bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of inositol phosphate formation in ros 17/2.8 cell membranes by guanine nucleotide, calcium, and parathyroid hormone
Authors: Cosman Felicia , Morrow Bruce , Kopal Marcela , Dr. Bilezikian John P.
Published: February 2010

In addition to stimulation of cyclic AMP, parathyroid hormone (PTH) may influence cellular events by utilizing other pathways of hormone action, such as the generation of inositol phosphates (IPs). We sought to examine this potential action of PTH by assessing the formation of inositol phosphates in PTH-sensitive ROS 17/2.8 cells. The polyphosphoinositides were labeled by growing the cells with [3H]inositol following which cell homogenates were prepared. The nonhydrolyzable guanine nucleotide, GTPγS, and calcium ion, alone and together, stimulated all three IPs, IP1, IP2, and IP3. IP1 formation was linear over 30 minutes but IP2 and IP3 accumulated more rapidly peaking by 5 minutes for all agonist conditions. The proportion of total P as IP3 was enhanced when the cells were grown with retinoic acid (1 μM) or when the assay was conducted at pH 4.5. In addition, the lower pH was associated with much more enzyme activity. PTH agonists, bPTH-(1–84) and bPTH-(1–34), both caused a small but significant stimulation of IP3 formation. When bPTH-(1–84), and the analog bPTH-(3–34)amide, that inhibits PTH-mediated adenylate cyclase activity were present together, there was additive stimulation of IP3 formation compared with that with either agent alone. The results demonstrate that inositol phosphate formation can be stimulated directly in a membrane preparation of ROS cells by GTPγS, calcium ion, and PTH and that the enzyme mediating this activity, phospholipase C, is regulated by a guanine nucleotide binding protein.
One-day test using stable isotopes to measure true fractional calcium absorption
Authors: Eastell Richard , Riggs B. Lawrence , Vieira Nancy E. , Yergey Alfred L.
Published: February 2010

True calcium absorption can only be measured by the time-consuming and expensive metabolic balance method and is not predicted well by measuring the fractional absorption (FA) of radiocalcium from a fixed calcium carrier. We describe here a 1 day method for measuring the actual fraction of calcium absorbed from the habitual diet (true fractional calcium absorption, TFCA) using stable isotopes of calcium. Oral and intravenous isotopes were administered with each of the three daily meals, and then the ratio of the two isotopes in the urine was measured by mass spectroscopy. In 12 subjects, TFCA determined from stable Ca correlated well with TFCA measured by the balance method (r = 0.71, P < 0.01), and the mean values were not different (0.26 and 0.26). In contrast, no significant correlation was found between FA and TFCA. The weak relationship between FA and TFCA underscores the importance of tracing dietary calcium rather than a fixed calcium carrier in tests of calcium absorption. Using the new method, TFCA was inversely related to dietary calcium (r = −0.45, P < 0.05), demonstrating that it could detect physiological changes in calcium absorption. Thus, this test has two important advantages: (1) it provides a simple way to measure TFCA and true fractional calcium absorption (the product of TFCA and dietary calcium), the physiologically relevant variables, and (2) because there is no radiation exposure, the test can be used in pregnant women and children, the isotopes can be prepared in advance, and several isotopes can be used simultaneously.
Parabiosis suggests a humoral factor is involved in X-linked hypophosphatemia in mice
Authors: Dr. Meyer Ralph A. , Meyer Martha H. , Gray Richard W.
Published: February 2010

Reduced renal tubular reabsorption of phosphate of unknown etiology is characteristic of X-linked hypophosphatemia in both humans and mice. To test whether a humoral abnormality is involved in the renal effect, parabiosis was performed between Hyp and normal mice at 4 weeks of age. The normal mice joined to Hyp mice showed a progressive diminution of plasma phosphate over the next 3 weeks to approach the level of the Hyp mice. These normal mice had a greater renal phosphate excretion index (urine P/plasma P/urine creatinine) than normal-normal pairs, thus suggesting a reduced renal tubular reabsorption of phosphate. At the same time the expected rises in plasma calcium and plasma 1,25-dihydroxyvitamin D did not occur. There was a significant reduction in their femoral mineral content but not in their femoral length or body growth relative to normal-normal pairs. This change in renal handling of phosphate was specific since the urinary losses of potassium and magnesium were not significantly changed. Separation of normal-Hyp pairs 3 or 6 weeks after parabiosis caused the normal mice to achieve normal plasma phosphate levels within 24 h. At 48 h and 7 days after separation these normal mice had plasma phosphate levels higher than normal mice separated from normal-normal pairs. In summary, the data suggest the presence of a phosphaturic factor in the Hyp mice that can cross a parabiotic union into normal mice and induce many of the symptoms of X-linked hypophosphatemia.
Measurement of bone in the os calcis: A clinical evaluation
Authors: Lancaster Elizabeth K. , Dr. Evans Richard A. , Kos Susan , Hills Ellen , Dunstan Colin R. , Wong Stanley Y.P.
Published: February 2010

Bone mineral content (BMC) was measured in the os calcis of 232 normal subjects aged 17–82 years. The mean reproducibility (coefficient of variation) of the measurement was 1.8%. Substantial bone loss occurred between the ages of 20 and 50 years, and in females the menopause was associated with additional bone loss. There was no significant difference in the rate of bone loss in females and males, but the mean BMC was greater at all ages in males than in females.

