bluuebunny/crossref_metadata_2025 huggingface dataset:
The calcium-buffering phase of bone mineral: Some clues to its form and formation
Authors: Neuman Margaret W. , Imai K. , Kawase T. , Saito S.
Published: February 2010

Devitalized, neonatal, rat calvariae incubated in a buffered solution release Ca2+ and P1 during the first day into the medium until levels reach 1.2 mM and 2.6 mM, respectively. Thereafter levels gradually decrease to stabilize by the fifth day at 0.5 mM and 2 mM. Comparison of these solubility changes with those of known calcium phosphate salts suggests that calvarial solubility is controlled by a mixture of impure, soluble, apatite precursors, which at cell death transform to an impure apatite. Increasing concentrations of Mg2+ increased the rate of Ca2+ release and, at 3 mM, stabilized Ca2+ by the fifth day at 0.9 mM. Decreasing the pH from 7.4 to 7.2 produced similar solubility changes as did the addition of citrate at 0.3 mM, phosphocitrate at 0.3 mM, ATP at 0.1 mM, and the bisphosphonate, HEBP, at 0.1 mM. Osteocalcin did not increase calvarial solubility but was able to slow the rate of calcium phosphate phase transition if present at the time of in vitro crystal formation. Phosphocitrate and HEBP were also more effective when present during in vitro crystal formation. HEBP was most effective when present during biological crystal formation, as shown by the increased solubility of devitalized calvariae removed after in vivo administration of HEBP.

In vivo manipulations of osteoclast activity produced changes in plasma calcium which correlated with the solubility of the corresponding calvariae after removal and devitalization. Low milk intake increased calvarial solubility. Increasing doses of 1,25(OH)2D3 increased plasma calcium and calvarial solubility, both of which were reversed by injection of acetazolamide.

It was concluded from this survey of devitalized bone solubility that calcium exchange between bone and body fluids can buffer calcium homeostasis in the young rat. The exchange is passive. The active components appear to be osteoblastic formation of soluble apatite precursors and their stabilizers and, in reverse, osteoclastic transformation of apatite to precursors by H2CO3 secretion.
Inhibition by citrate of spontaneous precipitation of calcium oxalate in vitro
Authors: Nicar Michael J. , Hill Kathy , Pak Charles Y.C.
Published: February 2010

Effect of citrate on the spontaneous precipitation of calcium oxalate was examined in synthetic media. Citrate significantly increased the formation product of calcium oxalate. This “direct” measure of inhibitor activity, representing activity product at the point of nucleation, rose by 76% by the addition of citrate sufficient to provide trivalent citrate concentration of 1.49 mM. Moreover, citrate inhibited calcium oxalate crystallization by complexing calcium and lowering calcium oxalate saturation. This “indirect” measure of inhibitor activity was assessed from the concentration product of calcium oxalate at the point of nucleation, since this measure should provide a reflection of both ion pair formation and direct inhibitor activity of citrate. The concentration product exceeded the formation product at all ionic (trivalent) citrate concentrations, particularly at high ionic citrate levels. At the ionic citrate concentration of 1.49 mM, the rise in the concentration product was 373%, which was nearly fivefold that observed for the formation product. The presence of ferric or aluminum cations at a physiologic concentration of 2 mg/l did not modify the increase in formation product produced by citrate. Thus, citrate inhibits calcium oxalate crystallization, largely by complexing citrate, but also by directly affecting nucleation. Presence of ferric or aluminum cations at a physiological concentration does not modify the inhibitor action of citrate.
The proportion of trabecular bone in human vertebrae
Authors: Nottestad Sheri Y. , Baumel Julian J. , Kimmel Donald B. , Recker Robert R. , Heaney Robert P.
Published: February 2010

The proportion of trabecular bone in human cadaver vertebrae was assessed by anatomic dissection. Thirty-two whole thoracic and lumbar vertebrae were obtained from 10 normal human postmenopausal female cadavers, 14 from 4 normal adult human male cadavers of similar age, and 8 from one female osteoporotic cadaver. Each vertebra was opened by saw cuts and separated into four tissue types: (1) body trabecular bone and marrow; (2) body cortical bone; (3) vertebral arch trabecular bone and marrow; and (4) vertebral arch cortical bone. Calcium was determined in each tissue type for each vertebra by ashing and atomic absorption spectrophotometry.

Trabeculae accounted for 24.4 ± 4.5% of the total calcium in whole female vertebrae, and 18.8 ± 4.4% in whole male vertebrae (p < 0.001). The body averaged 41.8% trabecular bone in females and 33.5% in males. The arch averaged 9.7% trabecular bone in females and 4.9% in males. The proportion of trabecular bone in the whole vertebrae in the single osteoporotic spine was 28.5 ± 3.2%, a value not significantly different from the trabecular fraction in normal females.

These data indicate that whole human thoraco-lumbar vertebrae are composed of a substantially lower proportion of trabecular bone than is usually assumed, and they suggest that cortical and trabecular bone are eventually lost in equal proportion from the vertebrae during the development of spinal osteoporosis. These results are important for the interpretation of data from noninvasive bone measurement techniques that evaluate the spine, and they suggest that studies of this type are important for any site where noninvasive bone mass measurement is done.
Intravenous aminopropylidene bisphosphonate (APD) in the treatment of paget's bone disease
Authors: Vega Eduardo , Gonzalez Diana , Ghiringhelli Gualterio , Mautalen Carlos
Published: February 2010

