1,25（OH）2D3对大鼠肾小球系膜细胞凋亡的影响

目的探讨1,25一二羟基维生素D3[1,25(OH)2D3]对大鼠系膜细胞增殖与凋亡及其相关基因Fas表达的影响。方法体外培养的大鼠系膜细胞,分为正常对照组(0 mol/L)和不同浓度1,25(OH)2D3干预组(10-10mol/L;10-8mol/L;10-6mol/L),采用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪,检测不同作用时间(24 h、48 h)下,系膜细胞增殖及凋亡情况,免疫荧光染色法观察干预24 h后,凋亡相关基因Fas的表达情况并测定各组荧光强度。结果与正常对照组相比,1,25(OH)2D3干预组,可明显抑制系膜细胞增殖,促进凋亡,并呈作用时间依赖性。同时免疫荧光染色证实干预浓度越大,系膜细胞内Fas表达增多,荧光强度增高,差异有统计学意义(P<0.01)。结论 1,25(OH)2D3干预大鼠肾小球系膜细胞,能抑制细胞增殖,促进细胞凋亡,Fas的表达增多,呈浓度和时间依赖性。     

Differentiation of bone marrow cells from myelodysplastic patients in the presence of 1,25 dihydroxyvitamin D3 or 13-cis retinoic acid

The separate effects of vitamin D3 (1,25(OH)2D3) and 13-cis retinoic acid on the differentiation in liquid culture of marrow cells from seven patients with myelodysplastic syndrome (MDS) were studied. Following incubation with 1,25(OH)2D3, an increasing number of myeloid cells acquired the morphological appearance of mature monocyte-macrophages and reacted positively to fluoride-sensitive naphthyl acetate esterase and specifically bound My4 monoclonal antibody (McAb). Incubation of bone marrow cells with 13-cis retinoic acid enhanced the number of cells with the morphological appearance of metamyelocytes and mature granulocytes as well as those that reacted positively with AS-D naphthol chloroacetate esterase. The results suggest that the differentiation pattern of myeloid precursor cells from MDS patients can be modulated by 1,25(OH)2D3 and 13-cis retinoic acid.     

Influence of parathyroid hormone, calcitonin, 1,25(OH)2 cholecalciferol, calcium, and the calcium ionophore A23187 on erythrocyte morphology and blood viscosity

Parathyroid hormone and calcitonin, both endocrine modulators of calcium homeostasis, may influence blood rheology. Parathyroid hormone is known to reduce erythrocyte survival, leading to anemia. Calcitonin has been found to have some vascular effects. We have analyzed the Influence of parathyroid hormone (10(-7) to 10(-10) mol/L), calcitonin (10(-6) to 10(-12) mol/L), 1,25(OH)2 cholecalciferol (10(-7) to 10(-10) mol/L), additional calcium in plasma (+1 and 2 mmol/L), and the calcium lonophore A23187 (50 micromol/L) on erythrocyte morphology and blood viscosity at high shear rate (94 s(-1)) and low shear rate (0.1 s(-1)) in vitro. The loading of erythrocytes with calcium by the ionophore A23187 produced a marked echinocytic shape transformation, an increased blood viscosity at high shear rate caused by decreased deformability of these cells, and a decreased viscosity at low shear rate caused by decreased aggregation of echinocytes. In contrast, increasing plasma calcium concentrations, parathyroid hormone, calcitonin, and 1,25(OH)2 vitamin D3 had no effect on erythrocyte morphology and blood viscosity. We conclude that an increase in intraerythrocytic calcium leads to severe echinocytosis and altered blood viscosity. The endocrine modulators of calcium homeostasis--namely, parathyroid hormone, calcitonin, and 1,25(OH)2 vitamin D3--apparently do not influence intraerythrocytic calcium to a significant degree and have, therefore, no influence on cell morphology and blood viscosity.     

Vitamin D-dependent rickets type II. Defective induction of 25-hydroxyvitamin D3-24-hydroxylase by 1,25-dihydroxyvitamin D3 in cultured skin fibroblasts.