We also compared os calcis BMC with spinal bone mineral density (BMD), measured by quantitative computed tomographic (CT) scanning, in 85 subjects: 33 were normal controls, 19 had osteoporosis defined by the presence of one or more pathological fractures, and in the remainder the CT examination was performed at the patient's request. Os calcis BMC correlated with spinal BMD in both females (r = 0.69, p < 0.001) and males (r = 0.84, p < 0.001). However, the os calcis BMC did not reliably predict spine values around the CT “fracture threshold” of 90–100 mg/cm3 and did not correlate with osteoporotic fracture as well as did spinal BMD. It is concluded that measurement of the os calcis BMC is of limited clinical usefulness for the early diagnosis of osteoporosis.
The renal phosphate transport defect in normal mice parabiosed to X-linked hypophosphatemic mice persists after parathyroidectomy
Authors: Dr. Meyer Ralph A. , Meyer Martha H. , Tenenhouse Harriet S. , Klugerman Abbey H.
Published: February 2010

The X-linked hypophosphatemic (Hyp) mouse is a model for human X-linked hypophosphatemia. Surgical joining of normal to Hyp mice by parabiosis results in the normal mice developing low renal retention of phosphate and hypophosphatemia. These results suggest a humoral component to the renal defect. To test whether this component could be parathyroid hormone, surgical parathyroidectomy (PTX) or sham surgery was performed in mice 3 weeks after parabiotic union (n > 20 per group). After an overnight fast, PTX mice were hypocalcemic and hyperphosphatemic relative to sham-operated control mice. PTX normal mice joined to PTX Hyp mice were significantly lower in plasma phosphate and higher in fractional excretion of phosphate [U/P phosphate/(U/P creatinine)] when compared with PTX normal mice joined to other PTX normals. To test for more specific evidence of altered renal transport function, renal brush-border membrane vesicles (BBMV) were prepared from these mice, and phosphate and glucose uptakes were measured. The phosphate/glucose transport ratio was lower in BBMV from Hyp mice, joined to either normal mice or to Hyp mice, when compared with that from normal-normal pairs. Moreover, BBMV from normal mice joined to Hyp mice had a significantly lower phosphate/glucose uptake ratio than BBMV from normal mice joined to other normal mice, and their activity approached that of BBMV derived from Hyp mice. Glucose uptake in BBMV was unaffected by parabiosis or genotype. In summary, parabiosis of normal mice to Hyp mice resulted in the development of phosphaturia and decreased BBMV phosphate transport in the normal mice. The persistence of the phosphate transport defect in parathyroidectomized mice suggests that parathyroid hormone is not the humoral factor contributing to these results.
Age and bone mass in premenopausal women
Authors: Rosenthal Daniel I. , Mayo-Smith William , Hayes Curtis W. , Khurana Jasvir S. , Biller Beverly M.K. , Neer Robert M. , Klibanski Anne
Published: February 2010

It is important to determine the bone mass in normal premenopausal women because increasing numbers of conditions have been identified that result in premenopausal osteoporosis. The relationship between age and bone density was evaluated in 57 carefully characterized normal, premenopausal women using both single energy quantitative computed tomography (SEQCT) and dual energy (DEQCT). The mean bone density measurements were 172 mg/ml K2HPO4 SEQCT and 185 DEQCT. Bone density showed no statistically significant decline between age 18 and age 44. Single- and dual-energy data were highly correlated with each other (r = 0.89), and dual energy appeared to confer no advantage. There was an inverse relation between density and age of menarche. Bone density did not correlate with ideal body weight, percentage fat, or subcutaneous fat area.
Differential effects of gonadal function on bone histomorphometry in male and female rats
Authors: Turner Russell T. , Hannon Kathleen S. , Demers Laurence M. , Buchanan James , Bell Norman H.
Published: February 2010

The effects of castration on bone histomorphometry and mineral homeostasis were compared in male and female rats. Measurements were performed 4 weeks after sham operation or gonadectomy. Orchiectomy produced increases in serum calcium and decreases in serum testosterone and androstenedione, whereas ovariectomy produced decreases in serum estradiol and testosterone. Orchiectomy did not alter static bone histomorphometric measurements of the tibial diaphysis, whereas ovariectomy increased cross-sectional and medullary areas, lowered endosteal tetracycline-labeled surface length, and markedly increased endosteal nonlabeled surface length. Orchiectomy decreased mean periosteal bone formation rate and mean periosteal bone apposition rate, whereas ovariectomy increased both measurements. Orchiectomy and ovariectomy markedly diminished trabecular area and trabecular surface length at the tibial metaphysis. Orchiectomy did not alter the number of osteoclasts per mm trabecular surface or the percentage of trabecular surface covered by osteoclasts, whereas ovariectomy increased both measurements. These findings indicate that gonadal hormones produce separate and distinct effects on bone metabolism as determined by histomorphometry in male and female rats.
Immunoreactive calbindin-D9K localization in matrix vesicle-initiated calcification in rat epiphyseal cartilage: An immunoelectron microscope study
Authors: Balmain N. , Hotton D. , Cuisinier-Gleizes P. , Mathieu H.
Published: February 2010

