The role of the vitamin D endocrine system in health and disease.
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Bone Diseases↗
Biomedical subjects
H Reichel
Publications and source records attributed to H Reichel.
Systemic effects of vitamin D.
Traditionally perceived as a seco-steroid hormone involved in the regulation of calcium metabolism, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] is now known also to be active in tissues not directly contributing to mineral metabolism. New data show that 1 alpha,25(OH)2D3 is produced by and interacts with hematopoietic cells. The hormone promotes myeloid differentiation and modulates the function of activated lymphocytes. Another new target tissue for 1 alpha,25(OH)2D3 is the skin, where 1 alpha,25(OH)2D3 enhances differentiation of epidermal cells. Therapeutic application of 1 alpha,25(OH)2D3 or suitable analogues in differentiation disorders of hematopoietic and skin cells is currently under investigation.
Calcitriol↗
1,25-Dihydroxyvitamin D3 inhibits myelopoiesis but not B-lymphopoiesis in long-term bone marrow cultures.
The biologically active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) has been demonstrated to have differentiative and antiproliferative effects on myeloid tumors of human or murine origin. Its effects on normal murine hemopoiesis were tested by addition of the seco-steroid to long-term bone marrow cultures optimized for either myelopoiesis or B-lymphopoiesis. The addition of 10(-8) M 1,25(OH)2D3, but not 10(-8) M 25(OH)D3, to myeloid bone marrow cultures (MBMC) resulted in a complete cessation of hemopoiesis by 4 weeks, because no hemopoietic cells or colony-forming units were detected. This result was observed whether or not the cultures were initiated and maintained in hydrocortisone. A potential effect of 1,25(OH)2D3 on the production of myeloid growth factors by adherent layer cells in the cultures was examined, but this function was not affected by 1,25(OH)2D3 treatment. Further, adherent layers that had been treated with 1,25(OH)2D3 for 3 weeks were capable of supporting myelopoiesis upon seeding with a stromal cell-depleted population of bone marrow cells. Transfer of MBMC to lymphoid bone marrow culture (LBMC) conditions results in the cessation of myelopoiesis and the initiation of B-cell production. Lymphopoiesis did not initiate in 1,25(OH)2D3-pretreated MBMC that were transferred to LBMC conditions, indicating that the pool of B-cell precursors present in MBMC had been depleted by exposure of MBMC to 1,25(OH)2D3. When 1,25(OH)2D3 (10(-8) M) was added to MBMC at the time of transfer to LBMC conditions, the seco-steroid did not affect induction of B-lymphopoiesis, although the overall cellularity was less in 1,25(OH)2D3-treated cultures than in control cultures.
Animals↗
Differential effects of 1,25-dihydroxyvitamin D3 upon intestinal vitamin D3-dependent calbindin (a 28,000-dalton calcium binding protein) and its mRNA in D-replete and D-deficient chickens.
