bluuebunny/crossref_metadata_2025 huggingface dataset:
A new sensitive homologous radioimmunoassay for amino-terminal parathyroid hormone in the rat
Authors: Finch Jane L. , Rapp Neville , Martin Kevin J. , Slatopolsky Eduardo
Published: February 2010

The determination of circulating biologically active PTH in the rat has been difficult due at least in part to the inability to develop an antibody suitable for RIA of rat PTH. However, since the amino acid sequence of the rat PTH molecule has been deduced by molecular techniques, corresponding synthetic peptides have made it possible to produce such an antibody. A total of 12 roosters were immunized with synthetic rat PTH-(1–34), and one animal, RD1, developed a sensitive antibody against this amino-terminal region of the rat PTH molecule. To further increase the sensitivity of the RIA, we utilized an analog of rat PTH, Tyr1 rat PTH-(2–34), as the radioligand, which can be iodinated to high specific activity (450 μCi/μg). The iodinated peptide was purified by HPLC using a C18 Nova Pak HPLC column and a 20–60% acetonitrile gradient in 0.1% TFA. Synthetic rat PTH-(1–34) was used as the standard. To validate the RIA, we measured PTH under a variety of metabolic conditions. Normal values for PTH were 55.6 ± 3.9 pg/ml (n = 26). Levels in parathyroidectomized (PTX) rats (n = 9) were undetectable, but renal insufficiency and vitamin D deficiency increased PTH to 587.4 ± 141.3 pg/ml (n = 73) and 1662.0 ± 137.8 (n = 27), respectively. Intraperitoneal (IP) administration of ethylenediaminetetraacetic acid (EDTA), 200 mg/kg, was used to decrease ionized calcium (ICa) from 4.75 ± 0.07 to 3.55 ± 0.10 mg/dl, which increased PTH from 51.3 ± 5.9 to 109.3 ± 13.4 pg/ml (n = 12). During calcium administration (30 mg/kg) IP, serum ICa increased from 4.69 ± 0.05 to 7.26 ± 0.24 mg/dl and PTH became rapidly undetectable (n = 14). Dietary manipulation (low Ca 0.02% and high PO4 2.0%) for 3 weeks increased PTH from 43.2 ± 3.2 to 71.9 ± 11.4 pg/ml (n = 13). In conclusion, we have developed a specific, sensitive, homologous RIA directed against the biologically active region of the rat PTH molecule. Rapid changes in PTH in response to physiologic stimuli are readily quantitated with this assay, which will be a useful tool for the assessment of parathyroid function in the intact rat.
Sequential and precise in vivo measurement of bone mineral density in rats using dual-energy x-ray absorptiometry
Authors: Ammann Patrick , Dr. Rizzoli René , Slosman Daniel , Bonjour Jean-Philippe
Published: February 2010

In the design of new strategies for the treatment of osteoporosis, noninvasive, precise, and sensitive bone mass measurement capable of detecting changes over short periods of time in small animals is essential. Most of the models described thus far require the sacrifice of the animals and/or display low reproducibility. Using a dual-energy x-ray absorptiometer (DEXA; Hologic QDR-1000) in an ultrahigh-resolution mode, we measured bone mineral density (BMD) in rats at the levels of lumbar spine (L1–4), proximal tail (caudal vertebrae C2–4), and tibia. Accuracy was evaluated by measuring the mineral content of bone powder capsules (within the range of rat vertebrae BMD), under 0.5–3 cm water to mimic variations in soft tissue thickness. The bone powder capsule mineral content was highly correlated with chemically determined hydroxyapatite content (r = 0.999). In vivo reproducibility was evaluated by calculating the coefficient of variation (CV = 100 × SD/mean) of four to six BMD measurements, each time with repositioning, in seven rats (220–500 g body weight). CV was 1.36 ± 0.32% (x ± SD) for lumbar spine, 0.66 ± 0.50% for proximal tail, and 1.12 ± 0.45% for tibia. The ability to detect BMD changes was investigated by measuring BMD before and every 4 weeks after ovariectomy (OVX) in 270 g rats, pair fed during the whole experiment. Compared with sham-operated control animals, a highly significant difference in lumbar spine BMD was observed 4 weeks after OVX, which reached a maximum by 8 weeks and remained stable thereafter. At the level of the proximal tibia, the difference was maximal 4 weeks after OVX. In contrast, proximal tail BMD was only slightly affected by OVX (p < 0.05). In conclusion, BMD measurements by noninvasive DEXA is feasible in small experimental animals. It seems to be accurate and precise, allowing the detection of BMD changes over short-term periods, particularly at lumbar and peripheral sites with high trabecular bone content. This technique should be useful for the development of therapies aimed at increasing bone mass.
Rat osteoclasts adhere to a wide range of rgd (arg-gly-asp) peptide-containing proteins, including the bone sialoproteins and fibronectin, via a β3 integrin
Authors: Helfrich Miep H. , Nesbitt Stephen A. , Dorey Elaine L. , Horton Michael A.
Published: February 2010