Calbindin-D9K immunoreactivity was localized by electron microscopy in rat calcifying epiphyseal plate cartilage. Antigen-antibody reaction sites were visualized by the presence of protein A-gold complex particles on undecalcified material embedded in Lowicryl K4M. Immunoreactive calbindin-D9K was found in the hyaloplasm of hypertrophic chondrocytes and inside and at the ends of their cell processes. It was localized outside the cells, inside matrix vesicles (MVs), often against the inner face of the delimiting membrane, and inside the trilaminar membrane. Immunoreactive calbindin-D9K appeared to be extruded from the chondrocytes into the matrix vesicles when the latter were formed during the budding of cell processes. In calcifying MVs, gold particles were detected over the needle-shaped crystallites and often over the crystallites lying against the inner leaflet of the vesicular membrane. At a later stage of matrix vesicle calcification after MV membrane disruption, the number of gold particles remained unchanged over the clusters of crystallites at the loci from which the crystallites appeared to have grown and radiated. At a yet more advanced stage of calcification, they remained in the same areas, which were limited to the lateral edges of calcified cartilage longitudinal septa. These results suggest that immunoreactive calbindin-D9K plays a role in calcium input to matrix vesicles and may be involved in matrix vesicle calcification, perhaps in the initial event of matrix vesicle crystal nucleation.
Colles' fracture and bone density of the ultradistal radius
Authors: Eastell Richard , Riggs B. Lawrence , Wahner Heinz W. , O'Fallon W. Michael , Melton L. Joseph , Amadio Peter C.
Published: February 2010

To determine whether Colles' fracture, generally considered a manifestation of postmenopausal osteoporosis, is associated with a decrease in bone density at the site of fracture, we measured bone mineral density of the ultradistal radius (UDR-BMD) by single-photon absorptiometry with computer-assisted image processing. In 119 normal women (ages 22–92 years), UDR-BMD decreased by 17% between ages 30 and 75 years. From UDR-BMD measurements in these normal women and in 40 women (ages 53–80 years) with Colles' fracture alone, a population-based analysis was made to estimate fracture risk at different values of UDR-BMD. Colles' fracture was uncommon at UDR-BMD > 0.40 g/cm2 (the “fracture threshold”). As bone density decreased below this level, fractures became more frequent (a “gradient of risk”).
Interleukin-6 is produced by bone and modulated by parathyroid hormone
Authors: Dr. Feyen Jean H.M. , di Padova Franco E. , Trechsel Ulrich , Elford Peter
Published: February 2010

Interleukin-6 (IL-6) is a cellular regulatory molecule, the diverse functions of which relate to cells both within and outside the immune system. In this report we demonstrated that bone tissue, specifically osteoblasts, produce interleukin-6 and that this function can be modulated by the osteotrophic hormone parathyroid hormone (PTH). Given that the complex process of bone remodeling is now thought to be regulated not only by systemic hormones but also by locally produced factors, the existence of a parathyroid hormone-stimulated production of interleukin-6 by osteoblasts may have important physiological significance.
The effect of age and menopause on bone mineral density of the proximal femur
Authors: Hedlund Lars R. , Gallagher J.C.
Published: February 2010

Although the menopause has been associated with increased bone loss at several skeletal sites, it has not previously been noted in the hip, yet estrogen therapy has been reported to reduce the incidence of hip fractures. We investigated the effect of age and menopause on bone loss in the proximal femur by measuring bone mineral density (BMD) of the femoral neck, Ward's triangle, and trochanter by dual-photon absorptiometry in 263 normal women aged 20–84.

Multiple regression analyses revealed a significant decrease in BMD of the femoral neck and Ward's triangle with age in both pre- and postmenopausal women (p < 0.001). In the trochanter the decrease with age was significant only in postmenopausal women (p < 0.001).

Further analysis revealed that BMD decreased faster at all sites in the early postmenopausal years. During the first 6 years postmenopause, the decrease in BMD of the femoral neck and trochanter was 3–10 times higher than the change in the decade prior to menopause. About 20% of the lifetime femoral neck loss and 30% of the trochanteric loss occurred in the early postmenopausal period.

It is concluded that both age and menopause are major determinants of BMD in the proximal femur. These findings could explain why estrogen therapy has been reported to prevent hip fracture. The rapid early postmenopausal loss in BMD of the proximal femur demonstrates the importance of starting estrogen replacement therapy immediately after menopause for maximum effect.
Osteogenic response to marrow aspiration: Increased serum osteocalcin and alkaline phosphatase in human bone marrow donors
Authors: Foldes Josef , Statter Michael , Menczel Jacob , Naparstek Ella , Prof. Bab Itai
Published: February 2010

It has been shown recently in experimental animals that regeneration of bone marrow after ablation is associated with enhanced osteogenic growth factor activity and a systemic increase in bone formation. To assess the possible occurrence of a similar phenomenon in humans, serum markers of bone formation, osteocalcin and alkaline phosphatase, were measured in marrow donors before the aspiration of large amounts of iliac marrow and 1 day to 5 weeks thereafter. Both osteocalcin and alkaline phosphatase showed significant increases, with peak values 1–3 and 2–4 weeks postaspiration, respectively. The absolute maximal increase in osteocalcin was significantly higher in adolescent and child donors than in adults. When evaluated together with studies on systemic changes during fracture healing and marrow regeneration, these findings suggest that marrow aspiration in humans evokes a systemic osteogenic response.
Perspectives: Methodologic issues in evaluating risk factors for osteoporotic fractures
Authors: Ross Philip D. , Wasnich Richard D. , Davis James W. , Vogel John M. , Heilbrun Lance K.
Published: February 2010

Techniques for measuring bone mineral content (BMC) were developed for the purpose of providing an objective and noninvasive indication of bone strength (or lack thereof) and fracture risk, to the extent that strength relates to bone mass. As such, BMC measurements could help to (1) identify those who are most likely to experience nonviolent fractures in the future and who would therefore benefit most from preventive measures, (2) improve their treatment compliance, and (3) monitor the efficacy of treatments intended to reduce bone loss. All these potential uses require that the measurement provide an indication of fracture risk (probability of fractures).

