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1.
The field of Vitamin D assay technology has progressed significantly over the past 4 decades. Further, the clinical utility of these measurements has moved from esoteric into mainstream clinical diagnosis. This movement has been fueled by the realization that Vitamin D is involved in bodily systems beyond skeletal integrity. The clinical assay techniques for circulating 25(OH)D and 1,25(OH) 2D have progressed away from competitive protein binding assay (CPBAs) that utilize tritium reporters to radioimmunoassay (RIAs) that utilize both I 125 and chemiluminescent reporters. These advances have allowed direct serum analysis of 25(OH)D in an automated format that provides a huge sample throughput. Detection of circulating 25(OH)D can also be achieved utilizing direct high-performance liquid chromatographic (HPLC) or liquid chromatography coupled with mass spectrometry (LC–MS) techniques. These methods are accurate, however, they require expensive equipment and restrict sample throughput in the large clinical laboratory. Direct serum detection of 1,25(OH) 2D is unlikely to occur for many reasons as a sample pre-purification will always be required. However, a semi-automated chemiluminescent detection system with automated sample preparation is in final development for the determination of circulating 1,25(OH) 2D. These advances will allow both 25(OH)D and 1,25(OH) 2D to be detected in an accurate, rapid fashion to meet the clinical demands we see emerging. 相似文献
3.
Since the discovery of the Vitamin D receptor (VDR) in mammary cells, the role of the Vitamin D signaling pathway in normal glandular function and in breast cancer has been extensively explored. In vitro studies have demonstrated that the VDR ligand, 1,25(OH) 2D 3, modulates key proteins involved in signaling proliferation, differentiation and survival of normal mammary epithelial cells. Anti-proliferative and pro-differentiating effects of 1,25(OH) 2D 3 have also been observed in VDR positive breast cancer cells, indicating that transformation per se does not abolish Vitamin D signaling. However, many breast cancer cell lines are less sensitive to 1,25(OH) 2D 3 than normal mammary epithelial cells. Reduced sensitivity to 1,25(OH) 2D 3 has been linked to alterations in Vitamin D metabolizing enzymes as well as down regulation of VDR expression or function. In this report, we describe results from a proteomics screening approach used to search for proteins involved in dictating sensitivity or resistance to Vitamin D mediated apoptosis in breast cancer cells. Several proteins not previously linked to 1,25(OH) 2D 3 signaling were identified with this approach, and a distinct subset of proteins was linked to 1,25(OH) 2D 3 resistance. Follow-up studies to determine the relevance of these proteins to Vitamin D signaling in general are in progress. 相似文献
7.
Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D 3 (1,25(OH) 2D 3) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH) 2D 3, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH) 2D 3 mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH) 2D 3 induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH) 2D 3 induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH) 2D 3 induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH) 2D 3 induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH) 2D 3 stimulated VDR protein expression and 1,25(OH) 2D 3 mediated actions in human osteoblast cells. 相似文献
8.
This review summarizes the reported effects of the menstrual cycle, pregnancy and lactation on serum concentration of the calciotropic hormones PTH and 1,25(OH) 2D. A midcycle rise in PTH and 1,25(OH) 2D has been observed, but in the majority of studies there was no change in PTH and 1,25(OH) 2D concentrations throughout the menstrual cycle. Both total and free 1,25(OH) 2D levels are increased during pregnancy. The renal 1,25(OH) 2D production is stimulated, and there is some evidence of 1,25(OH) 2D production by decidua/placenta and fetal kidney in vitro; the decidual/placental production should not be overestimated in vivo. The increased renal 1-hydroxylase activity is possibly mediated by estrogens and PTH, although the effect of pregnancy on PTH remains uncertain. Increased serum 1,25(OH) 2D concentrations probably result in a rise of intestinal calcium absorption during pregnancy. There is a postdelivery drop in PTH and 1,25(OH) 2D levels, but they are increased when lactation is prolonged, or in mothers nursing twins. The 1-hydroxylase activity during lactation may be stimulated by PTH, but also by prolactin. 相似文献
9.
Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH) 2D 3 on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH) 2D 3 (10 −6, 10 −7, 10 −10 M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH) 2D 3, although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH) 2D 3 in prostate cancer indicating that CLU interferes with vitamin D signalling pathways. 相似文献
10.
The ingestion of Solanum glaucophyllum ( SG) causes a calcinosis of cattle named Enteque Seco (ES). The toxic principle is the 1,25-(OH) 2D 3, mainly conjugated as glycoside. This study aims to validate a simple novel method of evaluation of the VDA of SG leaves. Aqueous extracts of SG were purified using C 18 minicolumns and assayed by RIA with an antibody raised in rabbits by injection of the acid—C22, 1-(OH)Vitamin D 3. Data were expresed as glycoside equivalent to 1,25-(OH) 2D 3 in ng/g of dry leaves. We compared this data with 1,25-(OH) 2D 3 levels measured, in the same samples, by liquid chromatography (HPLC) after enzyme cleavage. This procedure involved the incubation of SG leaves with rumen fluid, followed by C 18-OH solid phase extraction. The 1,25-(OH) 2D 3 fraction was run by HPLC and detection was achieved using a photodiode array detector. Data were expressed as micrograms of 1,25-(OH) 2D 3/g dry leaves. A significant regression of 1,25-(OH) 2D 3 levels ( Y) as a function of glycoside RIA 1,25-(OH) 2D 3 equivalents ( X) was found: Y = 12.02 + 0.35 X [ R = 0.81; P = 0,0002; N = 15], allowing us to conclude that this novel assay could be used to estimate the amount of this active principle contained in SG leaves. 相似文献
11.
In a double blind placebo controlled 3-year osteoporosis study in elderly women, we collected prospective data on falls. The study population comprised 489 normal elderly women aged 65–77 years randomized to four groups: placebo, calcitriol 0.25 mcg b.i.d., conjugated equine estrogens (0.625 mg/day) and calcitriol + estrogen. Falls occurred in 57% of all women. Using a Poisson regression model, the placebo group with low GFR-creatinine clearance (CrCl < 60 ml/min) had 60% more falls compared to the group with CrCl ≥ 60 ml/min. Further sub group analyses showed that there is no increased risk of falls with CrCl 60–70, 70–80 and >90 ml/min. Calcitriol treatment significantly reduced the number of falls by 50% (OR = 0.5, 95% CI: 0.4–0.9, p = 0.010) compared to placebo in the low CrCl group. The group with lower CrCl had lower calcium absorption (p < 0.001), lower serum 1,25-dihydroxyvitamin D (1,25(OH)2D) (p < 0.001) and normal serum 25OHD suggesting that there is decreased conversion of 25OHD to 1,25(OH)2D by the aging kidney. It is postulated that the decrease in falls on calcitriol therapy is related to an increase in serum 1,25(OH)2D, upregulation of VDR and improvement in muscle strength although one cannot exclude an effect on the central nervous system. 相似文献
12.