We studied the effect of the intravenous administration of the bisphosphonate APD in 9 patients with Paget's bone disease. The medication was given in a daily dose of 25 mg for 7 days in 0.9% saline infusion over 2 hours. At the end of treatment a significant fall of serum calcium and phosphate was observed. The urinary excretion of calcium decreased markedly and the serum levels of the mid-molecule PTH fragment increased from (mean ± SE) 85 ± 11 to 122 ± 16 pg/ml (p < 0.05). A marked and rapid decline in the hydroxyprolinuria was observed from 297 ± 61 mg/24 h to 194 ± 51 mg/24 h (p < 0.01); meanwhile the serum alkaline phosphatase decreased from 102 ± 22 to 84 ± 21 KAU (p < 0.05). The effect of ADP on suppression of hydroxyprolinuria varied markedly from +1 to -81% and was negatively related to the basal hydroxyprolinuria (r = -0.90; p < 0.001). The duration of the bone turnover suppression was short. A relapse greater than 30% in hydroxyprolinuria was observed in 6 of 8 patients 2 to 3 months after APD withdrawal.

The short-term intravenous administration of ADP is a useful means to rapidly suppress the activity of Paget's bone disease. However, further studies should determine the optimum dose, the length of treatment, and the need to associate oral therapy to induce a prolonged remission.
Calcium/phosphatidylserine-stimulated protein phosphorylation in bone: Effect of parathyroid hormone
Authors: Stewart Pamela J. , Stern Paula H.
Published: February 2010

The calcium/phosphatidylserine (PS)-stimulated phosphorylation of endogenous proteins in the 100,000 × g particulate fraction from neonatal mouse calvaria was investigated. EGTA selectively inhibited the phosphorylation of a 20K protein. The phosphorylation of this 20K protein was stimulated by calcium and by PS. The combination of calcium plus PS increased the phosphorylation of the 20K protein more markedly than either calcium or PS alone. Parathyroid hormone (PTH) (100 nM) treatment of calvaria rapidly altered the phosphorylation of the 20K protein in a time-dependent manner. The PTH treatment time course demonstrated that after 5 minutes the in vitro phosphorylation of the 20K protein was markedly enhanced, after 15 minutes the 20K protein was not as heavily phosphorylated, and after 30 minutes the in vitro phosphorylation of the 20K was less than control. Our results demonstrate the presence of calcium/PS-stimulated phosphorylation in bone tissue and a rapid effect of PTH on this phosphorylation.
Effect of dietary acid and calcium on 25-hydroxyvitamin D metabolism by chick kidney
Authors: Dr. Cunningham John , Bikle Daniel D. , Avioli Louis V.
Published: February 2010

Studies of the effects of acidosis and dietary calcium on 25-hydroxyvitamin D3 (25OHD3) metabolism were conducted utilizing assays of mitochondrial 1-hydroxylase and 24-hydroxylase activity following 96 hours of acid loading. The results showed decreased 1-hydroxylase (206 ± 6 vs. 132 ± 22 fmol min−1 mg/mitochondrial protein−1, p < 0.01), augmented 24-hydroxylase (48 ± 14 vs. 180 ± 30 fmol min−1 mg−1, p < 0.001) and increments in plasma calcium and phosphate following acidosis. High calcium diet also increased 24-hydroxylase activity (48 ± 14 vs. 160 ± 24 fmol min−1 mg−1, p < 0.001) and plasma calcium, but decreased plasma phosphate. The extramitochondrial concentrations of calcium, potassium, and phosphate which maximally stimulated the hydroxylases in vitro were not altered by the in vivo dietary perturbations. The increments in 1-hydroxylase and 24-hydroxylase resulting from the presence of calcium, potassium, or phosphate in optimal concentrations (10−5M, 100 mM, and 100 mM, respectively) were changed by both acid loading and high calcium diet, decreasing in the case of 1-hydroxylase and increasing in that of 24-hydroxylase. The data indicate that the suppression of 1-hydroxylase and augmentation of 24-hydroxylase following these dietary perturbations may depend on both changed total enzyme capacity and altered enzyme sensitivity to extramitochondrial ions and are consistent with mediation by calcium of the effects of acidosis.
A comparative immunocytochemical study on the subcellular distributions of 44 kDa bone phosphoprotein and bone γ-carboxyglutamic acid (Gla)-containing protein in osteoblasts
Authors: Mark Manuel P. , Prince Charles W. , Gay Steffen , Austin Ronald L. , Bhown Merra , Finkelman Richard D. , Dr. Butler William T.
Published: February 2010

Bone γ-carboxyglutamic acid (Gla)-containing protein (BGP or osteocalcin) and 44 kDa bone phosphoprotein (44K BPP, also called Sialoprotein I or osteopontin) have been localized at the ultrastructural level in osteoblasts from woven bones of newborn rats. Frozen, undecalcified sections of periodate-lysine-paraformal-dehyde fixed specimens were incubated with affinity purified, monospecific antibodies against BGP or 44K BPP. The sites of the antigen-antibody reaction were demonstrated by the avidin-biotin-peroxidase complex method using the Hanker-Yates reagent as a peroxidase substrate. In some cases immunostaining could only be achieved after detergent treatment. The immunostained sections were then flat-embedded in Epon 812 and processed for electron microscopy. Strong specific intracellular labeling was obtained with both antibodies, but the patterns of staining differed significantly: BGP antigenicity was mainly located in the endoplasmic reticulum (ER), whereas 44K BPP behaved as a Golgi-specific antigen. In both cases, however, we found no evidence for immunostained secretory vesicles. There was no correlation between the expression of BGP by osteoblasts and the morphological aspect of these cells, their apparent degree of polarization with respect to the bone matrix, or their relation with the mineralized phase.
Increase in serum parathyroid hormone concentration in the lactating rat: Effects of dietary calcium and lactational intensity
Authors: Garner Sanford C. , Peng Tai-Chan , Hirsch Philip F. , Boass Agna , Toverud Svein U.
Published: February 2010