During the past 10–15 years there have been conflicting reports regarding the association of reduced BMC with nonviolent fractures. Some authors have criticized the usefulness of BMC measurements, whereas others have questioned the value of one or more techniques. However, the epidemiology of osteoporosis has only recently been subjected to rigorous study. The use of appropriate statistical methods for relating fracture risk to bone mass may be no more widely practiced in osteoporosis epidemiology today than it was for studying risk factors (e.g., blood pressure) in cardiovascular epidemiology during the 1960s.

The intent of this article is to explore three areas that may have contributed to controversy in the study of bone mass and fracture occurrence: (1) perspective of the investigators, (2) study design, and (3) analytic methodology. Although the focus of this paper is on bone mass, these considerations are equally applicable to some investigations of other risk factors for osteoporotic fractures (e.g., bone architecture, bone turnover and loss rate, or biochemical markers of bone loss).
The effect of cross-gender hormonal treatment on bone metabolism in male-to-female transsexuals
Authors: Dr. Lips P. , Asscheman H. , Uitewaal P. , Netelenbos J.C. , Gooren L.
Published: February 2010

A deficiency of sex hormones leads in both sexes to increased bone loss, and hormonal substitution can prevent this. The effect of the change of hormonal environment on bone metabolism in transsexuals is unknown. Transilial bone biopsies were obtained from 23 male-to-female transsexuals (mean age ± SD, 38.0 ± 11.7 years) after estrogen (ethinylestradiol, 100 μg/day) and antiandrogen treatment (cyproterone acetate, 100 mg/day) for 8–41 months. Histomorphometric data were compared with those from 11 healthy men (39.6 ± 9.4 years). There was no difference in bone volume, bone surface, or trabecular thickness between transsexuals and controls. Eroded surface and osteoclast number were not different between the groups. The osteoid volume, surface, and thickness were significantly lower in the transsexuals than in the controls. The mineral apposition rate and adjusted apposition rate were normal, but mineralizing surface and bone formation rate were suppressed in the transsexuals compared with data reported from the literature. The results indicate that antiandrogen and estrogen treatment in male-to-female transsexuals may suppress bone turnover and is not associated with bone loss.
Calibration and standardization of bone mineral densitometers
Authors: Kelly T.L. , Slovik D.M. , Neer Robert M.
Published: February 2010

Measurements of bone mineral density (BMD) by four 153Gd and two x-ray bone densitometers were compared utilizing spine phantoms that simulated the human lumbar spine. The six instruments provided BMD values that differed by as much as 16%, due to differences as large as 8% in bone mineral content and as large as 7% in bone area. Instrument calibration curves, determined by measuring thin, medium, and thick hydroxyapatite blocks, were linear (r = 0.99) but had different (p < 0.0001) slopes and intercepts.

Serial measurements of spine and total body phantoms were employed to evaluate the long-term stability of 153Gd bone densitometry. These measurements of spine phantom BMD increased 1.0–2.6% (p < 0.0001) following a software change, and measurements of total body bone density increased 4–6% after 153Gd source replacement. The changes occurred at a time when serial measurements of cylindrical calibration standards were stable, indicating that such simple standards are unable to detect and correct for changes in instrumental response. We conclude that investigators, manufacturers, and government regulatory agencies must develop and implement the following: (1) effective calibration procedures that would assure comparability among instruments, and (2) appropriate quality control phantoms that would allow the confident interpretation of serial patient measurements.
The evolution of osteomalacia in the rat with acute aluminum toxicity
Authors: Rodriguez Mariano , Felsenfeld Arnold J. , Llach Francisco
Published: February 2010

Aluminum toxicity is the presumed cause of aluminum-associated osteomalacia. In animal models, osteomalacia has been produced after a prolonged course of aluminum. In the present study, rats with renal failure received 20 mg intraperitoneal aluminum during a 2 day period. This model allows sequential observations in the development of osteomalacia. Rats were sacrificed and studied 5, 12, 25, and 40 days after aluminum administration. No differences were observed in serum calcium, phosphorus, or creatinine as a consequence of aluminum administration. Compared with control rats, parathyroid hormone was decreased at 12 and 25 days. A direct correlation was present between plasma and bone aluminum at 12 days (r = 0.92, p < 0.01), 25 days (r = 0.85, p < 0.005), and 40 days (r = 0.88, p < 0.001) but not 5 days after aluminum administration. Plasma aluminum peaked at 5 days (727 ± 89 μg/liter, mean ± SEM) and bone aluminum at 40 days (273 ± 40 μg/g). Aluminum had profound effect on bone histology. At 5 days there was a decrease in osteoblast surface and osteoid surface; at 12 days osteoblast surface and osteoid surface returned to normal but osteoclast surface decreased. Subsequently there was a progressive increase in osteoid surface and osteoid volume. Bone formation rate measured at 12, 25, and 40 days was decreased at these intervals.

