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With the knowledge that estrogen replacement therapy can circumvent postmenopausal osteoporosis and with the discovery of estrogen receptors (ER) in cultures of normal osteoblast-like cells, extensive investigations have been directed toward understanding the role of the ER in normal bone homeostasis. ROS 17/2.8 and UMR-106-01, two established osteoblast-like cell lines derived from rat osteosarcomas, have been shown to have estrogen-regulated biologic responses. Only the ROS 17/2.8 cell line has been reported to contain ER. In this study, high-affinity, saturable binding sites characteristic of the ER were detected in UMR-106-01 cells by binding assays with the high-affinity ligand, [125I]17 beta-estradiol. An initial immunoconcentration step before western blot analysis also allowed detection of the full-length ER protein. In addition, northern blot analysis indicated that the entire ER transcript was expressed and that the half-life of the ER message was increased following cycloheximide treatment. Message levels were also regulated by removal of serum and treatment with estradiol. An estrogen-regulated reporter vector, ERET81CAT, was transfected into the UMR-106-01 cells to determine whether the detected level of ER was transcriptionally functional. Using this assay, estrogen responsiveness was evident; however, the response was inconsistent. Multiple factors, such as serum, estradiol, and cell density, influence the ER levels in these cells and probably cause fluctuations in the abundance of receptors available to induce the CAT response. When the cells are responsive, the ICI 164,384 antagonist could block the estrogen-induced activation of CAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Fast cameras already installed on the National Spherical Torus Experiment (NSTX) have be equipped with near-infrared (NIR) filters in order to measure the surface temperature in the lower divertor region. Such a system provides a unique combination of high speed (> 50 kHz) and wide fi eld-of-view (> 50% of the divertor). Benchtop calibrations demonstrated the system's ability to measure thermal emission down to 330 oC. There is also, however, signi cant plasma light background in NSTX. Without improvements in background reduction, the current system is incapable of measuring signals below the background equivalent temperature (600 - 700 oC). Thermal signatures have been detected in cases of extreme divertor heating. It is observed that the divertor can reach temperatures around 800 oC when high harmonic fast wave (HHFW) heating is used. These temperature profiles were fi t using a simple heat diffusion code, providing a measurement of the heat flux to the divertor. Comparisons to other infrared thermography systems on NSTX are made.

Annual Target: Conduct experiments on major fusion facilities to improve understanding of the heat transport in the tokamak scrape-off layer (SOL) plasma, strengthening the basis for projecting divertor conditions in ITER. The divertor heat flux profiles and plasma characteristics in the tokamak scrape-off layer will be measured in multiple devices to investigate the underlying thermal transport processes. The unique characteristics of C-Mod, DIII-D, and NSTX will enable collection of data over a broad range of SOL and divertor parameters (e.g., collisionality ν*, beta β, parallel heat flux q||, and divertor geometry). Coordinated experiments using common analysis methods will generate a data set that will be compared with theory and simulation.

ELMs reduce the stored energy achieved with HHFW heating compared with the ELM free case as also occurs for NBI heating alone. This reduction can be attributed both to direct ELM ejection of stored energy and to an increase in edge density with ELMs that exceeds the onset density for perpendicular wave propagation near the antenna [1,2], and leads to significantly more edge RF power deposition. This latter effect causes a more intense RF “hot” zone in the lower divertor scrape off region due to an increase in edge RF power propagating to the divertor from the antenna region along the magnetic field lines. Fast IR measurements of the direct ELM heat deposition at the lower divertor shows it to be peaked in the vicinity of the outer strike radius and to fall off strongly as the “hot” zone is approached, indicating little direct ELM effect on the “hot” zone heating. Physics studies of the “hot” zone have begun with sweeping the RF “hot” zone spiral over Rogowski instrumented divertor region tiles to show tha...

A mitogenic response to transforming growth factor beta (TGF) occurred in the UMR106 cells cultured in serum-free medium and exposed serially to estradiol and TGF. This mitogenic response was lost when insulin was removed from the medium. TGF inhibited growth and increased the alkaline phosphatase content in the UMR106 cells cultured in medium lacking insulin. Prior exposure of the cells to estradiol enhanced this response. Monoclonal antibodies against TGF blocked the estradiol induced inhibition of growth after a two day incubation in medium devoid of insulin.