1,25(OH)2D3 induces 25(OH)D3-24-hydroxylase (24-OHase) in cultured skin fibroblasts from normal subjects. We evaluated 24-OHase induction by 1,25(OH)2D3 in skin fibroblasts from 10 normal subjects and from four unrelated patients with hereditary resistance to 1,25(OH)2D or vitamin D-dependent rickets type II (DD II). Fibroblasts were preincubated with varying concentrations of 1,25(OH)2D3 for 15 h and were then incubated with 0.5 microM [3H]25(OH)D3 at 37 degrees C for 30 min; lipid extracts of the cells were analyzed for [3H]24,25(OH)2D3 by high performance liquid chromatography and periodate oxidation. Apparent maximal [3H]24,25(OH)2D3 production in normal cell lines was 9 pmol/10(6) cells per 30 min and occurred after induction with 10(-8) M 1,25(OH)2D3. 24-OHase induction was detectable in normal fibroblasts at approximately 3 X 10(-10) M 1,25(OH)2D3. [3H]24,25(OH)2D3 formation after exposure to 1,25(OH)2D3 was abnormal in fibroblasts from all four patients with DD II. In fibroblasts from two patients with DD II, [3H]24,25(OH)2D3 formation was unmeasurable (below 0.2 pmol/10(6) cells per 30 min) at 1,25(OH)2D3 concentrations up to 10(-6) M. Fibroblasts from the other two patients with DD II required far higher than normal concentrations of 1,25(OH)2D3 for detectable [3H]24,25(OH)2D3 induction. In one, [3H]24,25(OH)2D3 production reached 2.9 pmol/10(6) cells per 30 min at 10(-6) M 1,25(OH)2D3 (30% normal maximum at 10(-6) M 1,25(OH)2D3). In the other, [3H]24,25(OH)2D3 production achieved normal levels, 7.3 pmol/10(6) cells per 30 min after 10(-6) M 1,25(OH)2D3. The two patients whose cells had a detectable 24-OHase induction by 1,25(OH)2D3 showed a calcemic response to high doses of calciferols in vivo. Our current observations correlate with these two patients' responsiveness to calciferols in vivo and suggest that their target organ defects can be partially or completely overcome with extremely high concentrations of 1,25(OH)2D3. The two patients whose cells showed no detectable 24-OHase induction in vitro failed to show a calcemic response to high doses of calciferols in vivo. In conclusion: (a) the measurement of 24-OHase induction by 1,25(OH)2D3 in cultured skin fibroblasts is a sensitive in vitro test for defective genes in the 1,25(OH)2D effector pathway. (b) This assay provides a useful tool for characterizing the target tissue defects in DD II and predicting response to calciferol therapy.     

Cytogenetic studies in 174 consecutive patients with preleukemic or myelodysplastic syndromes

Routine cytogenetic studies were done in 174 consecutive patients with preleukemic or myelodysplastic syndromes (PL/MDS): 5 had the 5q - syndrome, 2 had refractory cytopenia, 43 had refractory anemia, 38 had refractory anemia with ringed sideroblasts, 69 had refractory anemia with excess blasts, 6 had refractory anemia with excess blasts in transition, and 11 had chronic myelomonocytic leukemia. Successful chromosome studies were accomplished in 167 patients (96%); 64 (37%) had a chromosomally abnormal clone. Abnormal clones were most common among patients who had refractory anemia with excess blasts (45%), refractory anemia with excess blasts in transition (60%), and chronic myelomonocytic leukemia (45%); they were least common among patients with refractory anemia (32%) and refractory anemia with ringed sideroblasts (21%). The two patients with refractory cytopenia had normal cytogenetic results. Each patient with the 5q - syndrome had a 5q-chromosome, as this is a prerequisite for the diagnosis. The two most common structural abnormalities were deletion of part of a chromosome 5 long arm (17 patients) and deletion of part of a chromosome 20 long arm (8 patients). Nonspecific structural abnormalities of chromosomes 1, 3, 6, and 17 were also common. The most common numeric abnormalities were monosomy 5 (7 patients), monosomy 7 (4 patients), loss of the Y chromosome (9 patients), and trisomy 8 (20 patients). No chromosome abnormalities were specifically associated with any PL/MDS classification.     