In conclusion, (1) high plasma aluminum may be directly toxic to the osteoblast; (2) progressive osteoid accumulation is secondary to matrix (osteoid) deposition, which exceeds the depressed bone formation rate; (3) the progressive decrease in plasma aluminum and increase in bone aluminum suggest that bone has a high affinity for aluminum but may have a relatively slow rate of uptake at any given time; (4) aluminum may directly decrease parathyroid hormone; (5) the correlation between plasma and bone aluminum suggest an exchange is present; and (6) aluminum toxicity may independently affect the osteoblast and bone mineralization.
Cigarette smoking, obesity, and bone mass
Authors: Slemenda Charles W. , Hui Siu L. , Longcope Christopher , Johnston C. Conrad
Published: February 2010

This study was designed to assess the effects of smoking on bone mass and bone loss and to ascertain whether these effects are independent of effects on adiposity and hormone concentrations.

A total of 84 healthy, peri- and postmenopausal women were studied prospectively over 3½2 years. Heavy smokers had significantly (p < 0.05) lower radial and vertebral bone mineral content than light or nonsmokers (who did not differ from each other). In regression models, which contained measurements of obesity, pack-years smoking remained a significant predictor of bone mass. However, there were no detectable effects of smoking on rates of bone loss at any site.

Smokers appear to be at greater risk of osteoporosis due to their lower bone mass. However, this reduced bone mass is already present around the time of menopause, and rates of bone loss during this period do not appear to be influenced by smoking. Furthermore, we have previously shown in this population that menopausal serum estrogen concentrations (which determine rates of bone loss) do not differ between the smokers and nonsmokers. Further studies of larger groups are required to determine whether small differences in bone loss may exist, since the power to detect such differences was not ideal in this study.
Application of an in vitro model and a clinical protocol in the assessment of the potency of a new bisphosphonate
Authors: Papapoulos Socrates E. , Hoekman Klaas , Löwik Clemens W.G.M. , Vermeij Pieter , Bijvoet Olav L.M.
Published: February 2010

The development of new bisphosphonates for clinical use requires congruence between the results of basic and clinical investigations. We have previously shown that this can be achieved with the use of an in vitro coculture mouse metacarpal resorption system sensitive to the activation of osteoclast precursors together with a clinical protocol in which the rate of decrease in urinary hydroxyproline excess with bisphosphonate treatment is assessed in patients with Paget's disease. In these studies bisphosphonates of known potencies were used. In the present study we have evaluated these approaches prospectively in the assessment of the antiresorptive potency of the new bisphosphonate (3-dimethylamino-1-hydroxypropylidene)-1,1-bisphosphonate (dimethyl-APD). A total of 42 patients with Paget's disease of bone received dimethyl-APD in doses predicted from the in vitro system. A total of 24 patients received the bisphosphonate intravenously (2, 4, and 8 mg/day) in groups of 8 patients each and 18 orally (100, 200, and 400 mg/day) in groups of 6 patients each for 10 days. Dimethyl-APD therapy was highly effective in inhibiting bone resorption. Urinary hydroxyproline excretion reached 30.9 ± 5.6, 17.1 ± 3.1, and 2.1 ± 5.3% of initial excess after 10 days treatment with intravenous dimethyl-APD, 2, 4, and 8 mg/day, and 37.4 ± 18, 10.4 ± 8.5, and 13 ± 4.1% with oral therapy, 100, 200, and 400 mg/day, respectively. Comparison of the antiresorptive potency of dimethyl-APD with that of APD showed that the former is roughly five times more potent, as predicted in the in vitro study. These results demonstrate the predictive power of the coculture mouse metacarpal resorption assay and the use of a standardized protocol in Paget's disease for the introduction of new bisphosphonates into clinical practice. Full treatment efficacy in a simple and convenient way can be achieved with oral dimethyl- APD, which can be considered representative of third-generation bisphosphonates.
Relationship between interleukin-1 and prostaglandins in resorbing neonatal calvaria
Authors: Dr. Garrett I. Ross , Mundy Gregory R.
Published: February 2010

The relationship between the cytokine interleukin-1 and prostaglandins in resorbing bone cultures is very confusing. In some reports, the effects of interleukin-1 are described as prostaglandin dependent, but in some they appear prostaglandin independent. Recent data suggest that events occurring during the preculture period may be important for understanding later effects of any factor on bone resorption in neonatal mouse calvaria. In this study, we examined neonatal mouse calvaria morphologically for indices of resorption to determine the importance of this preculture period to their subsequent response to interleukin-1. Our results show that after a preculture period of 24 h in control media, osteoclast numbers and resorption areas are markedly enhanced. The numbers of osteoclasts fall when the calvaria are transferred to fresh control media for the following 72 h. This increase in osteoclast formation was inhibited by the addition of indomethacin (10−6 M) during the preincubation period. When interleukin-1 was added to bones after the preincubation period, osteoclast numbers present at this time were maintained and indomethacin had no effect on this response. Furthermore, the inhibitory effect of indomethacin on osteoclast formation during the preculture period could be reversed by adding prostaglandins of the E series in low concentrations together with indomethacin. The effects of interleukin-1 did not appear to be related to osteoclast precursor proliferation, since hydroxyurea (which inhibits DNA synthesis in these cultures) had no effect on the response of calvarin to interleukin-1. These results indicate that prostaglandin production by mouse calvaria increases osteoclast numbers during the preincubation period. The effects of interleukin-1 were not dependent on prostaglandin synthesis, and this factor may stimulate bone resorption by acting at later stages in osteoclast formation and activation in these bones.
The effect of tartrate on bone cell acid phosphatase activity: A quantitative cytochemical study
Authors: Webber D. , Dr. Braidman I. P. , Robertson W. R. , Anderson D. C.
Published: February 2010