The ligand binding ability of rat osteoclast adhesion receptors was investigated in an attachment assay using osteoclasts disaggregated from bone. Osteoclasts adhered well to the Arg-Gly-Asp (RGD)-containing proteins osteopontin (bone sialoprotein I) and BSP (bone sialoprotein II), vitronectin, fibrinogen, von Willebrand factor, and fibronectin. Osteoclasts also adhered, but less strongly, to type I collagen. No attachment of osteoclasts was observed to thrombospondin, tenascin, laminin, or a range of non-RGD-containing bone proteins and proteins from other sources. The attachment of osteoclasts to all ligands was abolished in the presence of GRGDSP peptide, indicating the involvement of the RGD cell binding sequence in ligand binding. Attachment of osteoclasts to all substrates, with the exception of type I collagen, was also strongly inhibited by the addition of monoclonal antibody F11 to the β3 integrin subunit, indicating that a β3 integrin, probably the vitronectin receptor, was involved. Attachment to type I collagen was blocked by EDTA chelation of divalent cations and was not significantly affected by anti-β3 or anti-β1 antibodies; when taken with the inhibition by RGD peptide, this suggests the involvement of various receptors, possibly including nonintegrin collagen receptors, in the binding of osteoclasts to this protein. These results define the wide range of ligands for extracellular matrix receptors in osteoclasts in vitro. It remains to be established which of these proteins are important in osteoclast adhesion and osteoclastic bone resorption in vivo.
Performance of dual-energy X-ray absorptiometry in evaluating bone, lean body mass, and fat in pediatric subjects
Authors: Chan Gary M.
Published: February 2010

We studied the performance of the dual-energy x-ray absorptiometry method in evaluating bone mineral, fat, and lean soft tissue mass. This method was accurate in quantifying known small amounts of calcium, lard, and lean tissue mass. It was also accurate in evaluating small animal ashed bones, fat, and muscle mass. The analytic sensitivity of the method was 40 mg for calcium, 180 mg for fat, and 270 mg for lean tissue mass. The method was highly correlated to the single-photon absorptiometry method in measuring bone mineral content at the radius bone in 32 children, r = 0.998. There was a difference between the two methods in older children. Long-term precision for a small bone phantom was 2.0%. Total-body, lumbar, and radial bone scans were performed on 14 newborn infants whose gestational ages ranged from 28 to 41 weeks. Both total-body bone mineral and fat mass increased with gestational age and weight. The infant's total-body calcium was also associated with length and lumbar and radial bone densities. The lumbar bone density was associated with birth weight, gestational age, length, body mass index, body fat, and radial bone density. Male infant's lumbar bone density to total-body calcium ratio was higher than female infant's lumbar bone density ratio. Dual-energy x-ray absorptiometry may be used in pediatrics with high accuracy, sensitivity, and precision.
Bone mass measurements in the distal forearm using dual-energy X-ray absorptiometry and γ-ray computed tomography: A longitudinal, in vivo comparative study
Authors: Dr. Overton Thomas R. , Wheeler Garry D.
Published: February 2010