1,25-DIHYDROXYCHOLECALCIFEROL INDUCES AN INCREASE IN PGE1 - AND FORSKOLIN-STIMULATED CYCLIC-AMP PRODUCTION IN T47D HUMAN BREAST CANCER CELL LINE

The effect of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], the active form of vitamin D3, on cell growth, clonogenicity, and cyclic adenosine monophosphate (cAMP) production was examined in human breast cancer cell line T47D. 1,25(OH)2D3 markedly inhibited proliferation of T47D cells in a time- and concentration-dependent manner. 1,25(OH)2D3 5 X 10(-7) reduced to 70% [3H]thymidine incorporation into DNA. Specific high affinity nuclear receptors for 1,25(OH)2D3 were present in this cell line. The cAMP produced by T47D cells was measured during 10 min stimulation by effectors (prostaglandin E1 or forskolin). Without effector, T47D cells produced similar amounts of cAMP in control and 1,25(OH)2D3-treated cells. After 3 days in the presence of 1,25(OH)2D3, cAMP production was significantly increased compared to control cells when stimulated by 10(-4) M prostaglandin E1 or 5 X 10(-7) M forskolin (3.2- and 2.4-fold increase, respectively). This cAMP increase was concentration dependent within the same range that inhibited cell growth and clonogenicity. These results suggest that 1,25(OH)2D3 may indirectly affect cAMP production by modulating the target cell response to stimulatory agents of cAMP production.     

1,25-Dihydroxyvitamin D3 maintains adherence of human monocytes and protects them from thermal injury.

Adherence to a substratum is a characteristic feature of monocyte-macrophages which may be required for several effector functions. Human peripheral blood monocytes selected by adherence were found to readhere preferentially at 1 h to fibronectin or to a biological matrix. There was then a progressive decrease in the number of adherent cells, and by 48 h only 8-20% of monocytes remained adherent. This loss of adherence occurred while monocytes remained viable by criteria such as exclusion of trypan blue or release of lactate dehydrogenase. 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) maintained the adherence of cultured monocytes to tissue culture plastic as well as to the biological matrix. This effect was concentration- and time-dependent, and suppressed by inhibitors of protein synthesis. Cellular proteins were labeled after incubation with [35S]methionine. Analysis by two-dimensional gel electrophoresis revealed increased labeling of several distinct proteins in 1,25-(OH)2D3-treated monocytes compared with control monocytes. The increased loss of adherence and decreased overall protein synthesis observed in monocytes incubated at 45 degrees C was partially prevented by preincubation of the cells with 1,25-(OH)2D3. We further evaluated the effects of thermal stress and 1,25-(OH)2D3 on protein synthesis by monocytes, and found that 1,25-(OH)2D3 increased the synthesis of heat shock proteins, protected normal protein synthesis, and increased the rate of recovery of normal protein synthesis after the thermal stress. These observations suggest that 1,25-(OH)2D3 influences monocytes by preserving the synthesis of proteins, including those critical for the maintenance of cell adherence.     

Vitamin D compounds. Effect on clonal proliferation and differentiation of human myeloid cells.

We examined the effect of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) and a variety of vitamin D analogs on proliferation and differentiation of normal and leukemic myeloid clonogenic cells. Only cells from myeloid leukemic lines that contained relatively mature cells (HL-60, U937, THP, HEL, M1) were induced to differentiate and were inhibited in their clonal growth by exposure to 1 alpha,25(OH)2D3 (50% inhibition, 3 X 10(-8)-8 X 10(-10) M). A fluorinated analog of vitamin D was 5-10-fold more potent than 1 alpha,25(OH)2D3. Cells from a human myeloblast line (KG-1) and normal human granulocyte-monocyte stem cells (GM-CFC), both of which depend on colony-stimulating factor (CSF) for clonal growth, were stimulated in their clonal proliferation by 1 alpha,25(OH)2D3 in the presence of suboptimal concentrations of CSF. Leukemic cells from 10 of 14 patients with myeloid leukemia, but not normal GM-CFC from 12 patients in remission, were markedly inhibited in their clonal proliferation by 1 alpha,25(OH)2D3. Our results suggest that 1 alpha,25(OH)2D3 may be a cofactor in hematopoiesis and that vitamin D analogs may have a differential effect on normal versus leukemic growth.     