Tartrate-resistant acid phosphatase activity (TRAPase) is widely used as a cytochemical marker to distinguish osteoclasts from macrophages and other related cell types. The degree of tartrate resistance, however, may depend on which reaction methods, tissues, or species are used. To investigate this further, we have measured the amount of cytochemical reaction product by microdensitometry. We compared osteoclast acid phosphatase (APase) activity in fresh frozen sections of neonatal rat calvaria using two different reaction methods; one is commonly employed for qualitative histochemistry and includes α naphthyl phosphate as substrate, simultaneous coupling to the chromagen Fast Garnet, and a 30-minute reaction time (method A). The other may be used to measure enzyme reaction rates in cells in situ and employs conditions suitable for initial velocity kinetics, namely naphthol-ASBI phosphate as substrate, post coupling to Fast Garnet, and a 2-minute reaction time. Although enzyme reaction rates differed greatly between the two methods, significant inhibition of APase activity by tartrate was observed in calvarial osteoclasts (69% and 59% with methods A and B, respectively), osteoblasts, and spleen macrophages. Using method B, mouse calvarial osteoclasts had similar APase activity to that seen in the rat. Tartrate produced little inhibition in these mouse cells, in contrast to the observations made with rat tissue, but murine spleen macrophages were significantly tartrate sensitive (40% inhibition with tartrate). On this basis, conclusions regarding the cell specificity of TRAPase should be treated cautiously.
Organization of osteoclast microfilaments during the attachment to bone surface in vitro
Authors: Lakkakorpi Päivi , Tuukkanen Juha , Hentunen Teuvo , Järvelin Kaisa , Väänänen Kalervo
Published: February 2010

Rat and chicken osteoclasts were cultured on bone slices, where they showed active resorption with resorption lacunae, which cold be seen by toluidine blue staining or with a scanning electron microscope. Osteoclast microfilaments, F-actin, vinculin, and talin were studied by immunofluorescence. In attached osteoclasts, vinculin appeared as a double circle in the periphery of the cell, and the most intense F-actin staining was located between these vinculin zones. Some chicken osteoclasts showed also intense F-actin staining throughout the center of the cell. Talin appeared in a similar double circle to vinculin. This kind of distribution of microfilaments was always associated with resorption lacunae, and F-actin, vinculin, and talin zones correspond roughly to the edge of lacunae. Osteoclasts showing a diffuse staining pattern were not associated with a resorption pit. The results suggest that this specific microfilament arrangement is located at the attachment zone of the osteoclast and is obligatory for the attachment and resorption. However, this arrangement of microfilaments is quite different from the one that has been previously described in osteoclasts cultured on glass.
Rapid, simple identification of individual osteoblastic cells and their specific products by cell blotting assay
Authors: Murray Elsa , Martin Estralita , Burton Douglas , Deftos Leonard J.
Published: February 2010

Biochemical and molecular biological studies of osteoblastic cell function and hormonal regulation are frequently confounded by the inherent cellular heterogeneity and phenotypic instability of existing in vitro and in vivo model systems. A new technique (derived from Western blotting or antibody-based detection of protein molecules bound to nitrocellulose paper) is described for identification of individual cells which synthesize osteoblast-specific gene products (bone Gla-protein, type I collagen, and alkaline phosphatase) or produce cAMP in response to parathyroid hormone (PTH) or isoproterenol. Dispersed primary neonatal rat calvariae or osteogenic sarcoma cells were “plated” on Immobilon-P (a hydrophobic transfer membrane with very high protein-binding capacity) for 30 minutes to several hours, followed by agonist treatment, formalin fixation, hematoxylin staining, and immunostaining with a battery of antibodies specific for osteoblastic products. Individual cells and their secretory zones were visualized by light microscopy and counted. Treatment with PTH with or without isoproterenol resulted in increases in the percentages of osteoblastic cells elaborating cAMP, as well as the intensity of immunostaining, but had no effects on MCF-7 cells, a nonosteoblastic breast carcinoma control line. The percentage of cells within each primary osteoblastic cell population isolated or rat osteogenic sarcoma cell clone (G2 or C12) that elaborated bone-specific proteins or that generated cAMP in response to PTH varied with time and the individual cellular preparation, reconfirming the cellular heterogeneity of these systems. This method, in conjunction with techniques such as in vitro hybridization, should prove useful in characterizing discrete osteoblastic bone cell subpopulations and in clarifying mechanisms of hormonal regulation by local and systemic agents.
Bone remodeling in predialysis chronic renal failure: How does the choice of index for mineralizing surface influence the interpretation?
Authors: Dr. Dahl Eilif , Nordal Knut P. , Aksnes Lage , Halse Johan
Published: February 2010