Vitamin D is produced by exposure of 7-dehydrocholesterol in the skin to UV irradiation (UVR) and further converted in the skin to the biologically active metabolite, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) and other compounds. UVR also results in DNA damage producing cyclobutane pyrimidine dimers (CPD). We previously reported that 1,25(OH) 2D 3 at picomolar concentrations, protects human skin cells from UVR-induced apoptosis, and decreases CPD in surviving cells. 1,25(OH) 2D 3 has been shown to generate biological responses via two pathways—the classical steroid receptor/genomic pathway or a rapid, non-genomic pathway mediated by a putative membrane receptor. Whether the rapid response pathway is physiologically relevant is unclear. A cis-locked, rapid-acting agonist 1,25(OH) 2lumisterol 3 (JN), entirely mimicked the actions of 1,25(OH) 2D 3 to reduce fibroblast and keratinocyte loss and CPD damage after UVR. The effects of 1,25(OH) 2D 3 were abolished by a rapid-acting antagonist, but not by a genomic antagonist. Skh:hr1 mice exposed to three times the minimal erythemal dose of solar-simulated UVR and treated topically with 1,25(OH) 2D 3 or JN immediately after UVR showed reduction in UVR-induced UVR-induced sunburn cells ( p < 0.01 and <0.05, respectively), CPD ( p < 0.01 for both) and immunosuppression ( p < 0.001 for both) compared with vehicle-treated mice. These results show for the first time an in vivo biological response mediated by a rapid-acting analog of the vitamin D system. The data support the hypothesis that 1,25(OH) 2D 3 exerts its photoprotective effects via the rapid pathway and raise the possibility that other D compounds produced in skin may contribute to the photoprotective effects. 相似文献
13.
The Vitamin D International External Quality Assessment Scheme (DEQAS) was established in 1989 to monitor the performance of assays for 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D (I,25(OH) 2D). This is achieved through the quarterly distribution of five samples of human serum. Results are used to calculate an All-Laboratory Trimmed Mean and a Method Mean for each of the methods used by participants. In July 2005, participants were asked to assay serum to which 50.9 nmol of either 25-OHD 3 or 25-OHD 2 had been added as ethanolic solutions. The final concentration of ethanol in the serum was 0.7%. The distribution also included a sample of the original serum (OS) containing 0.7% pure ethanol. The percentage recoveries of exogenous 25-OHD 3 (R1) and 25-OHD 2 (R2) were calculated for each method. Results (OS nM, R1 and R2) were as follows: DiaSorin RIA ( n = 53); 39.2, 82.1%, 83.3%, DiaSorin Liason ( n = 16); 36.8, 81.4%, 88.6%, IDS RIA ( n = 21); 36.4, 54.2%, 29.1%, IDS OCTEIA ( n = 16); 47.3, 78.8%, 56.4%, Nichols Advantage ( n = 21); 58.9, 46.4%, 43.2%, HPLC ( n = 9); 42.6, 112.2%, 97.1%, LC–MS ( n = 4); 34.0, 111.5%, 118.1%. The IDS RIA and Nichols assays gave unexpectedly low recoveries. This does not appear to be a calibration problem or the effect of ethanol. 相似文献
14.
We employed genetically modified mice to examine the role of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] on skeletal and calcium homeostasis. In mice expressing the null mutation for 25-hydroxyvitamin D 1 hydroxylase (1OHase −/−), or the vitamin D receptor (VDR −/−), 1,25(OH) 2D 3 and calcium were both required for optimal epiphyseal growth plate development, serum calcium and phosphorus alone were sufficient to mineralize skeletal tissue independent of 1,25(OH) 2D 3 and the VDR, and endogenous 1,25(OH) 2D 3 and the VDR were essential for baseline bone formation. In 2-week-old 1OHase −/− mice and in 2-week-old mice homozygous for the PTH null mutation(PTH −/−), PTH and 1,25(OH) 2D 3 were each found to exert independent and complementary effects on skeletal anabolism, with PTH predominantly affecting appositional trabecular bone growth and 1,25(OH) 2D 3 influencing both endochondral bone formation and appositional bone growth. Endogenous 1,25(OH) 2D 3 maintained serum calcium homeostasis predominantly by modifying intestinal and renal calcium transporters but not by producing net bone resorption. Administration of exogenous 1,25(OH) 2D 3 to double mutant PTH −/−1OHase −/− mice produced skeletal effects consistent with the actions of endogenous 1,25(OH) 2D 3. These studies reveal an important skeletal anabolic role for both endogenous and exogenous 1,25(OH) 2D 3 and point to a potential role for 1,25(OH) 2D 3 analogs in the treatment of disorders of bone loss. 相似文献
15.