The change in circulating levels of immunoreactive parathyroid hormone (iPTH), measured with an N-terminal specific radioimmunoassay, was examined during lactation in rats. In lactating rats consuming a diet containing 0.4% Ca (basic diet), serum iPTH was a) increased by an average of 53% between days 10 and 18 compared to the level of age-matched nonlactating rats (24.7 ± 2.1 pg/ml vs 16.1 ± 0.8 pg/ml, mean ± SE, p < 0.01) and b) significantly higher in dams suckling large litters (10–15 pups) than in dams suckling small litters (3 pups) over the period 3–13 days of lactation. Lactating rats consuming a low calcium diet (0.04% Ca), had serum iPTH levels on days 16–18 of lactation approximately twice those of nonlactating rats fed the same diet and 73% higher than those of lactating rats fed the basic diet. Serum Ca concentrations were 22% and 10% lower in dams consuming the 0.04 and 0.4% Ca diets, respectively, than in the nonlactating controls fed the same diets. Regression analysis showed a significant (p < 0.001) negative correlation between iPTH and total serum calcium. Compared with nonmated controls, net mineral loss from femurs of dams consuming the 0.4% Ca diet was a) insignificant at day 6, b) 27% at day 15, and c) 34% at day 21 of lactation. Our data demonstrate that lactation in the rat is characterized by hyperparathyroidism that appears to be related to lactational intensity and that is accentuated when dietary calcium intake is restricted. The decrease in bone mineral during the period of elevated serum iPTH also suggests that parathyroid hormone plays a role in the bone loss normally seen in lactating rats.
Signal sequence of human preproparathyroid hormone is inactive in yeast
Authors: Dr. Born W. , Freeman M. , Bornstein W. , Rapoport A. , Klein R.D. , Hendy G.N. , Khorana H.G. , Rich A. , Potts J.T. , Kronenberg H.M.
Published: February 2010

The biosynthesis of human preproparathyroid hormone (hpreproPTH) and the processing to mature parathyroid hormone (hPTH) was investigated in yeast. Cells were transformed with a plasmid that carried a fusion gene made of the yeast pyruvate kinase promoter, complementary DNA (cDNA) encoding a slightly modified form of hpreproPTH and the transcription termination signal from yeast triosephosphate-isomerase. In transformed yeast cells we identified a protein that was recognized by a PTH antiserum and, on gel electrophoresis, comigrated with hpreproPTH marker. The amino-terminal sequence of the protein was consistent with that of hpreproPTH, indicating that the hormone precursor is not processed. It was localized inside the cell, when analyzed in pulse-chase experiments by trypsin accessibility in intact and lysed spheroplasts. In contrast, when mRNA from these yeast cells and from human parathyroid tissue was translated into preproPTH in a reticulocyte lysate supplemented with canine pancreatic microsomes, the preproPTHs from both mRNAs were transported and cleaved with identical efficiencies. We conclude that hpreproPTH is synthesized in yeast but not recognized and processed like a precursor of a secreted protein by the yeast secretory apparatus.
Effect of age on circulating immunoreactive and bioactive parathyroid hormone levels in women
Authors: Forero Martha Susana , Klein Robert F. , Nissenson Robert A. , Nelson Karen , Iii Hunter Heath , Arnaud Claude D. , Riggs Lawrence B.
Published: February 2010

Although levels of serum immunoreactive parathyroid hormone (iPTH) increase with age in women, this could be caused by retention of non-biologically active PTH fragments by the aging kidney. In 102 normal women, aged 30 to 89 yr, serum iPTH increased with age by 58% (r = 0.33, p < 0.001) with antiserum GP-1M (which has midmolecule specificity) and 43% (r = 0.32, p < 0.001) with antiserum CH-12M (which may have whole molecule specificity); urinary cAMP/GFR excretion increased by 29% (r = 0.22, p < 0.05). The results of these assays were validated by comparison with serum levels of biologically active PTH (BioPTH) in immunoextracts of serum followed by renal adenylate cyclase assay in a selected subgroup of 25 of the women. Serum BioPTH correlated with serum iPTH assessed by antiserum GP-1M (r = 0.48, p < 0.05) and antiserum CH-12M (r = 0.48, p < 0.05) but not with urinary cAMP. The data are consistent with an increase of parathyroid function with aging: clearly, we do not find decreased parathyroid function as would be expected if age-related bone loss was not mediated, in part, by PTH.
Radioimmunoassay for osteonectin. Concentrations in bone, nonmineralized tissues, and blood
Authors: Malaval Luc , Fournier Brigitte , Delmas Pierre D.
Published: February 2010

We describe a double antibody radioimmunoassay for the bone protein osteonectin using a polyclonal antibody raised against bovine fetal bone osteonectin. This assay, which cross-reacts with human osteonectin, was used to explore the tissue distribution of the protein. Osteonectin was detected in extracts of nonmineralized tissues from bovine fetuses, with concentrations ranging from 10 ng/100 μg of proteins in the lens to about 80 ng/100 μg in collagen-rich tissues (e.g., tendon), which represents respectively from 0.01% to 0.5% of the concentrations found in bone extracts. Osteonectin is thus a widespread component of extracellular matrix, although its concentration is minute in tissues other than bone and dentin. Adult human serum contains 200 ng/ml of osteonectin and values obtained in plasma prepared without platelet activation are about one-third of those in matched sera. This platelet-independent fraction might be used as a marker for bone metabolism.
The assessment of bone formation and bone resorption in osteoporosis: A comparison between tetracycline-based iliac histomorphometry and whole body 85Sr kinetics
Authors: Dr. Reeve J. , Arlot Monique E. , Chavassieux Pascale M. , Edouard Claude , Green J.R. , Hesp R. , Tellez Marisol , Meunier P.J.
Published: February 2010