Phagocytic cells, including normal human blood neutrophils and monocytes, metabolize 25-hydroxyvitamin D3 in vitro to more polar metabolites. Cells of the human monoblastic cell line U937 produced three metabolites when incubated with 25-hydroxyvitamin D3. One of these metabolites, previously designated peak III, has its maximal absorbance at 310 nm. Mass spectral analysis of the trimethylsilylated derivatives of peak III revealed a spectral pattern very similar to that published for the trimethylsilylated derivatives of 19-nor-10-keto-25-hydroxyvitamin D3. The biosynthetic 19-nor-10-keto-25-hydroxyvitamin D3 was active in the induction of differentiation of U937 cells.

Human blood monocytes cultured in the presence of 1,25(OH)2D3 developed enhanced competence for secretion of H2O2 relative to cells suspended in media. This effect was maximal at a concentration of 10(-8) M 1,25(OH)2D3. After 3 days of incubation, monocyte-derived macrophages (MDM) exposed to 1,25(OH)2D3 demonstrated competence for secretion of H2O2 equivalent to cells exposed to recombinant IFN-gamma. Both IFN-gamma and 1,25(OH)2D3 offset decay of this function among cells in culture after 7 days. Simultaneous exposure of cells to 1,25(OH)2D3 and IFN-gamma did not activate competence for H2O2 secretion more than either agent alone. 24,25(OH)2D3 and 25(OH)2D3 activated MDM but at higher concentration than required for 1,25(OH)2D3. Progesterone did not affect H2O2 production. Incubation of MDM with a monoclonal antibody directed against IFN-gamma inhibited activation induced by lymphokine, and to a lesser extent by cells activated with IFN-gamma; this antibody had an insignificant effect on cells treated with 1,25(OH)2D3. These results suggest that 1,25(OH)2D3 exerts a receptor-mediated effect on monocyte function that results in cellular activation as manifested by enhanced competence for secretion of H2O2. It is possible that smaller concentrations of 1,25(OH)2D3 present in serum are permissive for macrophage activation, or that monocytic phagocytes are exposed to high concentrations of vitamin D metabolites under some clinical circumstances.

UMR106 cells, a rat osteosarcoma derived clonal line, secreted insulin-like growth factors (IGF) in vitro. The IGF-II levels corrected for the cell numbers were 7-8 times higher than the IGF-I levels in the medium. Both growth factors were higher by 4-5 fold in medium conditioned by rapidly growing cells than in medium conditioned by confluent cells. The addition of 17-beta-estradiol (E) to the culture medium was associated with a statistically significant increase in the IGF concentrations. This increment was metabolite specific, not occurring with 17-alpha-E, the inactive epimer of E. 1,25(OH)2D3 also increased the IGF-I concentration but prior treatment with E blocked the response to 1,25(OH)2D3, demonstrating antagonistic actions of these two hormones on IGF secretion by osteoblast-like cells.

The question of whether the skeleton metabolizes 25-hydroxycholecalciferol [25(OH)D3] to more-polar products was studied. Calvarial cells were dispersed from 16-day old chicken embryos by using collagenase and then grown in culture in serum-free medium. Confluent cell cultures were incubated with 7 nM 25(OH)[3H]D3 for 2 hr, and the vitamin D metabolites were then extracted. At least four polar metabolites were produced. Based on separation by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography, two of these metabolites were identified as 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3]. These metabolites were also produced by cultured kidney cells but not by liver, heart muscle, or skin cells isolated from the same embryos. The specific activities of the calvarial 1- and 24-hydroxylases were similar in magnitude to those in isolated kidney cells. The specific activity of the calvarial 25(OH)D3:1-hydroxylase was inhibited by an 8-hr preincubation with 1,25(OH)2D3, whereas the 24-hydroxylase was enhanced. It is concluded that (i) vitamin D metabolism by isolated cells is organ-specific, (ii) calvarial cells produce active metabolites of vitamin D in significant amounts, (iii) vitamin D metabolism by calvarial cells is regulated by 1,25(OH)2D3, and (iv) locally produced, active metabolites could act locally, thereby adding a new dimension to the regulation of mineral metabolism by vitamin D metabolites.