The effect of vitamin D3 status upon the responsiveness of chick intestinal epithelium to exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was studied. Intestinal calbindin [A recent consensus decision was made to redesignate the vitamin D-dependent calcium binding protein as "calbindin-D28K" (R.H. Wasserman (1985) in Vitamin D: Chemical, Biochemical, and Clinical Update (Norman, A.W., Schaefer, K., Grigoleit, H.-G., and Herrath, D.V., Eds.), pp. 321-322, de Gruyter, Berlin/New York).] protein and intestinal calbindin mRNA were quantitated in birds which had been raised on a vitamin D3-deplete (-D) or on a vitamin D3-replete (+D) diet. 1,25(OH)2D3 stimulated intestinal calbindin mRNA levels in -D chickens in a proportional dose-dependent manner, when measured at both 12 and 48 h after administration of the hormone. A first increase was observed with 1,25(OH)2D3 concentrations between 0.065 and 0.65 nmol. The maximal stimulation achieved by 1,25(OH)2D3 (6.5-18 nmol) in -D tissue was approximately 10-fold over the calbindin mRNA levels present in vehicle-treated birds. The increase of calbindin mRNA in -D birds was associated with a similar dose-dependent increase in calbindin protein in 1,25(OH)2D3-treated -D birds after 12 or 48 h. In +D intestine, while exogenous 1,25(OH)2D3 also increased calbindin mRNA levels in a dose-dependent fashion, the maximal stimulation observed after 5 h (1.2- to 2-fold) was clearly less than that observed in -D intestine. In contrast to -D birds, intestinal calbindin levels in +D birds were decreased by administration of exogenous 1,25(OH)2D3. Administration of 32.5 to 65 nmol 1,25(OH)2D3 resulted in an approximately 1.8-fold repression compared to vehicle-treated birds. This differential responsiveness between +D and -D intestines with respect to 1,25(OH)2D3 was not explained either by differences in the uptake in the chromatin fractions of these tissues or by metabolism of radiolabeled 1,25(OH)2D3. Dietary withdrawal of vitamin D3 led to a gradual decline in ambient intestinal calbindin levels, while intestinal sensitivity to 1,25(OH)2D3 was restored. These findings suggest that vitamin D3 status regulates intestinal responsiveness to the seco-steroid 1,25(OH)2D3.
Animals↗
[New aspects of endocrine regulation of calcium metabolism].
The maintenance of circulating calcium levels within the narrow physiological range requires the action of two hormones, the polypeptide hormone parathyroid hormone and the steroid hormone 1-alpha-25-dihydroxyvitamin D3. These two hormones act on bone, kidney and intestine to regulate calcium homeostasis. Disorders of mineral metabolism are frequently associated with abnormal regulation of the metabolism of parathyroid hormone or 1-alpha-25-dihydroxyvitamin.
Calcitriol↗
Metabolism of the vitamin D3 derivative (24R)-hydroxycalcidiol by human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z) and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol.
Human promyelocytic leukemia cells (HL-60 cells) incubated with (24R)-hydroxy[26,27-methyl-3H]calcidiol (0.2 microCi) or non-radioactive (24R)-hydroxycalcidiol (370 micrograms) produced significant quantities of two new vitamin D3 (calciol) metabolites. The metabolites were isolated from HL-60 cell culture media by methanol/chloroform extraction and a series of chromatographic procedures. The two new metabolites were identified as (5Z)- and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol by HPLC analysis, ultraviolet absorption spectrophotometry, mass spectrometry and Fourier-transform infrared spectrophotometry. According to the isolation and purification procedures, the total amounts of 3.04 micrograms (5Z)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 310 nm, epsilon = 17070 M-1 cm-1) and 8.89 micrograms (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 312 nm, e = 24,500 M-1 cm-1) were calculated, assuming an Mr of 418. The activity of 19-nor-10-oxo-(24R)-hydroxycalcidiol to promote HL-60 cell differentiation was higher than the activity of the precursor (24R)-hydroxycalcidiol suggesting a possible biological action of this metabolite in HL-60 cells.
24,25-Dihydroxyvitamin D 3↗
Synthesis in vitro of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by interferon-gamma-stimulated normal human bone marrow and alveolar macrophages.
Cultured human macrophages from normal donors were examined for their capability to metabolize 25-hydroxyvitamin D3 (25-(OH)D3). Upon exposure to recombinant human interferon-gamma (IFN-gamma) both bone marrow-derived macrophages (BMM) and pulmonary alveolar macrophages (PAM) produced a polar 25-(OH)D3 metabolite which was purified from conditioned media and unequivocally identified as 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by UV-absorbance spectrophotometry and mass spectrometry. The BMM and PAM also synthesized a second 25-(OH)D3 metabolite which was structurally identified as 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). The time course of 25-(OH)D3 metabolism by macrophages suggested that the production of 24,25-(OH)2D3 was stimulated by high intracellular levels of 1,25-(OH)2D3 and not by IFN-gamma. The 1,25-(OH)2D3 obtained from BMM and PAM promoted macrophage-like differentiation of promyelocytic HL-60 leukemia cells and inhibited IFN-gamma production by normal human lymphocytes. Our data suggest that locally high levels of 1,25-(OH)2D3 in the microenvironment of IFN-gamma-stimulated BMM and PAM may modulate the function of hormone-responsive cells.
