-related 5 homologue, combined with the chemical inhibitors of autophagic flux, bafilomycin A₁, an inhibitor of autophagosome-lysosome fusion and subsequent acidification, and SID 26681509 an inhibitor of the lysosome hydrolase cathepsin L, we show that the 1,25D3-mediated inhibition of HIV replication and mycobacterial growth during single infection or dual infection is dependent not only upon the induction of autophagy, but also through phagosomal maturation. Moreover, through the use of RNA interference for CAMP, we demonstrate that cathelicidin is essential for the 1,25D3 induced autophagic flux and inhibition of HIV replication and mycobacterial growth. The present findings provide a biological explanation for the benefits and importance of vitamin D sufficiency in HIV and M. tuberculosis-infected persons, and provide new insights into novel approaches to prevent and treat HIV infection and related opportunistic infections.
Luo, Wei; Johnson, Candace S; Trump, Donald L
The antiproliferative and pro-apoptotic effects of 1α,25-dihydroxycholecalciferol (1,25(OH)2D3, 1,25D3, calcitriol) have been demonstrated in various tumor model systems in vitro and in vivo. However, limited antitumor effects of 1,25D3 have been observed in clinical trials. This may be attributed to a variety of factors including overexpression of the primary 1,25D3 degrading enzyme, CYP24A1, in tumors, which would lead to rapid local inactivation of 1,25D3. An alternative strategy for improving the antitumor activity of 1,25D3 involves the combination with a selective CYP24A1 inhibitor. The validity of this approach is supported by numerous preclinical investigations, which demonstrate that CYP24A1 inhibitors suppress 1,25D3 catabolism in tumor cells and increase the effects of 1,25D3 on gene expression and cell growth. Studies are now required to determine whether selective CYP24A1 inhibitors+1,25D3 can be used safely and effectively in patients. CYP24A1 inhibitors plus 1,25D3 can cause dose-limiting toxicity of vitamin D (hypercalcemia) in some patients. Dexamethasone significantly reduces 1,25D3-mediated hypercalcemia and enhances the antitumor activity of 1,25D3, increases VDR-ligand binding, and increases VDR protein expression. Efforts to dissect the mechanisms responsible for CYP24A1 overexpression and combinational effect of 1,25D3/dexamethasone in tumors are underway. Understanding the cross talk between vitamin D receptor (VDR) and glucocorticoid receptor (GR) signaling axes is of crucial importance to the design of new therapies that include 1,25D3 and dexamethasone. Insights gained from these studies are expected to yield novel strategies to improve the efficacy of 1,25D3 treatment. © 2016 Elsevier Inc. All rights reserved.
Full Text Available The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1 to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes. Since nuclear factor-kappaB (NF-kappaB plays a pivotal role in the regulation of cell proliferation, differentiation and apoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-kappaB, using 1,25-dihydroxycholecalciferol (calcitriol as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFkappaB DNA binding activity as well as NF-kappaB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-kappaB inhibitor protein, IkappaB alpha, in a time dependent manner, while no changes in total NF-kappaB-p65 mRNA or protein levels were observed. Another measure of NF-kappaB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IkappaB alpha was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, as determined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR, 20-hydroxycholecalciferol did not affect IkappaB alpha mRNA levels, indicating that it requires VDR for its action on NF-kappaB activity. Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-kappaB. Since NF-kappaB is a major transcription factor for the induction of inflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases.
Triennial Growth Symposium--Effects of dietary 25-hydroxycholecalciferol and cholecalciferol on blood vitamin D and mineral status, bone turnover, milk composition, and reproductive performance of sows.