Bone formation and resorption have been measured in patients with idiopathic osteoporosis by histomorphometry of 7.5-mm trephine biopsies and in the whole body by 85Sr radiotracer methodology and calcium balances. The studies were synchronized and most were preceded by double in vivo tetracycline labeling. Correlations between histological and kinetic bone formation indices were better when based on the extent of double tetracycline labels than on measurements of osteoid by visible light microscopy. Correction of the kinetic data for long-term exchange, using 5 months' serial whole body counting of retained 85Sr, improved the fit of the kinetic to the histological data. A statistical analysis of the measurement uncertainties showed that the residual scatter in the best correlations (between exchange-corrected bone formation rates and double-labeled osteoid surface indices) could be attributed to measurement imprecision alone.

The exchange-corrected resorption rate correlated fairly well with iliac trabecular resorption surfaces, and using a volume referent rather than a surface referent for the histological index improved the statistical fit when patients with therapeutically accelerated bone turnover were included. A much better correlation was obtained by including osteoid volume acting as an independent predictor of bone resorption in a bivariate regression with a resorption surface index. The residual errors could then be accounted for by known measurement uncertainties. Whereas osteoid taking a double label closely predicted the kinetic rate of bone formation, further analysis suggested that osteoid that took no label or a single label was more closely related to bone resorption, presumably as a secondary result of the coupling of bone formation to bone resorption. The idea that continued bone loss in some patients is associated with defective osteoblastic bone formation is supported by the low rates found in some patients by both techniques. Heuristically these studies validate both in vivo tetracycline labeling for dynamic histomorphometry and corrections for long-term exchange in kinetic studies of bone formation, providing a quantitative framework for the design and analysis of future studies of bone remodeling in the osteoporoses.
1,25(OH)2D levels in dihydrotachysterol-treated patients: Influence on 1,25(OH)2D assays
Authors: Taylor Arlene , Norman Michael E.
Published: February 2010

Many clinicians continue to prefer dihydrotachysterol (DHT) as the initial vitamin D agent of choice in hypoparathyroidism and renal osteodystrophy because of its long history of efficacy and safety. Assessment of the factors influencing the clinical response to DHT treatment should include measurement of vitamin D metabolite profiles, but investigators have heretofore been unable to measure 1,25(OH)2D because levels have been found to be falsely elevated when employing the chick intestinal cytosol receptor assay. After converting from the chick cytosol receptor assay to the calf thymus receptor assay for measuring 1,25(OH)2D, we did not note falsely elevated levels of 1,25(OH)2D in DHT-treated patients. The design of this study, therefore, was aimed at determining whether or not the calf thymus receptor measured authentic 1,25(OH)2D in such patients. We controlled for the possibility that freezing and thawing or prolonged storage might have either lowered 1,25(OH)2D levels or degraded a metabolite(s) of DHT that would have otherwise been recognized as “1,25(OH)2D” by the calf receptor. Similarly, technical differences between the two assays, source of thymus, and potential interference by other cytosolic proteins were eliminated as causes for the difference between the 1,25(OH)2D levels in the two assays. Our experiments do not provide an explanation for why the thymus receptor does not “see” the interfering metabolite(s) of DHT. This could reflect either a tissue difference or perhaps a species difference. Our results do provide the first opportunity to expand the investigation of the metabolic effects of DHT therapy to include changes in intrinsic 1,25(OH)2D metabolism.
Two forms of parathyroid hormone-like adenylate cyclase-stimulating protein derived from tumors associated with humoral hypercalcemia of malignancy
Authors: Stewart Andrew F. , Burtis William J. , Wu Terence , Goumas Douglas , Broadus Arthur E.
Published: February 2010

Tumors associated with humoral hypercalcemia of malignancy (HHM) contain parathyroid hormone-like adenylate cyclase-stimulating proteins (hACSPs). We previously purified a 17,000 MW hACSP from an HHM-associated breast carcinoma. This report describes the characterization of hACSPs from three additional HHM-associated tumors: two typical HHM-associated tumors (squamous carcinomas) and a third unusual tumor type (pheochromocytoma).

Each tumor was extracted in acid-urea/ethanol-sodium chloride, and adenylate cyclase-stimulating activity (ACSA) was examined following reverse-phase and size-exclusion HPLC and isoelectric focusing. Each tumor contained a high molecular weight form of ACSA which co-eluted with the 17,000 molecular weight breast carcinoma hACSP in each of the three separation procedures. Each also contained a lower molecular weight form(s) of ACSA (6,000–9,000 molecular weight). Both forms were inhibited by Nle8,18Tyr34bPTH(3–34) amide. The high molecular weight form was not changed to the lower molecular weight form by a reducing agent.