We studied the effect of 17 beta-estradiol (E) on the proliferation and alkaline phosphatase activity of cultured UMR106 cells, a clonal osteoblastic cell line. Growth rates were reduced and alkaline phosphatase activity was increased in cells incubated for 2 days in medium containing E (10(-8) M). In contrast, E had no effect on the growth rates or alkaline phosphatase of a human fibroblastic cell line, S90E. The effect of E was not observed with low cell density or at confluence. 1,25-Dihydroxyvitamin D3 antagonized the response to E. Preincubation of the cells with dexamethasone, a potent inducer of differentiation, reversed the effect of E or 1,25-dihydroxyvitamin D3. These results indicate that cellular and/or extracellular factors such as cell density, the phase of the cell cycle, the state of differentiation, and the presence or absence of other steroids influenced the response of UMR106 cells to E. Serum was removed from the culture medium to minimize the effect of the steroids, growth factors, and nutrients present in serum. A striking stimulation of alkaline phosphatase by E occurred with serum-free conditions. This stimulation was biphasic over an E concentration from 10(-12) to 10(-8) M, with the peak response at 10(-10) M. The action of E on UMR106 cells was metabolite-specific, since the isomer 17 alpha-estradiol produced no effect on proliferation rates or alkaline phosphatase activity. The cyclic AMP response to parathyroid hormone (residues 1-34) was not altered by E treatment of these cells. In contrast, dexamethasone exposure did increase the cyclic AMP response to parathyroid hormone. These results demonstrate a direct effect of E on an osteoblastic cell line. They also raise the possibility that similar or identical actions of E occur in cultured normal osteoblasts.

Metabolism of 3H-25OHD3 was studied in pregnant, D-deprived rats and their fetuses at 21 days’ gestation. Significant changes in the circulating levels of 3H-24,25(OH)2D3 and 3H-1,25(OH)2D3 in maternal and fetal plasma occurred from 4 to 12 h after the administration of 223 ng of 3H-250HD3. 3H-1,25(OH)2D3 appeared before 3H-24,25(OH)2D3 in both the mother and fetuses. Maternal plasma contained more 3H-1,25(OH)2D3 than fetal plasma and, contrariwise, fetal plasma contained more 3H-24,25(OH)2D3 than maternal plasma. Maternal nephrectomy studies showed that the in vivo synthesis of 3H-24,25(OH)2D3 at 6 h was dependent on circulating levels of 3H-1,25(OH)2D3. These results revealed aspects of maternal-fetal metabolism of 250HD3 similar to those in the non-pregnant state as well as others unique to pregnancy.

Thyrocalcitonin (TCT) is useful as a diagnostic and therapeutic agent in selected human diseases. Elevated plasma levels of TCT occur in patients with medullary carcinoma of the thyroid gland. Asymptomatic relatives of these patients harboring microscopic foci of tumor may have abnormal plasma levels of TCT in the basal state or after provocative stimuli. In both instances the plasma levels of TCT can be used in the diagnosis and management of this thyroid neoplasm. Elevated plasma levels of TCT have also been described recently in subjects with certain extrathyroidal neoplasms or renal failure. Moderate elevations are seen during normal pregnancy and in the neonatal period. Although exogenous TCT has actions on several organs, including bone, the kidneys and the gastrointestinal tract, its physiological role in man, if any, is still unknown. The recently reported measurements of TCT in normal subjects should facilitate the clarification of this issue. TCT has been used as a therapeutic agent in Paget's disease of bone, hypercalcemia of diverse etiologies, osteopenia and several other skeletal disorders. The dramatic improvement of patients with Paget's disease has been a unique therapeutic action of TCT. The therapeutic responses in hypercalcemic subjects given TCT are encouraging but more information is needed about the pharmacology of the hormones in these subjects before conclusions can be formed. At present the therapeutic role of TCT in osteopenia is hypothetical and the results of ongoing and future studies are needed to determine its effects.

SummaryVitamin D metabolism was studied in pregnant, D-deficient rats and their fetuses. D-depleted, pregnant rats were supplemented with [3H]25OHD3 on the 19th day of pregnancy. The distribution and metabolism of radiolabeled D metabolites was different in maternal and fetal blood, kidneys, and small intestine. 24,25(OH)2D3 was the predominant dihydroxylated D metabolite in the fetus, whereas 1,25(OH)2D3 was the predominant dihydroxylated D metabolite in the mother. The ratio of 24,25(OH)2D3:1,25-(OH)2D3 was 12-fold greater in fetal plasma than maternal plasma. Maternal nephrectomy reduced the metabolism of [3H]25OHD3 to 24,25(OH)2D3 (43%) and 1,25(OH)2D3 (75%). However, plasma levels of these two metabolites were unchanged in the fetuses of these animals when compared with levels observed in fetuses from mothers with intact kidneys. These results suggest the possibility of independent control of 25OHD3 metabolism by the feto-placental unit and raise questions as to the possible role of 24,25(OH)2D3 in f...