良性原发性甲状旁腺功能亢进症患者血清1,25-二羟基维生素D3水平变化

目的探讨良性原发性甲状旁腺功能亢进症(primary hyperparathyproidism,PHPT)患者血清1,25-二羟基维生素D3[1,25-dihydroxyvitamin D3,1,25(OH)2D3]水平变化及与甲状旁腺激素(parathyroid hormone,PTH)、血钙、血磷的关系。方法良性PHPT患者56例为观察组,同期体检健康者1118例为对照组,采用电化学发光法检测2组血清1,25(OH)2D3、PTH水平,比色法测定血钙水平,磷钼酸盐法测定血磷水平。比较2组维生素D缺乏[1,25(OH)2D3<20μg/L]、严重缺乏[1,25(OH)2D3<10μg/L]的比率及不同年龄分层患者血清1,25(OH)2D3水平变化,Pearson法分析观察组维生素D缺乏、严重缺乏患者血清1,25(OH)2D3与PTH、血钙及血磷的相关性。结果观察组维生素D缺乏比率、严重缺乏比率(94.64%、46.43%)高于对照组(62.79%、14.13%)(P<0.05);Pearson相关分析显示,观察组血清1,25(OH)2D3与PTH、血钙及血磷均无线性相关性(r=-0.226,P=0.352;r=-0.274,P=0.256;r=0.073,P=0.593)。观察组年龄18~40岁、>40~60岁、>60岁患者血清1,25(OH)2D3[(10.76±3.17)、(10.61±5.01)、(10.72±4.85)μg/L]低于对照组[18~40岁:(18.19±9.86)μg/L,>40~60岁:(17.18±9.19)μg/L,>60岁:(17.91±10.52)μg/L](P<0.05);观察组维生素D缺乏患者血清PTH[(818.86±233.49)ng/L]、血钙[(2.98±0.59)mmol/L]、血磷[(0.78±0.17)mmol/L]与维生素D严重缺乏患者[(640.09±622.69)ng/L、(2.96±0.69)mmol/L、(0.75±0.20)mmol/L]比较差异无统计学意义(P>0.05);观察组维生素D缺乏、严重缺乏患者血清1,25(OH)2D3与PTH(r=-0.360,P=0.060;r=0.071,P=0.723)、血钙(r=-0.225,P=0.250;r=-0.228,P=0.252)、及血磷(r=0.239,P=0.221;r=-0.208,P=0.297)均无线性相关。结论良性PHPT患者血清1,25(OH)2D3低于正常人群,维生素D缺乏比率较高,且血清1,25(OH)2D3与PTH、血钙及.血磷无线性相关。     

1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation.

Human foreskin keratinocytes in vitro metabolize 25-hydroxyvitamin D3 to a number of metabolites, including 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This metabolite remains mostly within the cell and does not accumulate in the medium under the conditions of these experiments. With time, 1,25(OH)2D3 is catabolized, and more polar metabolites appear in both the cells and the medium. The production of 1,25(OH)2D3 has an apparent Michaelis constant (Km) for 25-hydroxyvitamin D3 of 5.4 X 10(-8) M. The levels of 1,25(OH)2D3 within the cell are increased both by increased production and decreased catabolism when parathyroid hormone(1-34) and isobutylmethylxanthine are added. Exogenously added 1,25(OH)2D3 at concentrations as low as 10(-12) M reduces endogenous 1,25(OH)2D3 production, increases 1,25(OH)2D3 catabolism, and increases 24,25-dihydroxyvitamin D3 production by an actinomycin D-sensitive process. These data indicate that the regulation of 1,25(OH)2D3 production by keratinocytes is similar to, but not identical to the regulation of 1,25(OH)2D3 by the kidney.     

Defective binding and function of 1,25-dihydroxyvitamin D3 receptors in peripheral mononuclear cells of patients with end-organ resistance to 1,25-dihydroxyvitamin D.