Some HHM-associated tumors contain two forms of hACSP, one with a molecular weight of 17,000 and another with a molecular weight of 6,000–9,000, which appears to be an amino-terminal cleavage product of the larger protein.
Bone density of the radius, spine, and proximal femur in osteoporosis
Authors: Mazess Richard B. , Barden Howard , Ettinger Mark , Schultz Eloy
Published: February 2010

Bone mineral density (BMD) was measured in 140 normal young women (aged 20 to 39 years) and in 423 consecutive women over age 40 referred for evaluation of osteoporosis. Lumbar spine and proximal femur BMD was measured using dual-photon absorptiometry (153Gd), whereas the radius shaft measurement used single-photon absorptiometry (125I). There were 324 older women with no fractures, of which 278 aged 60 to 80 years served as age-matched controls. There were 99 women with fractures including 32 with vertebral and 22 with hip fractures. Subsequently, another 25 women with hip fractures had BMD measured in another laboratory; their mean BMD was within 2% of that of the original series. The mean age in both the nonfracture and fracture groups was 70 ± 5 years. The BMD in the age-matched controls was 20% to 25% below that of normal young women for the radius, spine, and femur, but the Ward's triangle region of the femur showed even greater loss (35%). The mean BMD at all sites in the crush fracture cases was about 10% to 15% below that of age-matched controls. Spinal abnormality was best discriminated by spine and femoral measurements (Z score about 0.9). In women with hip fractures, the BMD was 10% below that of age-matched controls for the radius and the spine, and the BMD for the femoral sites was about 25% to 30% below that of age-matched control (Z score about 1.6). Femoral densities gave the best discrimination of hip fracture cases and even reflected spinal osteopenia. In contrast, neither the spine nor the radius reflected the full extent of femoral osteopenia in hip fracture.
Bone resorption by isolated chick osteoclasts in culture is stimulated by murine spleen cell supernatant fluids (Osteoclast-Activating Factor) and inhibited by calcitonin and prostaglandin E2
Authors: de Vernejoul Marie-Christine , Horowitz Mark , Demignon Josiane , Neff Lynn , Baron Roland
Published: February 2010

The question of whether any of the agents known to activate bone resorption in vivo or in organ cultures acts directly on the osteoclast or via intermediate target cells that secondarily secrete locally paracrine factors is important for our understanding of bone remodeling. In an attempt to clarify this issue for some of the agents, we have taken advantage of the recent progress in obtaining and culturing relatively pure populations of osteoclasts. We performed an in vitro bone-resorbing assay in which isolated and partially purified chick osteoclasts were cultured on devitalized, paired and standardized bone disks prepared from rat calvaria prelabeled with both 45Ca and 3H-proline. Some of the isolated osteoclasts attached to the devitalized bone matrix, formed a ruffled border, and acidified the bone-resorbing compartment that they established with the matrix, thereby indicating that they resorbed bone in a physiologic manner. Salmon calcitonin added to these cultures (0.3 U/ml = 60 ng/ml) and prostaglandin E2 (PGE2) (10−6M) inhibited both basal and stimulated 45Ca and 3H-proline release. Neither parathyroid hormone (PTH) 1–34 (1 U/ml), 1,25-(OH)2-D3 (10−8 and 10−9M), nor interleukin 1 (IL-1) (purified from P388D1 macrophage culture supernatant fluids or recombinant murine IL-1-alpha) (100 ng/ml) stimulated bone resorption in these cultures. In contrast, supernatant fluids from concanavalin A (Con-A)-activated murine spleen cell cultures (murine osteoclast-activating factor; OAF) consistently and significantly induced a 3- to 5-fold stimulation of bone resorption in this system.
Does Interleukin-1 affect intracellular calcium in osteoblast-like cells (UMR-106)?
Authors: Pacifici Roberto , Civitelli Roberto , Rifas Leonard , Halstead Linda , Avioli Louis V.
Published: February 2010

Interleukin-1 (IL-1) enhances bone resorption and formation in vitro, presumably through a primary action on osteoblasts, but the mechanism by which IL-1 activates bone cells is unknown. We investigated the possibility that the effect of IL-1 on osteoblasts is mediated through an increase in intracellular calcium [Ca++]i by studying the effects of purified human monocyte-derived IL-1 (hIL-1) and recombinant human IL-1α (rhILα) and β (rhIL-1β) on [Ca++]i in the rat osteogenic sarcoma cell line UMR 106 using indo-1, a new-generation fluorescent Ca++-sensitive probe. hIL-1 (1 U/ml) resulted in an 85.5% rise in [Ca++]i over baseline that reached a peak after 30 seconds and returned to basal levels within 60 seconds. A similar transient rise in calcium was obtained upon exposure of the UMR cells to both the hIL-1 suspension buffer and to the concentration of fetal bovine serum present in the hIL-1 buffer. This effect was not abolished either by heat inactivation of both hIL-1 and serum or by pretreatment of hIL-1 with specific rabbit antihuman-IL-1 antibody. Moreover, exposure of the UMR cells to either rhIL-1α or rhIL-1β or to a mixture of both at concentrations of 1 to 100 U/ml was not followed by any change in [Ca++]i. Our data do not support the idea that IL-1 can stimulate osteoblasts through a calcium-mediated pathway.
The effect of fluoride on bone histology in postmenopausal osteoporosis depends on adequate fluoride absorption and retention
Authors: Dr. Budden F.H. , Bayley T.A. , Harrison J.E. , Josse R.G. , Murray T.M. , Sturtridge W.C. , Kandel R. , Vieth R. , Strauss A.L. , Goodwin S.
Published: February 2010

