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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.

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.

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.

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.

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.

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.

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.