U937 cells respond to a variety of stimuli with increased differentiation as manifested by reduced growth, increased adherence, increased expression of several surface receptors, and increased capacity for phagocytosis and formation of reactive oxygen intermediates. In the present study the effects of lymphocyte conditioned media, recombinant interferon-gamma (IFN-gamma), and 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the ability to form reactive oxygen intermediates by U937 cells were measured by using the luminol-dependent luminescence (LDL) assay. Neither 1,25(OH)2D3 alone nor IFN-gamma alone enhanced competence for phorbol myristate acetate- stimulated LDL. Cells were capable of moderate LDL after exposure to lymphocyte conditioned media, and this was enhanced by 1,25(OH)2D3 (10(- 8) mol/L) and other vitamin D metabolites at higher concentrations. This effect was not secondary to accelerated production of myeloperoxidase, which is important in the LDL assay. Enhanced phorbol myristate acetate-stimulated phosphorylation of a 48-kd substrate was observed in 32P-labeled intact cells treated with 1,25(OH)2D3 alone or in combination with IFN-gamma. Treatment of cells with IFN-gamma or lymphocyte conditioned media did not alter phosphorylation. These results support the concept that 1,25(OH)2D3 plays a role in phagocyte differentiation and activation beyond the effects of lymphokines. Protein kinase C-mediated phosphorylation reactions may be necessary for the ability of U937 cells to reduce O2 and required for maximal activity under some conditions of incubation.

Tartrate-resistant acid phosphatase (TRAcP) is used as a marker for osteoclasts, which are believed to be derived from phagocytic cells or phagocyte stem cell precursors. To further investigate the relationship between monocytic phagocytes and osteoclasts, acid phosphatase (AcP) activity was measured by three different techniques in human peripheral blood monocytes, monocyte-derived macrophages, and the U937 cell line. We found that cytochemistry and gel electrophoresis led to similar results, but that the colorimetric assay was inconsistent. Normal human peripheral monocytes expressed both tartrate-sensitive and -resistant AcP. In culture these cells formed polykaryons and expressed TRAcP activity that was further identified as an isoenzyme associated with bone tissue. In contrast, the U937 cells did not express TRAcP activity as measured by gel electrophoresis. Both U937 cells and monocytes possess material that interferes with interpretation of the colorimetric assay of AcP. The presence of TRAcP in monocyte-derived macrophages further supports the relationship between phagocytic cells and bone osteoclasts.

The kidneys are thought to be the only organs capable of 1 alpha-hydroxylation of vitamin D and its metabolites. We have examined the in vivo conversion of 3H-(25,26)-25-hydroxyvitamin D3(25OHD3) to 3H-(25,26)-1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] in vitamin D-deficient, pregnant and nonpregnant rats. As expected, nephrectomy of nonpregnant, vitamin D-deficient rats prevented the conversion of 25OHD3 to 1 alpha,25(OH)2D3. In contrast, nephrectomy of pregnant, vitamin D-deficient rats reduced but did not abolish the formation of 1 alpha,25(OH)2D3 from its precursor. The identity of the radioactive metabolite formed from 3H-25OHD3 which circulated in nephrectomized, pregnant rats was established as 1 alpha,25(OH)2D3 by comigration with synthetic 1 alpha,25(OH)2D3 on high-pressure liquid chromatography. The simultaneous absence of 1 alpha,25(OH)2D3 in the fetal kidneys indicated that the site of 1 alpha-hydroxylation after nephrectomy of the pregnant rat was probably extra-renal in origin. Two sites of 1 alpha-hydroxylation of 25OHD3, one renal and the other extra-renal, either fetoplacental or maternal, may exist in the pregnant, vitamin D-deficient rat.

The effect of 17 beta-estradiol (E) on the oxidative metabolism of U937 cells was studied. E had no direct effect on the proliferation, surface adherence, or luminol-enhanced luminescence (LEL) of the U937 cells. Exposure of U937 cells to lymphocyte-conditioned medium (LCM) and 1,25-dihydroxyvitamin D3 allowed a maximal LEL response by cells stimulated with phorbol myristate acetate. In contrast, LCM from lymphocytes exposed to E (LCM-E) did not stimulate LEL to the same extent as did an equal volume of LCM. Increasing the E concentration in the lymphocyte medium was associated with a dose-dependent reduction in the LEL response of the U937 cells. Mixing equal quantities of LCM and LCM-E significantly decreased LEL levels. LEL stimulated by LCM, gamma-interferon, or differentiation-inducing factor was reduced by the presence of LCM-E. The inhibitory action of LCM-E was reversible and metabolite specific. 17 alpha-E produced an effect that was only one tenth the magnitude of the E effect. These findings indicate that E can modulate the differentiation of phagocytes indirectly by altering the synthesis and/or secretion of lymphokines.