To study how calculating bone dynamics with different indices of mineralizing surface (MS) may influence the interpretation of bone remodeling in chronic renal failure (CRF) and to discuss which of three often used indices may be closest to the true MS, we compared bone histomorphometry in predialysis patients with moderate (se-creatinine < 400 μmol/liter) and advanced CRF. All were double-labeled with tetracycline and had no stainable bone aluminum. Bone dynamics were calculated with MS = double-labeled (dLS) + single-labeled surface (sLS), MS = dLS + sLS/2, and MS = dLS. As sLS was twice that of dLS in both groups and the label interval was only 10 days, most single labels were probably double labels, but unseparable due to wide and unsharp labels resulting from high rather than low bone turnover. Bone volume was the same in both groups, while osteoid and resorption indices, sLS, and bone formation rate (with MS = dLS + sLS) were increased in advanced CRF. dLS + sLS is higher than the true MS, but more representative for MS than dLS + sLS/2, with the true value between these two indices. Bone resorption, osteoid formation, and mineralization remain in balance even in advanced CRF. Osteomalacia is hardly the consequence of CRF alone.
WR-2721 reduces bone loss after hindlimb tenotomy in rats
Authors: Shaker Joseph L. , Fallon Michael D. , Goldfarb Stanley , Farber Joshua , Attie Maurice F.
Published: February 2010

WR-2721 is a thiophosphate analog of cysteamine that produces hypocalcemia in vivo. Previous studies suggest that WR-2721 produces hypocalcemia by independent inhibitory effects on parathyroid hormone (PTH) secretion, osteoclastic bone resorption, and tubular reabsorption of calcium. We sought to determine if WR-2721 would decrease bone loss in an animal model of disuse osteoporosis produced by unilateral knee tenotomy in the rat. Tenotomy significantly increased osteoclast number in tibias on the side of the procedure compared with tibias on the opposite side which had not undergone the procedure at 3 and 14 days. Femoral weight of tenotomized limbs were also reduced significantly compared with the contralateral limb at 3 and 14 days. WR-2721 treatment (240 mg/kg daily) prevented 26% of the loss of femoral dry weight and 29% of the loss of femoral ashed weight produced 14 days after tenotomy. In addition, WR-2721 treated (240 mg/kg daily) animals had fewer osteoclasts in tenotomized tibias than control animals at 3 days (6.6 ± 0.7/mm versus 10.3 ± 0.9/mm, p < 0.02) and at 14 days (5.8 ± 0.3/mm versus 8.7 ± 0.4/mm, p < 0.02). These data suggest that WR-2721 decreases bone loss in this model by decreasing osteoclastic bone resorption.
Inhibition of bone resorption by α-difluoromethylornithine may not be mediated by polyamine depletion
Authors: Dr. Lucas Rita C. , Seidenfeld Jerome , Krieger Nancy S. , Stern Paula H.
Published: February 2010

We have examined the effect of α-difluoromethylornithine (DFMO) on bone polyamine content and parathyroid hormone (PTH)- and calcitriol-stimulated bone resorption in cultures of neonatal mouse calvaria. Polyamine content in bone homogenates was determined by reverse-phase paired-ion HPLC. Treatment with 5 mM DFMO for 48 h reduced putrescine from 0.4 nmol/bone to nondetectable levels, slightly decreased spermidine, and did not affect spermine. Bone resorption elicited by 48 h of treatment with PTH or calcitriol was inhibited by concentrations of DFMO ≥ 5 mM added 48 h prior to hormone. This observation supported the concept that polyamines may play a role in bone resorption. However, other observations cast uncertainty on this conclusion. Measurement of calvarial polyamine content at 2 h intervals revealed no increase in endogenous polyamines for up to 10.5 h after calcitriol addition. Although addition of putrescine restored bone polyamine content, exogenous polyamines failed to reverse the inhibitory effects of DFMO on calcitriol-stimulated resorption. These results suggest that a mechanism other than depletion of polyamines could be contributing to the inhibitory effect of DFMO on resorption.
Biochemical characterization of an electrogenic vacuolar proton pump in purified chicken osteoclast plasma membrane vesicles
Authors: Bekker Petrus J. , Dr. Gay Carol V.
Published: February 2010

A well-characterized chicken osteoclast plasma membrane vesicle preparation manifested Mg2+-dependent ATP hydrolyzing activity of 0.213 μmol inorganic phosphate released per mg protein per minute (n = 7). The Mg2+ dependence showed a high-affinity component with a Kmg of 1.293 μM and Vmax of 0.063 μmol Pi per mg protein per minute, and a low-affinity component with a KMg of 297.6 μM and a Vmax of 0.232 μmol Pi per mg protein per minute. The Mg2+-ATPase activity was inhibited by N, N1-dicyclohexylcarbodiimide (DCCD, 0.2 mM, 50.7%), N-ethylmaleimide (0.5 mM, 34.6%), nolinium bromide (1 mM, 29.9%), 4,4′-di-isothiocyano-2,2′-stilbene sulfonic acid (DIDS, 1 mM, 45.1%), and p-chloromercuribenzoic acid (PCMB, 0.1 mM, 33.8%). Sodium orthovanadate (Na3VO4) at 1 μM had no effect but caused 29.5% inhibition at 1 mM. Na+ could substitute for K+ without loss of activity, NO3 caused 19.5% inhibition when substituted for CI−, and acetate replacement of CI− resulted in 36.4% stimulation of Mg2+-ATPase. ATP, GTP, ITP, CTP, and ADP were all hydrolyzed effectively.