Forty-one women with idiopathic postmenopausal osteoporosis have been followed for 2 years after initiation of sodium fluoride at 40–50 mg/day, given together with a daily calcium supplement of 1 gram and vitamin D2, at 50,000 IU weekly. Histological and histomorphometric analyses were done on bone biopsies taken prior to and after 1 year of treatment (mean 1.25 ± 0.35 years). Thirty patients (74%) developed the histological fluoride effect of hyperosteoidosis, while the remaining 11 patients (26%) had no change from pretreatment biopsies. Hyperosteoidosis was based on increased values for osteoid volume and/or thickened osteoid with >3 lamellar bands. Based on previously reported findings, this histological evidence of hypersoteoidosis within 12–18 months of initiation of therapy provides a useful predictor of ultimate satisfactory fluoride response in terms of bone mineral accretion. No increases in bone mass (measured by neutron activation analysis) were observed at the time of the posttreatment biopsy but, according to this previous work, increases are anticipated over a further 2–3 years of treatment. Factors affecting the development of hyperosteoidosis were analyzed. Hyperosteoidosis was associated with a significantly higher dose of sodium fluoride and a significantly higher level of bone fluoride retention but without significant increase in fasting serum fluoride. Results suggest that fluoride retention depends not only on fluoride dose but also on body size, renal function, and intestinal absorptions of calcium and fluoride. There were no differences in the initial investigations between patients with and without hyperosteoidosis, with respect to age, years of postmenopause, estrogen use, initial biochemistry, or initial bone histology. In this study, fluoride dose and bone fluoride retention were the only identified factors that influenced the histological fluoride response of hyperosteoidosis.
Increased intestinal absorption of calcium in young and adult x-linked hypophosphatemic mice after the administration of 1,25-dihydroxyvitamin D3
Authors: Meyer Martha H. , Dr. Meyer Ralph A.
Published: February 2010

We have reported that X-linked hypophosphatemic (Hyp) and normal mice respond equally to the administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] with uptake of 45Ca from an oral test meal as judged by the isotope remaining in the fecal samples. To determine whether this was due to specific stimulation of calcium absorption, as opposed to changes in calcium secretion or transit time, 1,25(OH)2D3 was administered to 4-week-old (young) and 13-week-old (adult) normal and Hyp mice at a dose of 0.12 μg/kg per day by continual infusion from an Alzet minipump. After 3 days of infusion, absorption of 45Ca from the isolated duodenum was measured in situ. Malabsorption of calcium was shown in vehicle-treated 4-week-old Hyp mice by significantly more 45Ca remaining in the intestinal segment and by significantly reduced plasma levels and reduced skeletal levels of 45Ca. Treatment of the young mice, both normal and Hyp, with 1,25(OH)2D3 resulted in increased absorption of 45Ca, increased plasma 45Ca, and increased incorporation of 45Ca into the femur. The young Hyp mice treated with 1,25(OH)2D3 showed a significant increase in femoral ash weight. At 13 weeks of age both normal and Hyp vehicle-treated mice showed equivalent absorption of calcium, and both responded to 1,25(OH)2D3 administration with enhanced calcium absorption. At both ages plasma phosphate rose in only the Hyp mice treated with 1,25(OH)2D3, whereas plasma and urine calcium were increased in only the hormone-treated normal mice. In conclusion, 1,25(OH)2D3 stimulates the absorption of calcium in the isolated duodenum of the young Hyp mouse with equal potency to that of young normal mice.
Interferon-inhibited human osteosarcoma xenografts induce host bone in nude mice
Authors: Forster Simon , Triffitt James T. , Bauer Henrik C.F. , Brosjö Otte , Nilsson Olle S. , Smith Roger , Sykes Bryan
Published: February 2010

The growth of human osteosarcoma xenografts in nude mice can be inhibited by human interferon-alpha (IFN-α). Histologic examination of growth-inhibited tumors has revealed mineralization and partial replacement of the tumor by normal bone tissue. We have investigated whether the normal bone tissue was formed by differentiated tumor cells or by induction of host stroma to differentiate into bone tissue. Employing antibodies to both murine and human type I collagen, it was found that the normal bone produced in IFN-inhibited osteosarcomas was host derived. These results suggest that IFN induced the osteosarcoma cells to produce a bone-inductive agent that interacts with the host cells, and leads to the formation of mature normal bone tissue in a heterotopic site.
Effect of vitamin a on bone resorption: Evidence for direct stimulation of isolated chicken osteoclasts by retinol and retinoic acid
Authors: Oreffo Richard O.C. , Teti A. , Triffitt J.T. , Francis M.J.O. , Carano A. , Dr. Zallone Zambonin A.
Published: February 2010

The effects of retinol (vitamin A) and retinoic acid on primary cultures of isolated chicken osteoclasts have been studied. The experiments were performed to establish the direct actions of these two agents on the organization of cytoskeletal structures, on the acid phosphatase contents, and on the bone resorption activities of these cells. The results showed that by treating the cultures with retinol or retinoic acid, from 10−8 to 10−5M, there were dose-related responses of the osteoclasts. Adhesion to the substratum was stimulated by increasing the number of cells exhibiting the specialized dot-like adhesion structures, or podosomes, which represent the active part of the sealing zone. The treatments also induced rearrangement of the microtubular patterns with reversible depolymerization of microtubules. Acid phosphatase activity was significantly higher both in vitamin A-treated osteoclasts and in their media. When [3H]proline-labeled bone particles were added to the retinoid-treated osteoclasts, the release of [3H]proline was increased significantly compared to controls. These results suggest that the two vitamin A metabolites cause several modifications of the metabolic status of isolated osteoclasts that result in augmented rates of bone resorption.
Inhibition by phenytoin ofin vitrosecretion of calcitonin from rat thyroid glands and cultured rat C cells
Authors: Cooper Cary W. , Yi Su-Jin , Seitz Patricia K.
Published: February 2010