Lectin-induced DNA synthesis by peripheral mononuclear cells from 17 normal donors was inhibited (40-60%) by 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) at physiological concentrations (10(-10)-10(-9) M). The lymphocytes acquire specific receptors for 1,25(OH)2D3 upon activation by the lectins. This process precedes the inhibitory effect of 1,25(OH)2D3. We studied lymphocytes from six patients from four different kindreds with the syndrome of hereditary end-organ resistance to 1,25(OH)2D (the so-called vitamin D-dependent rickets type II). In five patients (three kindreds) peripheral blood mononuclear cells did not acquire receptors for 1,25(OH)2D3 upon phytohemagglutinin-induced activation. Moreover, in contrast to normal lymphocytes, the mitogenic stimulation of these patients' lymphocytes by phytohemagglutinin and concanavalin A was not inhibited by 1,25(OH)2D3. Activated lymphocytes of the sixth patient from a fourth kindred exhibited normal binding of [3H]1,25(OH)2D3 but the hormone failed to inhibit the mitogenic stimulation. A similar pattern of the vitamin D effector system was previously observed in fibroblasts cultured from skin biopsies of the same group of patients. The conclusions from these findings are: (a) the inhibition of mitogenic stimulation by 1,25(OH)2D3 is mediated by specific functional receptors to the hormone; and (b) the receptors for 1,25(OH)2D3 in mononuclear cells are probably controlled genetically by the same mechanisms as the effector system in well-characterized target organs of the hormone, such as intestine and kidney.     

No 1,25-dihydroxyvitamin D3 receptors on osteoclasts of calcium-deficient chicken despite demonstrable receptors on circulating monocytes.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is known to stimulate osteoclastic bone resorption in vivo and whole organ bone culture systems in vitro. It has not been established whether 1,25(OH)2D3 acts directly on osteoclasts or whether its action on osteoclasts is mediated via other bone cells (e.g., osteoblasts) or recruitment of osteoclast precursor cells. Circulating monocytes have been characterized as osteoclast precursors. In the present study, vitamin D3-replete chicken on a calcium-deficient diet were studied. Circulating monocytes, whole bone cell preparations, and isolated osteoclasts (differential sedimentation) were examined for presence of 1,25(OH)2D3 receptors. Reversible, specific, and saturable binding of [3H]-1,25(OH)2D3 to a 3.5 S macromolecule was demonstrated in nuclear fractions of monocytes (maximal binding capacity, 48 fmol/mg protein; dissociation constant, 1.3 X 10(-10) M) and of whole bone cell preparations. 1,25(OH)2D3 receptors were not demonstrable in osteoclast preparations (70% pure; detection threshold, 2 fmol/mg protein). Data are consistent with indirect action of 1,25(OH)2D3 on osteoclastic bone resorption.     

Serum 1,25 dihydroxy vitamin D (1,25(OH)2D3), 25 hydroxy vitamin D (25(OH)D) and parathormone levels in diabetic retinopathy

OBJECTIVES: To investigate whether there is a relationship between serum 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], which is an inhibitor of angiogenesis, concentrations and severity of diabetic retinopathy (DR). DESIGN AND METHODS: Serum 1,25(OH)2D3, 25 hydroxy vitamin D [25(OH)D] and parathormone (PTH) concentrations were measured in diabetic patients (n = 66) and nondiabetic healthy subjects (n = 20). RESULTS: The mean serum 1,25(OH)2D3 concentration in diabetic patients was lower than that in nondiabetics (57.3+/-21.44 vs. 89.4+/-18.01 pmol/L, p<0.001); mean 1,25(OH)2D3 concentrations fell with increasing severity of DR [being 63.4+/-17.26 pmol/L for background DR (BDR), 47.7+/-13.27 pmol/L for preproliferative DR (pre-PDR), and 43.1+/-19.45 pmol/L for proliferative DR (PDR)]. Compared with the control group, serum 25(OH)D concentrations were found to be decreased in diabetic patients (p<0.001).There were negative correlations between 1,25(OH)2D3 and age (r = -0.331, p<0.01) and duration of diabetes (r = -0.255, p<0.05). CONCLUSION: From these findings, it was found that there was an inverse relationship between the severity of the retinopathy, i.e., neovascularization, and serum 1,25(OH)2D3 concentrations, being the lowest in PDR and the highest in diabetic patients without retinopathy (NDR) patients. The measurement of serum 1,25(OH)2D3 concentrations might be helpful to predict severity of DR in patients with diabetes mellitus.     