24,25-Dihydroxyvitamin D 3↗
Synthesis and biological activity of 1 alpha,23,25,26-tetrahydroxyvitamin D3.
The metabolic pathway from 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone includes the formation of 1 alpha,23,25-26-tetrahydroxyvitamin D3 [1 alpha,23,25,26-(OH)4D3]. The aim of the current study was to explore the as yet unknown biological properties of this vitamin D3 sterol. The four diastereoisomers of 1 alpha,23,25,26-(OH)4D3 were chemically synthesized. They were compared to 1 alpha,25-(OH)2D3 in terms of their affinity for the chick intestinal 1 alpha,25-(OH)2D3 receptor and their biologic activity in vivo (stimulation of intestinal calcium absorption and mobilization of calcium from bone in vitamin D-deficient rats). The 1,25-(OH)2D3 receptor binding affinities of 1 alpha,23(R)25(R)26-(OH)4D3, 1 alpha,23(S)25(S)26-(OH)4 D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 1 alpha,23(R)25(S)26-(OH)4D3 were 11, 100, 216, and 443 times weaker than the binding affinity of 1 alpha,25-(OH)2D3, respectively. Compared to 1 alpha,25-(OH)2D3, the relative capacities of the 1 alpha,23,25,26-(OH)4D3 compounds to stimulate intestinal calcium absorption were 1/4 for 1 alpha,23(R)25(R)26-(OH)4D3; 1/19 for 1 alpha,23(S)25(S)26-(OH)4D3; 1/90 for 1 alpha,23(S)25(R)26-(OH)4D3; and 1/136 for 1 alpha,23(R)25(S)26-(OH)4D3. Maximal stimulation of intestinal calcium transport occurred 8 h after administration of vitamin D3 metabolites. Mobilization of calcium from bone was quantitated by serum calcium concentration measurements. The activities of 1 alpha,23(R)25(R)26-(OH)4D3, 1 alpha,23(S)25(S)26-(OH)4D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 1 alpha,23(R)25(S)26-(OH)4D3 to increase serum calcium were estimated to be 4, 13, 43, and 69 times weaker than that of 1 alpha,25-(OH)2D3, respectively. These results illustrate the stereospecificity of the chicken intestine 1 alpha,25-(OH)2D3 receptor for binding of 1 alpha,23,25,26-(OH)4D3 and suggest that the 1 alpha,23,25,26-(OH)4D3 exerts its biological activity in the rat through an interaction with 1,25-(OH)2D3 receptors. In summary, the 1 alpha,23,25,26-(OH)4D3 had a markedly lower biological activity than 1 alpha,25-(OH)2D3.
Animals↗
1 alpha,25-Dihydroxyvitamin D3 inhibits gamma-interferon synthesis by normal human peripheral blood lymphocytes.
1 alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active metabolite of vitamin D3, inhibited synthesis of gamma-interferon (IFN-gamma) by phytohemagglutinin-activated peripheral blood lymphocytes (PBLs). A significant reduction of IFN-gamma protein levels in PBL culture medium was achieved with a physiologic 1,25-(OH)2D3 concentration (0.1 nM). 1,25-(OH)2D3 also inhibited accumulation of IFN-gamma mRNA in activated PBLs in a dose-dependent fashion. The ability of 1,25-(OH)2D3 to modulate IFN-gamma protein synthesis was unaltered in the presence of high concentrations of recombinant human interleukin 2. The suppression of IFN-gamma synthesis by PBLs was specific for 1,25-(OH)2D3; the potencies of other vitamin D3 metabolites were correlated with their affinities for the cellular 1,25-(OH)2D3 receptor. The time course of 1,25-(OH)2D3 receptor expression in phytohemagglutinin-activated PBLs was correlated with the time course of 1,25-(OH)2D3-mediated inhibition of IFN-gamma synthesis. In selected experiments, T-lymphocyte-enriched cell preparations were utilized. In these experiments, 1,25-(OH)2D3 was equally active as in PBL preparations. Finally, we examined the effects of 1,25-(OH)2D3 on the constitutive IFN-gamma production by two human T-lymphocyte lines transformed by human T-lymphotropic virus type I. The cell lines were established from a normal donor (cell line S-LB1) and from a patient with vitamin D-dependent rickets type 2 (cell line Ab-VDR). IFN-gamma synthesis by S-LB1 cells was inhibited in a dose-dependent fashion by 1,25-(OH)2D3, whereas IFN-gamma synthesis by Ab-VDR cells was not altered by 1,25-(OH)2D3. The data presented in this study provide further evidence for a role of 1,25-(OH)2D3 in immunoregulation.