The radiolabelled bone particle assay was used as an in vitro model of bone resorption. Normal human peripheral blood monocytes and the monoblastic cell line, U937, increased 45Ca release from the devitalized bone particles. In contrast, normal human peripheral blood neutrophils, resting or stimulated, did not increase 45Ca release. Exposure of monocytes to gamma interferon (INF-gamma) stimulated secretion of hydrogen peroxide and inhibited 45Ca release from bone particles. U937 cells incubated with 1,25(OH)2D3 and lymphokines had increased secretion of oxygen reduction products and increased 45Ca release. Medium conditioned by incubation with U937 cells stimulated 45Ca release to the same extent as the U937 cells. The 45Ca releasing activity in the medium was resistant to extremes of temperature and acidification. This activity is not dependent on the presence of disulfide bonds and was not inhibited by collagenase or trypsin inhibitors. Exposure to a proteolytic agent reduced the activity by over 50%. These findings are consistent with the concept that mononuclear phagocytes secrete a substance, presumably a protein, which acts directly on the bone particles. The isolation and identification of this substance may increase our understanding of the mechanisms of bone loss associated with inflammatory processes.

A model of the maternal-fetal metabolism of vitamin D3 is depicted in Fig. 2. 25-OHD3 of maternal origin is metabolized by the maternal kidneys to the potent metabolite, 1,25-(OH)2D3, which acts on the maternal intestine, kidneys, and skeleton. The maternal kidneys and other organs can produce 24,25-(OH)2D3, although this pathway may be suppressed near the end of gestation. The placenta has selective permeability to the vitamin D3 metabolites, with 25-OHD3 crossing from the mother to the fetus more readily than the dihydroxylated metabolites. The onset of the placental synthesis of 1,25-(OH)2D3 during gestation is unknown. Likewise the regulation of the placental 25-OHD3-1 alpha-hydroxylase is unknown. 1,25-(OH)2D3 of placental origin may enter the maternal or the fetal circulation or act locally on the placenta by inducing the synthesis of proteins involved in the cellular transport of Ca. Perhaps one placenta cell type synthesizes 1,25-(OH)2D3 and another cell type possessing a cytoplasmic receptor for 1,25-(OH)2D3 responds to this metabolite. The function of the 24,25-(OH)2D3 produced by the placenta is unknown. The concentration of free 25-OHD3 and free 1,25-(OH)2D3 in the fetal circulation exceeds the maternal levels due to the differences in the DBP concentrations of the two bloodstreams. The 1,25-(OH)2D3 in the fetal bloodstream may originate from either the placenta or the fetal kidneys. The latter site may not be active in utero due to the hypercalcemia and hyperphosphatemia relative to the maternal levels of these ions. 1,25-(OH)2D3 in the fetal bloodstream acts on those fetal tissues containing cytoplasmic receptors for this metabolite. The intestinal mucosa apparently lacks these receptors until sometime during neonatal life. In contrast, fetal bone cells possess receptors for the 1,25-(OH)2D3. The 24,25-(OH)2D3 in the fetal bloodstream may also be involved in the growth and differentiation of the fetal skeleton. However, the precise role of both metabolites in the fetus remains conjectural.

The placental translocation of calcium is a well-known process, but the underlying mechanisms remain unknown. Placental cytoplasmic proteins capable of binding calcium and the active metabolites of vitamin D3 were sought in these studies. Cytosol from full-term human placenta was prepared by ultracentrifugation of homogenates and tested for binding. Partial purification by column chromatography revealed the presence of two discrete proteins which bound specifically 45Ca and 3H-25-hydroxyvitamin D3 and having approximate molecular weights of 12,000 and 67,000, respectively. These placental proteins may be intermediaries in the process of placental calcium translocation.