Effects of dietary calcium restriction on 1,25-dihydroxyvitamin D3 net synthesis by rat proximal tubules

We measured in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production by kidney proximal tubules prepared by Percoll density centrifugation from male and female rats. 1,25(OH)2D3 in tubule extracts was determined by a sensitive and specific radioreceptor assay. Ingestion of diets adequate in vitamin D3 and containing either normal calcium (1.2% Ca, NC), reduced calcium (0.6% Ca, RCD) or low calcium (0.002% Ca, LCD) increased 1,25(OH)2D3 net synthesis (for male rats, NC vs. RCD vs. LCD 1.8 +/- 0.1 SEM vs. 9 +/- 2 vs. 17 +/- 2 pmol/mg protein/20 min; P less than 0.05 for all comparisons). At either level of reduced calcium intake, tubules from male rats produced more 1,25(OH)2D3 than tubules from females. Serum 1,25(OH)2D3 and tubule cyclic adenosine monophosphate (cAMP) content rose in parallel with progressive dietary calcium restriction, and males had higher circulating 1,25(OH)2D3 and tubule cAMP content than females at each level of reduced calcium intake. L-Epinephrine (10(-4) mol/L), in vitro, increased tubule accumulation of 1,25(OH)2D3 and cAMP. Yohimbine, and alpha 2-receptor antagonist, blocked this response, whereas prazosin was without effect. Increased 1,25(OH)2D3 net synthesis by tubules from male vs. female rats partly explains the higher serum levels and enhanced mineral conservation demonstrated previously in male rats. Preparation of proximal tubules from vitamin D-replete rats permits studies in vitro of 1,25(OH)2D3 production and regulation under more physiologic conditions in which parathyroid hormone, inorganic phosphorus, and calcium may be varied independently.     

1 alpha,25-dihydroxyvitamin D3 suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells.

Activated B and T lymphocytes from normal human subjects are known to have the specific high-affinity receptor for 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). In an attempt to determine a functional role for the sterol in such cells, we studied the effect of 1,25-(OH)2-D3 on DNA synthesis and Ig production by normal human peripheral blood mononuclear (PBM) cells activated in vitro by the polyclonal lymphocyte activators pokeweed mitogen and phytohemagglutinin, and the specific antigen dermatophyton O. A dose-dependent inhibition of [3H]thymidine incorporation was observed in cells incubated with 1,25-(OH)2-D3 in concentrations ranging from 10(-10) to 10(-7) M. Production of IgG and IgM, determined by enzyme-linked immunosorbent assay, was similarly inhibited by increasing concentrations of 1,25-(OH)2-D3. Half-maximal inhibition of DNA and Ig synthesis was found at 10(-10) to 10(-9) M 1,25-(OH)2-D3. This suppressive effect was specific for 1,25-(OH)2-D3; of the other vitamin D metabolites examined, only 10(-7) M 24R,25 dihydroxyvitamin D3 (24,25-(OH)2-D3) had a similar inhibitory effect. 1,25-(OH)2-D3 was not cytotoxic and did not affect unactivated PBMs. These data demonstrate that 1,25-(OH)2-D3 is a potent inhibitor of human PBM Ig production in vitro and suggest that this action is mediated through the hormone's antiproliferative effect on Ig-producing B cells and/or helper T cells.     

Metabolism of 25-hydroxyvitamin D3 by cultured pulmonary alveolar macrophages in sarcoidosis.