Induction of growth arrest and differentiation by 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the anti-proliferative action of 1,25-(OH) 2D 3. This appears to be due to increased 1,25-(OH) 2D 3-metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2 H-naphthalen-1-one (4) was found to be a potent inhibitor of Vitamin D 3 metabolising enzymes (IC 50 3.5 μM), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC 50 20 μM). The combination of the inhibitor and 1,25-(OH) 2D 3 caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH) 2D 3 plus inhibitor of Vitamin D 3 metabolising enzymes co-ordinately upregulated CYP24, p21 waf1/cip1 and GADD45. 相似文献
16.
1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR. 相似文献
17.
1,25-dihydroxyvitamin D 3 (1,25-(OH) 2D 3) is known to be involved in regulating the proliferation of parathyroid cells and PTH synthesis through reactions involving its nuclear receptor. We evaluated the effects of 1,25-(OH) 2D 3 and its hexafluorinated analog, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D 3 (26,27-F 6-1,25-(OH) 2D 3), on parathyroid cells. The 1,25-(OH) 2D 3 and 26,27-F 6-1,25-(OH) 2D 3 each inhibited [ 3H]thymidine incorporation and ornithine decarboxylase (ODC) activity, which is important in cell proliferation, in primary cultured bovine parathyroid cells. The inhibitory effect of 26,27-F 6-1,25-(OH) 2D 3 on PTH secretion from parathyroid cells was significantly more potent than that of 1,25-(OH) 2D 3 between 10 −11 M and 10 −8 M. Study of 26,27-F 6-1,25-(OH) 2D 3 metabolism in parathyroid cells in vitro elucidated its slower degradation than that of 1,25-(OH) 2D 3. After 48 h of incubation with [1 β- 3H]26,27-F 6-1,25-(OH) 2D 3, two HPLC peaks, one for [1 β- 3H]26,27-F 6-1,25-(OH) 2D 3, and a second larger peak for [1 β- 3H]26,27-F 6-1,23(S),25-(OH) 3D 3, were detected. No metabolites were detected after the same period of incubation with 1,25-(OH) 2[26,27- 3H]D 3. We observed that 26,27-F 6-1,23(S),25-(OH) 3D 3 was as potent as 1,25-(OH) 2D 3 in inhibiting the proliferation of parathyroid cells. Data suggest that the greater biological activity of 26,27-F6-1,25-(OH)2D3 is explained by its slower metabolisms and by the retention of the biological potency of 26,27-F6-1,25-(OH)2D3 even after 23(S)-hydroxylation. 相似文献
18.
The induction of antimicrobial peptides such as the human cathelicidin, CAMP/hCAP18, by 1,25(OH) 2D 3 provides a very exciting therapeutic approach in boosting immunity against infectious diseases. To explore the range of cell types and expand the number of cell models for studying the regulation of CAMP gene expression by 1,25(OH) 2D 3, we treated cell lines from various tissue types and determined CAMP gene expression. Also, we tested additional compounds together with 1,25(OH) 2D 3 to look for possible cooperative activation of the gene. We identified 1,25(OH) 2D 3-mediated induction of the CAMP gene in B-cell lymphomas, prostate and endometrial cancer lines and found cooperative activation with the histone deacetylase inhibitor sodium butyrate. The data suggest that regulation of CAMP by 1,25(OH) 2D 3 is potentially important in a wide range of tissues. 相似文献
19.
1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear. 相似文献
20.
The synthesis of 1,25(OH) 2D 3 is a critical control point in the regulation of calcium metabolism, and possibly in the growth and differentiation of a number of cell types. This paper reviews our current understanding of the regulation of this process at the cellular and molecular levels, with the emphasis on the mechanisms of feedback control 1,25(OH) 2D 3 itself, control of parathyroid hormone, the roles of cyclic AMP dependent protein kinase and protein kinase C, and the interaction between the various intracellular regulators of 1,25(OH) 2D 3 production. 相似文献
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