Weber, G M; Witschi, A-K M; Wenk, C; Martens, H
To evaluate the role of vitamin D3 during gestation and lactation of sows, 2 independent experiments were performed with the aim of investigating sow reproductive performance, milk composition (study 1 only), and changes in blood status of 25-hydroxycholecalciferol (25-OH-D3), 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3; study 2 only), minerals, and bone markers of sows during gestation and lactation. Study 1 comprised 39 primi- and multiparous crossbred sows fed 1 of 3 barley meal-based diets fortified with 200 IU/kg vitamin D3 (NRC, 1998; treatment DL), 2,000 IU/kg vitamin D3 (cholecalciferol; treatment DN), or 50 μg 25-OH-D3 (calcidiol; treatment HD)/kg feed. This study was conducted over a 4-parity period under controlled conditions. Study 2, running over 1 parity only, was performed in a commercial farm with 227 primi- and multiparous sows allocated to 2 dietary treatments: control (CON), receiving 2,000 IU vitamin D3/kg (equivalent to 50 μg/kg) feed (114 sows), and test (HYD), supplemented with 50 μg 25-OH-D3/kg feed (113 sows). Blood samples of sows were collected at 84 and 110d postcoitum and 1, 5, and 33 d postpartum (study 1) and at insemination and 28 and 80 d postinsemination as well as d 5 and 28 postpartum (study 2). Colostrum and milk samples in study 1 were obtained at 1, 9, and 33 d of lactation after oxytocin administration. Plasma 25-OH-D3 concentrations were increased (P < 0.05) in sows receiving 25-OH-D3 (HD and HYD) at any time of sampling whereas circulating plasma concentrations of 1,25-(OH)2-D3, Ca, and P were not affected by treatment. Milk concentrations of Ca and P were similar, but 25-OH-D3 content (except in colostrum) was clearly increased (P< 0.05) when 25-OH-D3 was fed. Most characteristics of sow reproductive performance responded similarly to the 2 sources and levels of vitamin D3, but weight gain of piglets between birth and weaning was decreased (P< 0.05) in offspring of DL and HD sows compared with animals of treatment DN
Djekic-Ivankovic, Marija; Weiler, Hope; Jones, Glenville; Kaufmann, Martin; Kaludjerovic, Jovana; Aleksic-Velickovic, Vesna; Mandić, Ljuba M; Glibetic, Maria
The objective of the present study was to determine if vitamin D intake and status are associated with pre-eclampsia in a country without a vitamin D fortification policy. A case-control study of pregnancies with (case) and without (control) pre-eclampsia was conducted from January to April when UVB is minimal. Maternal and cord blood obtained at delivery were measured for plasma 25-hydroxycholecalciferol (25-OH-D3), 3-epimer of 25-OH-D3 (3-epi-25-OH-D3) and 24,25-dihydroxycholecalciferol (24,25-(OH)2D3) by LC-MS/MS and maternal 1,25-dihydroxyvitamin D (1,25-(OH)2D). Differences between groups were tested with ANOVA and Bonferroni post hoc tests (Pcase: 11·2 (sd 5·1); control: 16·1 (sd 5·7) ng/ml; P=0·0006), 25-OH-D3 (case: 10·0 (sd 4·9); control: 14·2 (sd 5·8) ng/ml; P=0·002), 3-epi-25-OH-D3 (case: 0·5 (sd 0·2); control: 0·7 (sd 0·2) ng/ml; P=0·0007) and 1,25-(OH)2D (case: 56·5 (sd 26·6); control: 81·0 (sd 25·7) pg/ml; P=0·018), while 24,25-(OH)2D3 was not different between groups. Infants did not differ in total plasma 25-OH-D, 25-OH-D3, 3-epi-25-OH-D3 and 24,25-(OH)2D3, but the mean proportion of 3-epi-25-OH-D3 was higher in the infant case group (case: 7·9 (sd 1·1); control: 7·0 (sd 1·4) % of total 25-OH-D3; P=0·005). A high prevalence of vitamin D deficiency, as defined by plasma 25-OH-D<12 ng/ml, was observed in 47 % of all mothers and 77 % of all infants. These data underscore the need for prenatal vitamin D supplementation and a food fortification policy in Serbia.