The effect of 17β-estradiol (E) on an osteoblast-like cell line, UMR106, was studied in vitro. The concentrations of transferrin and seven enzymes (gamma glutamyl transferase, alkaline phosphatase, acid phosphatase, lactate dehydrogenase, creatine kinase, alanine aminotransferase and aspettate aminotransferase) were measured in these cells after incubation in culture medium containing either E or the vehicle. E treatment increased five of the seven enzymes and increased the transferrin concentration in the UMR106 cells white simultaneously reducing the proliferation rates, 4-Hydroxytamoxifen, an estrogen antagonist, produced a mild estrogen agonist action on growth rates and enzyme concentrations in the UMR106 cells, When E was present simultaneously, the agonist properties of 4-hydroxytamoxifen were enhanced. These studies show that E enhanced activity of five enzymes and the transferrin content of UMR106 cells after a 2-day incubation. 4-Hydroxytamoxifen enhanced the E effect, illustrating that estrogen antagonists may manifest agonist or antagonist properties depending on the model. These results extend our previous observations showing a direct effect of E in vitro on osteoblast-like cells.

Hypercalcemia is a frequent complication in patients with adult T-cell lymphoma. We measured serum calcitriol (1,25-dihydroxyvitamin D3) levels in five hypercalcemic patients with adult T-cell lymphoma and compared the values with those of five patients with mycosis fungoides, a T-cell neoplasm not associated with hypercalcemia. All five patients with adult T-cell lymphoma had calcitriol levels in or below the normal range. These data show that elevated calcitriol levels are not uniformly elevated in this disorder and may not be the usual cause of hypercalcemia in this subgroup of patients with lymphoma.

1. Intestinal phosphate absorption in human subjects was studied by the technique of triple lumen intestinal perfusion in vivo. 2. Ileal phosphate absorption increased as the intraluminal phosphate concentration was increased. 3. Ileal rates of phosphate absorption were lower at any given intraluminal phosphate concentration than previously described jejunal rates. Acidification of the ileal lumen did not increase phosphate absorption. 4. Phosphate absorption was shown in the jejunum to be dependent on the intraluminal sodium concentration. 5. Phosphate absorption in the human small intestine consists of at least two components, one directly proportional to water movement and the second apparently independent of water movement.

SummaryThe administration of SCT, natural and synthetic, had no apparent effect on the ileal water and electrolyte transport in the rabbit. The failure of SCT to influence ileal transport of water and electrolytes in the rabbit, as it does in man, may be due to differences in the rabbit intestinal response to a foreign peptide hormone.The authors wish to acknowledge the assistance of Kathy Dodson and Janet Patterson in the typing of this manuscript.

The analysis of the serum calcium and phosphate level changes in intact Vitamin D--dosed animals showed the increasing serum calcium values without any concomitant change in serum phosphate concentration. The observed discrepancy of intestinal phosphate transport in vitro and in vivo studies together with the effect of Vitamin D towards normalizing serum phosphate level in TPTX Vitamin D--dosed animals suggest the presence of Vitamin D as some phosphate regulatory factor. The participation of the possible role of 25 OH D3 in the observed phenomenon is under current investigations. In PTH supressed hypercalcemic conditions we did not reproduce, with the usage of 1alpha--OH D3, the regulatory effect of 1,25 (OH) 2D3 described by Garabedian et al. /1/.

The progress in knowledge about Vitamin D metabolism and mechanisms of its action in the last ten years has brought us to the formulation of several questions about hormonal factors involved in phosphate transport mechanisms, phosphate homeostasis and phosphate homeostasis hormonal regulation. Beside broadly described phosphaturic effect of parathyroid hormone also active metabolite of Vitamin D3, 1,25 (OH)2D3, in serum phosphate homeostasis recently has been suggested as an important factor /1/.

Patients with adult T-cell lymphoma frequently have hypercalcemia. Bone biopsies from these patients show increased numbers of osteoclasts. We hypothesized that substances produced by the malignant T-cell caused these phenomena by increasing the formation and/or activity of osteoclasts. To test this hypothesis, we cultured U937 cells in conditioned media from a clonal T-cell line derived from a patient with adult T-cell lymphoma and hypercalcemia. This conditioned media produced maturational changes in the U937 cells as evidenced by decreased proliferation, increased adherence, increased expression of complement receptors, and formation of multinucleated giant cells. These changes were synergistically enhanced by the addition of 1 alpha, 25-dihydroxyvitamin D3 which is known to promote monocyte differentiation. We also tested interleukin 2 and gamma- and alpha-interferon to see if they were responsible for the maturational changes. Although some effects were seen, these lymphokines could not account for all the changes induced by the T-cell conditioned media. These findings support the above hypothesis and suggest that other unidentified factors may promote the differentiation of osteoclast precursors and be involved in the pathogenesis of the hypercalcemia.