Metabolism of [3H]25-hydroxyvitamin D3(25-OH-D3) was studied in primary cultures of pulmonary alveolar macrophages (PAM) from seven patients with sarcoidosis and two patients with idiopathic pulmonary fibrosis. Production of a [3H]1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3)-like metabolite of [3H]25-OH-D3 was detected in lipid extracts of cells from five patients with sarcoidosis. Synthesis of this compound in vitro was limited to viable PAM and was greatest in cells derived from a patient with hypercalcemia and an elevated serum concentration of 1,25-dihydroxyvitamin D. The tritiated PAM metabolite coeluted with authentic 1,25-(OH)2-D3 in three different solvent systems on straight-phase high performance liquid chromatography (HPLC) and demonstrated binding to extracted receptor for 1,25-(OH)2-D3, which was identical to that of commercially available [3H]1,25-(OH)2-D3 of comparable specific activity. Incubation of PAM with high concentrations of 25-OH-D3 resulted in production of an unlabeled metabolite that co-chromatographed with the 3H-PAM metabolite on HPLC and that was bound with high affinity by both the specific receptor for 1,25-(OH)2-D3 and antiserum to 1,25-(OH)2-D3.     

Rapid diagnosis of vitamin D-dependent rickets type II by use of phytohemagglutinin-stimulated lymphocytes

The interactions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] with phytohemagglutinin (PHA)-stimulated lymphocytes from normal subjects and three patients with vitamin D-dependent rickets (DDR) type II were investigated. Impaired nuclear uptake and normal cytosol binding of [3H]1,25-(OH)2D3 were observed with PHA-stimulated lymphocytes of these patients as with their cultured skin fibroblasts. Furthermore, the incorporation of [14C]thymidine into PHA-stimulated lymphocytes of the patients was not reduced by 1,25-(OH)2D3, which is known to inhibit proliferation of various cells. These findings suggest that 1,25-(OH)2D3 receptors are reduced or absent in patients with DDR type II. Thus, the capacities of cytosol binding and nuclear uptake of 1,25-(OH)2D3 in PHA-stimulated lymphocytes seem to reflect those of endo-organs such as the intestine and bone. These findings show that a test of the effect of 1,25-(OH)2D3 on thymidine incorporation into PHA-stimulated lymphocytes is useful for rapid diagnosis of DDR type II.     

Impaired stimulation of 25-hydroxyvitamin D-24-hydroxylase in fibroblasts from a patient with vitamin D-dependent rickets, type II. A form of receptor-positive resistance to 1,25-dihydroxyvitamin D3.

We describe studies of the molecular defect in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action in cultured skin fibroblasts from a patient previously reported to have vitamin D-dependent rickets, type II. Binding of [3H]1,25-(OH)2D3 in fibroblast cytosol was normal with a Bmax (amount of high affinity binding) of 26 fmol/mg protein and a half-maximal saturation of 0.2 nM. Nuclear binding of [3H]1,25-(OH)2D3 following whole cell uptake was 1.5 fmol/micrograms DNA in patient fibroblasts compared with a range of 0.5-2.9 fmol/micrograms DNA in five control strains. The size of the [3H]1,25-(OH)2D3-receptor complex on sucrose density gradients, 3.8 S, was the same as in normal cells. This patient, therefore, appeared to have a receptor-positive form of resistance to 1,25-(OH)2D3. To document resistance to 1,25-(OH)2D3 in the fibroblasts we developed a method for detection of 1,25-(OH)2D3 action in normal skin fibroblasts. Following treatment of normal cell monolayers with 1,25-(OH)2D3 there was more than a 20-fold increase of 25-hydroxy-vitamin D-24-hydroxylase (24-hydroxylase) activity. Treatment of 10 control cell strains with 1,25-(OH)2D3 for 8 h increased the formation of 24,25-dihydroxy-vitamin D3 from 25-hydroxyvitamin D3 in cell sonicates from less than 0.02 to 0.11-0.27 pmol/min per mg protein. When cells from the patient with vitamin D-dependent rickets, type II were treated with 1,25-(OH)2D3 in a similar manner, maximal 24-hydroxylase activity was only 0.02 pmol/min per mg protein, less than a fifth the lower limit of normal. 24-Hydroxylase activity in fibroblasts from the parents of the patient increased normally following treatment with 1,25-(OH)2D3. We conclude that impaired induction of 24-hydroxylase in the presence of normal receptor binding is evidence for postreceptor resistance to the action of 1,25-(OH)2D3.