DCCD (0.2 mM), NEM (0.5 mM), nolinium bromide (1 mM), and DIDS (50 μM) almost completely abolished proton transport as measured spectrofluorometrically by acridine orange quenching. Na3VO4 (1 mM) had no effect, and duramycin (80 μg/ml) inhibited transport 52.7%. K+ replacement of Na+ caused a 79.2% increase in initial proton transport rate. NO3- and acetate substitution of CI− resulted in a 46.1 and 55.7% decrease in transport, respectively. ATP supports transport far more effectively than the other nucleotides tested. ADP was ineffective. Experiments using the potassium ionophore, valinomycin, indicated that the proton pump functions electrogenically, with CI− most likely cotransported by an anion transporter. The proton pump also seems to have at least one anion-sensitive site, elucidated by experiments in the presence of NO3- and CI−.
Muscle strength as a predictor of bone mineral density in young women
Authors: Snow-Harter Christine , Bouxsein Mary , Lewis Barbara , Charette Susan , Weinstein Pamela , Marcus Robert
Published: February 2010

It is widely accepted that physical activity is beneficial to bone. However, the specific relationships of muscle strength to bone mineral density (BMD) are poorly understood. We examined strength and BMD in 59 women aged 18-31 years who ranged in exercise patterns from sedentary to active. Mineral density of the right proximal femur (hip) and spine (L2-4) was evaluated by dual-energy x-ray absorptiometry. BMD at the midradius was measured by single-photon absorptiometry. Dynamic strength (one repetition maximum) was measured for the following muscle groups: back, elbow flexors (biceps), leg extensors (quadriceps), and the hip flexors, extensors, adductors, and abductors. Isometric grip strength was assessed by dynamometry.

Mineral density at the hip correlated independently with muscle strength and body weight, but not with age. Specifically, femoral neck BMD was significantly correlated with back strength and weight, whereas trochanter and overall hip mineral density were significantly related to biceps, back, and hip adductor strength. Hip mineral density was not related to strength of the quadriceps group or to that of the hip flexors, extensors, or abductors. In addition, muscle strength was an independent predictor of lumbar spine and midradius mineral density. In stepwise multiple regression analysis, biceps strength proved the most robust predictor of hip BMD and grip strength best predicted bone density at the lumbar spine and radius.

We conclude that muscle strength is an independent predictor of bone mineral density, accounting for 15-20% of the total variance in bone density of young women. In addition, it appears that muscle groups with attachments that are distant from the spine and hip may exert an important influence on bone mineral density at these sites.
Aluminum-induced neo-osteogenesis: A generalized process affecting trabecular networking in the axial skeleton
Authors: Quarles L. Darryl , Murphy Gayle , Vogler James B. , Drezner Marc K.
Published: February 2010

To determine if aluminum-induced neo-osteogenesis occurs in the axial skeleton, we compared spinal bone density and vertebral histology of beagles treated with aluminum for 8 and 16 weeks to that of untreated normals. Administration of aluminum (1.25 mg/kg) did not alter serum calcium, phosphorus, or creatinine but did result in a significant elevation of vertebral bone density, measured by quantitative computed tomography, after both 8 (286.7 ± 12.4 mg/ml) and 16 (361.7 ± 46.5 mg/ml) weeks of treatment compared with controls (212.2 ± 4.5 mg/ml). In accord with the increased bone density, biopsies from the spine displayed evidence of neo-osteogenesis, including the presence of woven bone, both mineralized and unmineralized, within the marrow space. The genesis of such woven bone units resulted after 16 weeks in a significant increase in trabecular bone volume, woven and lamellar (51.2 ± 4.4 versus 32.4 ± 1.2%; p < 0.05), woven bone volume (9.1 ± 3.6 versus 0 ± 0%; p < 0.05), and trabecular number (4.5 ± 0.3 versus 3.5 ± 0.2 per mm; p < 0.05). In addition, scanning electron microscopic evaluation of the bone biopsies confirmed the existence of new trabecular plates that provided interconnections between existent units.

These observations illustrate that aluminum-induced neo-osteogenesis positively influences trabecular networking in the axial skeleton. Such enhancement of bone histogenesis contrasts with the effects of other pharmacologic agents that solely alter the thickness of existing trabecular plates or rods within the vertebral spongiosa.
Influence of age and body weight on spine and femur bone mineral density in U.S. white men
Authors: Mazess Richard B. , Barden Howard S. , Drinka Paul J. , Bauwens Steven F. , Orwoll Eric S. , Bell Norman H.
Published: February 2010

Bone mineral density (BMD) was measured in normal white males using 153 Gd dual-photon absorptiometry. Measurements were made on the lumbar spine (n = 315) and on the proximal femur (n = 282) utilizing three regions of interest. There was a small but significant age-related decrease in spinal BMD (r = -0.11; -0.001 g/cm2 per year) and trochanteric BMD (r = 0.27; -0.002 g/cm2 per year). The BMD of the other femoral sites decreased more rapidly; the femoral neck (r = -0.58; -0.005 g/cm2 per year) and Ward's triangle (r = -0.69; -0.007 g/cm2 per year) declined by about 21 and 34%, respectively, from age 20 to age 70. These femoral BMD decreases were three to four times greater than those usually seen in the peripheral skeleton in males but less than the decreases of 25-30 and 40% in the femoral neck and Ward's triangle of white females. This pattern of aging bone loss may partially explain the paucity of spine fractures and the lower incidence of hip fractures in males versus females.