19 nor, 10 keto, 25-hydroxyvitamin D3 (19/10–25OHD3) is a metabolite of 25-OHD3 produced in vitro by various phagocytes including normal human blood monocytes and transformed cell lines, U937 and HL-60. We recently reported that 19/10–25OHD3 induced differentiation of U937 cells. In these studies, 19/10–25OHD3 alone produced no detectable effect on the growth rates, surface adherence, and oxidative metabolism of U937 and HL-60 cells. When combined with lymphocyte-conditioned medium (LCM), 19/10–25OHD3 reduced proliferation, increased surface adherence and stimulated luminol-dependent luminescence (LDL) of the U937 cells. In contrast, the combination of 19/10–25OHD3 and LCM had no effect on the growth of HL-60 cells but did increase the surface adherence and the expression of a complement receptor component. 19/10–25OHD3 competed for tritium-labeled 1,25(OH)2D3 binding to receptors extracted from cultured human skin fibroblasts. This displacement capacity was 600 times weaker than that of unlabeled 1,25(OH)2D3. Incubation of human skin fibroblasts for 24 hr with 19/10–25OHD3 induced 25OHD3-24-hydroxylase activity in the fibroblasts. The inductive potency of 19/10–25OHD3 was 1/50 that of 1,25(OH)2D3. These results demonstrate bioactivity of 19/10–25OHD3 in several systems. At least one of these responses, the induction of 25OHD3-24-hydroxylase, is a receptor-mediated event. Some of the other responses may be independent of the cellular receptor for 1,25(OH)2D3. Interestingly, the potency of 19/10–25OHD3 was highest in the receptor-mediated response (1:50) and lower in the other parameters, ranging from 1: 100 to 1:600 compared to 1,25(OH)2D3. This range of bioactivity in phagocytes and fibroblasts is presently unexplained.

U937 is a human-derived lymphoma cell line that has monoblastic properties and high-affinity receptors for 1 alpha,-dihydroxyvitamin D3. Incubation of these cells with the vitamin D metabolite at 10 nM for 5 days produced marked stimulation in adherence and ingestion of Staphylococcus aureus (645% of control) and of C3b receptor (CR1) expression (292% of control) and a slight increase in hexose monophosphate shunt activity without changing cell growth rates or Fc fragment receptor expression. The changes in cellular association of S. aureus and the CR1 were detected as early as 48 hr of incubation and peaked between 3 and 5 days. Similar changes in the CR1 were induced by 25-hydroxy- and 24,25-dihydroxyvitamin D3 at micromolar concentrations. Dexamethasone, hydrocortisone, and progesterone had no effect on CR1 expression. U937 cells incubated in the presence of vitamin D metabolites exhibited a change in their phenotype. These results suggest that vitamin D metabolites may contribute to monocyte/macrophage differentiation.

Phagocytic cells are widely distributed in tissues known to be important in the metabolism of vitamin D. Incubation of human polymorphonuclear leukocytes and monocytes and resident rat peritoneal macrophages with 3H-labeled 25-hydroxyvitamin D3 leads to the formation of three radioactive peaks. Peak I is most consistent with a lactone derivative of 25-hydroxyvitamin D3, and peak II has been identified as putative 24,25-dihydroxyvitamin D3. Peak III is a novel metabolite of 25-hydroxyvitamin D3 unlike any of the synthetic standards available in our laboratories. Human neutrophils converted more substrate than did the other phagocytes examined. The stimulation of neutrophils by opsonized zymosan or phorbol myristate acetate led to a 4-fold increase in synthesis of the metabolites. These results suggest that vitamin D metabolism by phagocytic cells may play a role in the microenvironmental events that surround bony metabolism and calcium homeostasis.

Previous studies have shown that synthetic salmon calcitonin (SCT) infused intravenously causes secretion of water and electrolytes in the jejunum of normal human subjects. The present experiments were carried out to learn more about the nature of this intestinal secretory process. During SCT- or synthetic human calcitonin (HCT)-induced intestinal secretion, the following observations were made: (1) There was no change in potential difference; (2) Cl was secreted against an electrochemical gradient; (3) unidirectional Na flux out of the lumen was decreased while the opposite flux was normal; (4) luminal pCO2 fell; (5) addition of glucose to the jejunal contents stimulated Na abdsorption, and this in turn counteracted the secretory effect of calcitonin. These findings suggest that calcitonin induces active Cl secretion and inhibits active Na absorption, and that HCO3 absorption is reduced by virtue of OH secretion; furthermore, jejunal glucose absorption and glucose-stimulated Na absorption are intact during calcitonin-induced secretion. Intravenous infusion of HCT caused intestinal secretion only when blood levels were much higher than occur physiologically; therefore, calcitonin is probably not a mediator of spontaneous variations of intestinal transport in normal people. However, because calcitonin induces secretion in the ileum as well as in the jejunum, hypercalcitonemia (within the range commonly found in patients with medullary carcinoma of the thyroid) could be a cause of severe secretory diarrhea.