Indices of bone mass were measured in 23 volunteers weekly over 14–16 weeks using dual-energy x-ray absorptiometry (DEXA) and special-purpose computed tomography (γ-CT). In vitro, the precision for both systems was excellent (coefficient of variation < 0.5%). Over 4 months, the precision in vivo (average CV for all subjects) for DEXA measures (BMD, g/cm2, and BMC, g/cm) varied between 0.6 and 1.1%; with γ-CT it varied from 1.1% for TBD (g/cm3) to 2.2% for CBD (g/cm3). Correlation between the indices of bone mass measured using DEXA and γ-CT at the ultradistal site was moderate, but these indices were not correlated at the distal third site. When BMD and BMC were derived from the CT index IBD, however, the correlation between these γ-CT indices and the corresponding DEXA indices was high for both ultradistal and proximal radial sites.
Isolation and purification of osteocytes
Authors: van der Plas A. , Nijweide P.J.
Published: February 2010

An isolation method for osteocytes is described. After removal of the periostea, bone cells were isolated from calvariae of 18-day-old chicken embryos by alternating treatments with collagenase and EDTA. Osteocytes were purified from the heterogeneous bone cell population with the help of the osteocyte-specific MAb OB 7.3 bound to protein G-conjugated magnetic beads. The purity of the osteocyte population ultimately obtained was more than 95%. Osteocytes were found to adhere rapidly to glass or plastic substrates. They showed numerous processes of various types. These processes could branch and make contact with those of other osteocytes. After 1–2 days of culture, the isolated osteocytes formed a network of apparently interconnected cell processes very similar to the osteocyte network in bone.
Identification of a functional mononuclear precursor of the osteoclast in chicken medullary bone marrow cultures
Authors: Prallet Bernard , Male Philippe , Neff Lynn , Baron Roland
Published: February 2010

Mononuclear cells were isolated from the peritrabecular bone marrow from the medullary bone of laying hens maintained on a calcium-deficient diet for 1 week. These cells were cultured for up to 7 days on devitalized bovine bone slices after removing the nonadherent fraction. The mononuclear precursors of the osteoclast that are present in such cultures adhere to bone matrix. These cells are TRAP+, express the vitronectin receptor at high levels, and also express high levels of sodium pumps and of carbonic anhydrase, enzymes that are characteristically enriched in the mature osteoclast. Finally, the most mature mononuclear precursors were found to be capable of resorbing the extracellular bone matrix before forming multinucleated osteoclasts.
Contrasting effects of parathyroid hormone and insulin-like growth factor I in an aged ovariectomized rat model of postmenopausal osteoporosis
Authors: Ibbotson Kenneth J. , Orcutt Christopher M. , D'Souza Sharyn M. , Paddock Carol L. , Arthur Jay A. , Jankowsky Maryann L. , Boyce Rogely W.
Published: February 2010

Agents that exert anabolic effects on bone have generally been tested in young or estrogen-replete animals. It is unclear whether these agents exert similar effects in older ovariectomized (Ovx) animals. In this single study we examined the effects of intermittent (daily) human PTH-(1–34) and continuous infusion of human recombinant IGF-I alone and in combination on bone resorption and formation over a 14 day period in an aged Ovx rat model of postmenopausal osteoporosis (2-year-old rats, Ovx at 1 year). Compared to Ovx controls, PTH treatment increased bone mineral content (BMC) and bone volume and stimulated bone formation but had no effect on bone resorption. In contrast, IGF-I treatment reduced BMC and stimulated resorptive activity as assessed by increases in marrow volume, cortical porosity, osteoclast-positive eroded surfaces, and urinary hydroxyproline excretion. IGF-I had no effect on bone formation, but when combined with PTH, IGF-I blunted the response to PTH on the periosteal and endocortical surfaces. In summary, PTH stimulated bone formation in a manner similar to that observed in younger animals and IGF-I stimulated bone resorption rather than formation and blunted the bone-forming response to PTH. The effects of IGF-I in older Ovx rats may differ from those observed in younger estrogen-replete animals.
Dietary restriction of calcium, phosphorus, and vitamin D elicits differential regulation of the mrnas for avian intestinal calbindin-D28K and the 1,25-dihydroxyvitamin D3 receptor
Authors: Meyer Julia , Fullmer Curtis S. , Wasserman Robert H. , Komm Barry S. , Haussler Mark R.
Published: February 2010