Calcitriol↗
Granulocyte-macrophage colony-stimulating factor. Sensitive and receptor-mediated regulation by 1,25-dihydroxyvitamin D3 in normal human peripheral blood lymphocytes.
We show that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), the most hormonally active metabolite of vitamin D3, modulates sensitively and specifically both the protein and messenger RNA accumulation of the multilineage growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). The regulation of GM-CSF expression is seen in both normal human mitogen-activated T lymphocytes and T lymphocytes from a line (S-LB1) transformed with human T cell lymphotropic virus 1 (HTLV-1). In contrast, cells from a HTLV-1 transformed T lymphocyte line (Ab-VDR) established from a patient with vitamin D-resistant rickets type II with undetectable 1,25(OH)2D3 cellular receptors are resistant to the action of 1,25(OH)2D3. Inhibition of GM-CSF expression by 1,25(OH)2D3 can occur independently of interleukin 2 regulation and is probably mediated through cellular 1,25(OH)2D3 receptors. We conclude that 1,25(OH)2D3 may be important in the physiology of hematopoiesis.
Calcitriol↗
25-Hydroxyvitamin D3 metabolism by lipopolysaccharide-stimulated normal human macrophages.
Cultured normal human pulmonary alveolar macrophages and peripheral blood monocyte-derived macrophages were studied for their capacity to metabolize [3H]25-hydroxyvitamin D3 (25OHD3). Incubation of macrophages with bacterial lipopolysaccharide (LPS) resulted in the conversion of [3H]25OHD3 to a more polar vitamin D3 metabolite (up to 15 pmol/10(6) cells). Untreated macrophages did not synthesize this metabolite. Several findings suggested that the metabolite was the biologically active form of vitamin D3, namely 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. (1) The metabolite comigrated with chemically synthesized 1,25-(OH)2D3 on four different high performance liquid chromatographic systems. (2) The metabolite had the same affinity for the chick intestinal 1,25-(OH)2D3 receptor as authentic 1,25-(OH)2D3. (3) The biological activity of the macrophage metabolite in vivo (stimulation of intestinal calcium absorption and bone calcium mobilization in rachitic chicks) was identical to the activity of chemically synthesized 1,25-(OH)2D3. The LPS-stimulated synthesis of the 1,25-(OH)2D3-like compound by macrophages was dose dependent in a linear fashion; a half-maximal response was typically found with 100-200 ng LPS/10(6) cells. Polymyxin B abolished the effects of LPS on 25OHD3 metabolism in macrophages. Our data suggest that LPS-stimulated macrophages can modulate, on a local level, the function of 1,25-(OH)2D3-responsive cells by releasing the 1,25-(OH)2D3-like metabolite.
Adult↗
25-Hydroxyvitamin D3 metabolism by human T-lymphotropic virus-transformed lymphocytes.