Baby rat thyroid glands and cultured rat medullary carcinoma C cells were incubated acutely with phenytoin (38–100 μM), and the calcitonin (CT) secreted into the serum-free medium was measured by radioimmunoassay (RIA). Phenytoin did not alter CT release from glands or C cells incubated in 1 mM Ca, but, when Ca was raised to 1.75 or 2.5 mM, a marked inhibitory effect of phenytoin was apparent. The inhibitory effect could be negated by including 10 μM BAY-K-8644 in the medium. Inhibitory effects on CT release also were obtained with 100 μM trifluoperazine or 100 μM nitrendipine, and these inhibitory effects also were counteracted by 10 μM BAY-K-8644. The results show that clinically relevant amounts of phenytoin can inhibit CT release, perhaps by interfering with C-cell Ca channels or by inhibiting calmodulin-dependent processes.
Factor derived from human lung carcinoma associated with hypercalcemia mimics the effects of parathyroid hormone on phosphate transport in cultured renal epithelia
Authors: Pizurki L. , Rizzoli R. , Caverzasio J. , Mundy G. , Bonjour J.-P.
Published: February 2010

A decrease in renal tubular reabsorption of inorganic phosphate (Pi) can be observed in hypercalcemia of malignancy. In the present study we investigated the effect of serum-free conditioned medium (CM) from cells, derived from a lung carcinoma (BEN) of a hypercalcemic patient, and of PTH on cyclic AMP (cAMP) production and sodium-dependent Pi transport (NaPiT) in epithelia of two renal cell lines. In opossum kidney cells (OK), PTH is known to enhance cAMP production and inhibit NaPiT; in contrast, in LLC-PK1 cells, PTH has no effect on NaPiT since this kidney cell line is devoid of PTH receptors. In OK cells, BEN CM induced a three- to fourfold increase of cAMP production, which was blunted by the PTH inhibitors bPTH(3–34) and bPTH(7–34). NaPiT, as assessed by measuring the initial rate of Pi uptake, was inhibited in a dose-dependent manner by BEN CM, with an effect maximal between 1h30 and 6 hr of incubation (40 ± 4% and 47 ± 4%, respectively), corresponding to the effect produced by 1–3 nM bPTH(1–34). The Na-dependent transport of a glucose analog was affected neither by BEN CM nor by PTH. In LLC-PK1 cells, neither BEN CM nor PTH altered cAMP production nor NaPiT after 1h30 of incubation. At 6 hr, BEN CM caused a slight decrease in NaPiT. In conclusion, these results constitute the first evidence of a direct and selective inhibition by tumor-derived factor(s) of NaPiT in cultured renal epithelia. Most of the renal NaPiT inhibitory activity produced by the lung tumor required the presence of a PTH receptor-adenylate cyclase system. This study also indicates that cultured renal epithelia can represent quite valuable tools for characterizing the activity of tumor-derived factors on kidney functions.
Changes in serum levels of type I and III procollagen extension peptides during infusion of human parathyroid hormone fragment (1–34)
Authors: Dr. Simon Lee S. , Slovik David M. , Neer Robert M. , Krane Stephen M.
Published: February 2010

Parathyroid hormone (PTH) inhibits collagen synthesis in vitro, in organ or cell culture and cell-free translation systems. We have designed studies to measure the effects of PTH on collagen synthesis in vivo in humans, utilizing measurements of the serum levels of procollagen extension peptides during and after infusion of synthetic human PTH (hPTH) fragment (1–34). Radioimmunoassays for the carboxy-terminal peptide of type I procollagen (pColl-C) and the amino-terminal peptide of type III procollagen (pColl-III-N) were used to measure acute changes in serum during and after hPTH(1–34). In all six osteoporotic subjects and two normal individuals, serum levels of pColl-I-C were decreased by 16 hr of infusion and returned towards normal 14 hr after the infusion was discontinued; serum levels of pColl-III-N did not change significantly during the infusion, but were increased at 14 hr after the infusion was discontinued. The PTH-induced decrease in levels of pColl-I-C correlated with an increase in blood levels of ionized calcium. In all but two subjects the serum levels of 1,25-dihydroxy vitamin D [1,25(OH)2D] were also increased during the period when serum levels of pColl-I-C were decreased. These results are compatible with the conclusion that infusion of PTH acutely inhibits type I (bone) collagen synthesis, but not type III collagen synthesis. These effects could be direct or indirect, related in part to PTH-induced increased 1-α-hydroxylation of 25-(OH) vitamin D and the resultant increased serum levels of 1,25(OH)2D.
One year's treatment of paget's disease of bone by synthetic salmon calcitonin as a nasal spray
Authors: Dr. Reginster J.Y. , Jeugmans-Huynen A.M. , Albert A. , Denis D. , Franchimont P.
Published: February 2010

The effectiveness of synthetic salmon calcitonin (SCT) administered as a nasal spray was assessed via clinical, biological, and radiological variables in 17 previously untreated Pagetic patients over a 1-year course of therapy. The results showed a highly significant decrease of serum alkaline phosphatase (S-ALP) (p < 0.05 after 1 month of treatment) and of the urinary hydroxyproline/creatinine ratio (OH/Cr) (p < 0.01 after 1 month of treatment). For the whole group, the mean decrease in S-ALP was 37 ± 4% (SEM) after 6 months (p < 0.01) and 31 ± 5% after 1 year (p < 0.01). The mean fall in OH/Cr was 35 ± 6% (SEM) (p < 0.01) and 37 ± 7% (p < 0.01) after 6 and 12 months, respectively. None of the usual side-effects of SCT were reported and local tolerance was excellent throughout the study.
Forearm bone loss in hemiplegia: A model for the study of immobilization osteoporosis
Authors: Dr. Prince R.L. , Price R.I. , Ho S.
Published: February 2010