A radioimmunoassay for 1,25-dihydroxycholecalciferol which did not cross react with 1,25-dihydroxyergocalciferol is described. IgG fractions were prepared from the serum of rabbits that had been immunized with 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine albumin. Radioligand binding by the IgG fractions was time-, temperature-, and pH-dependent. The IgG fractions had a high affinity for 1,25-dihydroxycholecalciferol but cross reacted with 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. Vitamin D2 metabolites did not cross react in the assay when amounts up to 9 ng per tube were tested. The determination of 1,25-dihydroxycholecalciferol in human serum required an organic extraction and chromatographic isolation of the metabolite. Radioligand binding was influenced by the presence and concentration of the proteins in the phosphate buffer. The mean concentration of 1,25-dihydroxycholecalciferol in serum from normal adults was 56 (SEM 5.7) ng/L. 1,25-Dihydroxycholecalciferol was not detectable in serum from a nephrectomized subject and the concentration in serum was lower than normal in hypoparathyroid patients. Ingestion of 1,25-dihydroxycholecalciferol by nephrectomized or hypoparathyroid patients restored the concentration of 1,25-dihydroxycholecalciferol in serum to the normal range. The stability of the IgG fraction, the relatively short incubation interval, and the ability to measure 1,25-dihydroxycholecalciferol without interference from 1,25-dihydroxyergocalciferol are unique aspects of this radioimmunoassay.

Metabolism of 3H-25OHD3 was studied in pregnant, D-deprived rats and their fetuses at 21 days' gestation. Significant changes in the circulating levels of 3H-24,25(OH)2D3 and 3H-1,25(OH)2D3 in maternal and fetal plasma occurred from 4 to 12 h after the administration of 233 ng of 3H-25OHD3. 3H-1,25(OH)2D3 appeared before 3H-24,25(OH)2D3 in both the mother and fetuses. Maternal plasma contained more 3H-1,25(OH)2D3 than fetal plasma and, contrariwise, fetal plasma contained more 3H-24,25(OH)2D3 than maternal plasma. Maternal nephrectomy studies showed that the in vivo synthesis of 3H-24,25(OH)2D3 at 6 h was dependent on circulating levels of 3H-1,25(OH)2D3. These results revealed aspects of maternal-fetal metabolism of 25OHD3 similar to those in the non-pregnant state as well as others unique to pregnancy.

vol. 27 p 460: The decimal in "4.0" is not clearly obvious in the body of Figure 4. p 462: Column one, third paragraph, line 24: "50 pg" should read "50 ng." p 580: Authors' corrections for this paper were received too late. The following corrections are needed. Abstract, last sentence: substitute "volatile profiles" for "these volatiles"; p 581, fourth paragraph, line 4: "0.5%" should read "0.2%"; column two, first line, "as a point" should read "or, equivalently, as points"; four lines further down, "X" should read "W"; three lines further down, remove hyphens from "sign of the dot"; in Table 1, p 582, remove "2-t-butylphenol" from bottom right. p 787: Boehringer Mannheim Corp. no longer supports the Award mentioned in column one. Boehringer Mannheim Diagnostics (formerly Hycel) supports the Award mentioned in column two. p 984: The authors of the abstract on this page (Ash) and that on p 985 (Ferrone) are interchanged. p 1041: The abstract numbered 084 here was printed again elsewhere. Abstract 084 should read: See table in the PDF file p 1045: The author of abstract no. 105 is V.B. Kambli (Norwalk Hospital, Norwalk, CT 06856). p 1190: Under Materials and Methods, line 7: change "µmol" to "mmol" in both mentions. p 1389: Column two, line 3: "Table 1" should read "Table 2." Table 2, not originally supplied, should have read as follows. See table in the PDF file p 1472: Column one, fifth full paragraph down: the word "restricted" should read "unrestricted."