Production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by human T-lymphotropic virus-I (HTLV-I)-infected lymphocytes may be the cause of the hypercalcemia frequently found in HTLV-I-associated adult T-cell lymphoma/leukemia. We examined three HTLV-I-transformed lymphocyte cell lines, two HTLV-II-transformed lymphocyte cell lines, and six HTLV-negative B and T-lymphocyte leukemia cell lines for metabolism of 25-hydroxyvitamin D3 (25OHD3). One HTLV-I-positive cell line, designated S-LB1, converted the substrate 25OH-[3H]D3 to several more polar metabolites, which were identified by high performance liquid chromatographic analysis as putative 1,25-(OH)2D3, 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], and 1,24,25-trihydroxyvitamin D3 [1,24,25-(OH)3D3]. The other cell lines gave no evidence of 25OH-[3H]D3 metabolism. Likewise, phytohemagglutinin-stimulated normal human lymphocytes did not metabolize 25OH-[3H]D3. The characteristics of 25OHD3 metabolism by S-LB1 cells were investigated in more detail. Kinetic studies revealed average Km values of 92 and 383 nM for 25OHD3 1-hydroxylase and 24-hydroxylase, respectively. Time-course studies showed that both 1,25-(OH)2-[3H]D3 and 24,25-(OH)2-[3H]D3 were further metabolized by S-LB1 cells to more polar compounds [primarily 1,24,25-(OH)3D3] and to compounds from which part of the side-chain had been cleaved. Exogenous 1,25-(OH)2D3 (1) inhibited endogenous 1,25-(OH)2D3 production, (2) stimulated 24,25-(OH)2D3 production, and (3) stimulated production of compounds more polar than 1,25-(OH)2D3. Bovine PTH-(1-34) had no effect on 25OH-[3H]D3 metabolism by S-LB1 cells. Our results indicate that the 25OH-[3H]D3-metabolizing system of cultured HTLV-I-transformed S-LB1 lymphocytes is similar but not identical to that of kidney cell culture systems. It appears, however, that infection of lymphocytes with HTLV does not uniformly result in acquisition of the competence to metabolize 25OHD3.
B-Lymphocytes↗
Regulation of 1,25-dihydroxyvitamin D3 production by cultured alveolar macrophages from normal human donors and from patients with pulmonary sarcoidosis.
Regulation of the production of the biologically active vitamin D3 sterol 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by cultured pulmonary alveolar macrophages (PAM) obtained from 6 patients with pulmonary sarcoidosis and from 9 normal subjects was studied. The sarcoid cells, all collected from patients with normal calcium metabolism, synthesized 1,25-(OH)2-[3H]D3 from the substrate 25-hydroxyvitamin [3H]D3 (25OH-[3H]D3), whereas in vitro incubation with recombinant human interferon-gamma (IFN gamma) or lipopolysaccharide (LPS) was required for induction of synthesis of the hormone by normal PAM. Exogenous 1,25-(OH)2D3 (10-100 nmol/L) decreased endogenous hormone production by normal PAM by approximately 45%. The relative inhibitory effect of 1,25-(OH)2D3 was less pronounced in sarcoid PAM, in which 10-100 nmol/L 1,25-(OH)2D3 inhibited 250HD3-1-hydroxylase by approximately 25%. An accompanying induction of the 250HD3-24-hydroxylase, which is typical for renal cells, was found at low levels in only 3 of 10 experiments; in this regard, no differences between sarcoid and normal PAM were apparent. PTH or forskolin did not influence 250HD3 metabolism by PAM. 1,25-(OH)2D3 production by sarcoid PAM was enhanced by lipopolysaccharide and IFN gamma. Likewise, recombinant human interleukin-2 stimulated 1,25-(OH)2D3 production by sarcoid PAM, suggesting a possible role for both IFN gamma and interleukin-2 in the induction of 1,25-(OH)2D3 synthesis by sarcoid PAM in vivo. Recombinant human IFN alpha, IFN beta, and granulocyte-macrophage colony-stimulating factor had little effect. Dexamethasone and chloroquine, which have in vivo antihypercalcemic activity in sarcoidosis, both inhibited 1,25-(OH)2D3 synthesis by sarcoid PAM; chloroquine simultaneously stimulated the 24-hydroxylase. Our studies suggest that the 250HD3-metabolizing system in PAM is in some respects different from renal metabolism of 250HD3.