Hemiplegia is associated with excessive bone loss in the paralyzed arm. The forearm bone mineral content was measured at a proximal cortical site and a distal trabecular site of both the normal and hemiplegic arms in 74 patients with hemiplegia. By comparing the ratios of bone mineral content in the two arms, the effect of immobilization could be quantitated independently of the large population variance in bone mineral content. Bone loss, estimated from this single comparison of bone mineral content in the normal and hemiplegic arms, at the trabecular and cortical sites was positively correlated with the duration of stroke and negatively correlated with reduction in forearm function. At the trabecular site females had a proportionately greater bone loss than males, indicating an interaction between gender and immobilization associated bone loss at this site. The regression line of excess bone loss in the hemiplegic limb against time had a negative slope of 1.3% and 1.5% a year of the starting value for the trabecular and cortical sites, respectively, over the 15 years studied. This study indicates that a reduction in function is associated with significant bone loss occurring over prolonged periods that may account, at least in part, for the significant osteoporosis seen in elderly people and in subjects with conditions resulting in reduced mobility such as arthritis, obstructive airways disease, and neurological disease.
Consistent patterns of changing hormone responsiveness during continuous culture of cloned rat calvaria cells
Authors: Dr. Aubin J.E. , Tertinegg I. , Ber R. , Heersche J.N.M.
Published: February 2010

The loss of responsiveness to hormones in cell populations is a fundamental problem in cell biology and aging. We have studied this process in cloned rat calvaria (RC) bone cell populations maintained in exponential growth in long-term culture in α-minimal essential medium supplemented with fetal bovine serum. At various times after cloning, the populations were tested for their ability to respond to parathyroid hormone (PTH), prostaglandin E2 (PGE2), and L-isoproterenol (IPT) with an increase in intracellular cAMP. Clone RCB 2.2, which was originally responsive to PTH but not PGE2, maintained this characteristic throughout 14 mo of culture, after which PTH responsiveness was gradually lost and a concomitant increase in responsiveness to PGE2 was observed. Subsequently, PGE2 responsiveness was also lost; however, continued response to IPT indicated the presence of a hormone-sensitive adenylate cyclase. A similar pattern of hormone responsiveness was observed when a number of frozen stocks of RCB 2.2 cells were thawed and the cells were again maintained in continuous culture. That this pattern of phenotypic change was not unique to clone RCB 2.2 was verified by assessing the hormone responses in other independently selected clones. Although the precise time sequence for hormone response changes was not constant, in all cases the pattern of hormone response changes was similar: i.e., PTH response was always lost and PGE2 response often first increased and then also was lost, despite the maintenance of response to IPT. These data indicate that clonal hormone-responsive populations can reproducibly give rise to unresponsive populations in an ordered series of phenotypic changes. Changes in response to the hormones tested did not appear to be concomitant with changes in other phenotypic properties, e.g., decrease in alkaline phosphatase activity.
Quantitative bone histomorphometry in nude mice bearing a human squamous cell lung cancer
Authors: Kukreja Subhash C. , Rosol Thomas J. , Shevrin Daniel H. , York Patricia A.
Published: February 2010

We performed quantitative bone histomorphometry on lumbar vertebrae in hypercalcemic tumor-bearing athymic mice carrying a human squamous cell carcinoma. For comparison, studies were also performed in athymic mice that received bovine 1–34 parathyroid hormone (PTH) infusion at the rate of 0.167 μg/hr for 7 days. In both the PTH-infused and tumor-bearing animals, percent cortical and total bone areas were significantly reduced as compared to controls, whereas trabecular bone was significantly reduced only in the tumor-bearing animals. Trabecular perimeter lined by osteoclasts was significantly increased in both tumor-bearing (1.7-fold) and PTH-infused animals (2.8-fold) compared to control mice. Trabecular perimeter lined by active osteoblasts was significantly reduced in the tumor-bearing animals (to 42% of control) and unchanged in the PTH-infused animals (97% of control). Tumor-bearing animals had significantly reduced resorptive as well as formative surfaces as compared to the PTH-infused animals. Dynamic histomorphometry revealed a marked reduction in bone formation rate (23% of control) in the tumor-bearing animals. The studies therefore demonstrate a marked inhibition of bone formation associated with increased bone resorption in this model of hypercalcemia of malignancy. These observations are similar to those seen in the human syndrome.
Effects of oxidation of human parathyroid hormone on its biological activity in continuously infused, thyroparathyroidectomized rats
Authors: Dr. Horiuchi Noboru
Published: February 2010

The effect of oxidation of human parathyroid hormone 1–34 (hPTH 1–34) on the hormone's biological activity was assessed in vivo using a multiparameter, thyroparathyroidectomized (TPTX) rat model. The peptide was oxidized by treatment with hydrogen peroxide, and the oxidized form (8,18-methionine sulfoxide) was isolated by reverse-phase HPLC. Vitamin D-deficienf rats were infused with either intact or oxidized hormone along with a 5 mM calcium chloride solution for 4 or 18 hr. Infusion of nonoxidized hormone (0.1–0.8 nmoles/hr) resulted in dose-dependent increases in serum calcium, decreases in serum phosphate, decreases in urine calcium, increases in urine phosphate and cAMP, and increased renal 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3) production. Oxidized PTH infused at doses up to 0.8 nmole/hr had no effect on any of these parameters. To assess the effect of oxidation on the ability of PTH to inhibit the production of the 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), the infusion protocol was performed in vitamin D-deficient rats repleted with 1,25(OH)2D3 by injection. In these experiments, intact hormone markedly suppressed 24,25(OH)2D3 production, whereas the oxidized form was without effect. We conclude that intact methionine residues at positions 8 and 18 of hPTH 1–34 are necessary for all its major biological actions, including its effect on the renal metabolism of 25-hydroxyvitamin D3(25(OH)D3).