Adult↗
Metabolism of 25-hydroxycholecalciferol in human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol.
Human promyelocytic leukemia cells incubated with 25-hydroxy[26,27-methyl-3H] cholecalciferol (1 microCi) or non-radioactive 25-hydroxycholecalciferol (550 micrograms) produced significant quantities of two vitamin D3 metabolites. The two metabolites were isolated and purified by methanol chloroform extraction and a series of chromatographic procedures. The metabolite purification and elution positions on these columns were followed by radioactivity and their ultraviolet absorption at 310 nm. The two metabolites have been unequivocally identified as (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol by ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry and co-chromatography with synthetic compounds on a high-performance liquid chromatograph. (5E)- but not (5Z)-19-nor-10-oxo-25-hydroxycholecalciferol was able to induce HL-60 cell phenotypic and functional differentiation. However, these two metabolites of 25-hydroxycholecalciferol did not bind specifically to the chick intestinal 3.7 S. receptor protein for 1 alpha,25-dihydroxycholecalciferol. The precise biological role of these metabolites is as yet unclear.
Animals↗
Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3.
The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.
Calcitriol↗
gamma-Interferon stimulates production of 1,25-dihydroxyvitamin D3 by normal human macrophages.
We show for the first time that normal human pulmonary alveolar macrophages (PAM) markedly enhance their basal rate of the production of [3H]-1,25(OH)2D3 when cultured in the presence of recombinant gamma-interferon (gamma-IFN). The rate of conversion of [3H]-25(OH)D3 to [3H]-1,25(OH)2D3 was dose-dependent in a linear fashion. A maximal production of 1,25(OH)2D3 by PAM occurred after exposure of PAM to gamma-IFN for one day. This maximum plateau-level was sustained for at least five days. The authenticity of the putative 1,25(OH)2D3 obtained from PAM was tested by demonstrating the exact comigration of [3H]-1,25(OH)2D3 with chemically synthesized 1,25(OH)2D3 in four different HPLC-systems.
Animals↗
A sensitive and simplified radioimmunoassay for 1,25-dihydroxyvitamin D3.
A sensitive radioimmunoassay system for 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] with an improved extraction procedure has been developed. Following one-step extraction and prepurification of 1,25(OH)2D3 by 'Extrelut-1' minicolumns final purification was achieved by high-performance liquid chromatography (HPLC) using a radial compression separation system equipped with a mu Porasil cartridge. The HPLC method applied allows the purification of 4 extracts/h. Recovery of 1,25(OH)2[3H]D3 after HPLC was 77 +/- 2.6% (mean +/- SD, n = 51). Since the recovery of 1,25(OH)2[3H]D3 was very reproducible, addition of labelled steroid to each single serum sample for monitoring recovery was omitted. The sensitivity of the assay was 0.8 pg/tube resulting in a detection limit of 3 ng/l, when 1 ml of serum was extracted. Intra-assay and inter-assay coefficients of variation were 12% and 16.8%, respectively. Serum 1,25(OH)2D3 concentration in 30 normal subjects (mean age: 25 yr) was 55 +/- 12 ng/l (mean +/- SD). In 55 elderly patients (mean age: 77 yr) the 1,25(OH)2D3 serum level was 32 +/- 12 ng/l (mean +/- SD) and in three patients with chronic renal failure on 1,25(OH)2D3 therapy 146 +/- 67 ng/l (mean +/- SD). Patients with chronic renal failure had reduced 1,25(OH)2D3 serum levels (mean 5.4 ng/l, range less than 3-11 ng/l, n = 10). In one patient with renal failure, following kidney transplantation the serum 1,25(OH)2D3 and creatinine levels were monitored from the 4th to the 12th post-surgical day: a highly significant negative correlation (r = 0.85) was found.
Adolescent↗
[Urgent need for hospital hygiene].
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Cross Infection↗