We investigated the regulation of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced calbindin-D28k (CaBP) and of the vitamin D receptor (VDR) by evaluating CaBP protein, CaBP mRNA, and VDR mRNA under conditions of altered intake of vitamin D, calcium, or phosphorus. Chickens were maintained for 10 days on one of four diets: vitamin D-deficient, normal (1.0% Ca and 1.1% P), low calcium (0.1% Ca and 1.2% P), and low phosphorus (1.1% Ca and 0.3% P). CaBP was undetectable in D-deficient duodena and was elevated above normal values by low-calcium (3.1-fold) and low-phosphorus (2.3-fold) intake. Contradictory to published data, we observed a correlation between CaBP protein and mRNA levels in that the CaBP mRNA was absent in D-deficient intestine and augmented threefold and twofold in low-calcium and low-phosphate duodena, respectively. In contrast, VDR mRNA concentrations were identical in vitamin D-deficient and normal duodena, implying that intestinal VDR is not dependent upon 1,25-(OH)2D3 for basal expression. Chickens fed a low-phosphorus diet displayed a twofold increase in VDR mRNA, but those fed a low-calcium diet exhibited a dramatic decrease in VDR mRNA. These data show that CaBP mRNA and protein levels are modulated in a tightly coupled fashion, and they are consistent with previous conclusions that augmented circulating 1,25-(OH)2D3 stimulates CaBP expression when dietary calcium or phosphorus is limiting. However, a more complex regulation of VDR expression occurs in that low-phosphorus restriction enhances VDR mRNA levels, possibly via increased circulating 1,25-(OH)2D3. Conversely, reduced dietary calcium diminishes VDR mRNA despite increased circulating 1,25-(OH)2D3, indicating that another factor, such as parathyroid hormone, is a predominant downregulator of VDR.
Developmental regulation of creatine kinase activity in cells of the epiphyseal growth cartilage
Authors: Dr. Shapiro Irving M. , Debolt Kristine , Funanage Vicky L. , Smith Susan M. , Tuan Rocky S.
Published: February 2010

During the process of endochondral bone formation, the maturing chondrocyte exhibits profound changes in energy metabolism. To explore the mechanism of energy conservation in cartilage we examined the expression of creatine kinase, an enzyme that catalyzes the formation of ATP in tissues under oxygen stress. Measurement of creatine kinase activity and cytochemical assessment of enzyme distribution clearly showed that the level of enzyme activity was related to chondrocyte maturation. Thus, as the cells hypertrophied, there was a progressive increase in creatine kinase activity. Similarly, an elevation in creatine kinase activity was noted in chondrocyte cultures as the cells assumed an hypertrophic state. When cartilage calcification was disturbed by rickets, there was a decrease in enzyme activity in the hypertrophic region. Studies were performed to examine the creatine kinase isozyme profile of cells of the epiphysis. In resting and proliferating cartilage, the isoform was MM. In hypertrophic cartilage, the predominant isoforms were MB and BB. In terms of the creatine phosphate content, the highest values were seen in the proliferative region; lower amounts were present in hypertrophic and resting cartilage; and no creatine phosphate was detected in calcified cartilage. These data suggest that turnover of creatine phosphate is greatest in the mineralized region of the epiphysis. The results of these investigations point to creatine kinase as being under developmental control. The activity of the enzyme in cartilage cells should serve as a marker of developmental events associated with chondrocyte proliferation, hypertrophy, and mineralization.
Identification of proteins secreted by human osteoblastic cells in culture
Authors: Johansen Julia S. , Williamson Matthew K. , Rice Jeffrey S. , Price Paul A.
Published: February 2010

To better understand the biochemistry of matrix-forming cells, we developed a simple and reproducible procedure for the isolation and identification by N-terminal sequencing of proteins secreted by cells into culture medium and applied this procedure to the analysis of the major Coomassie blue-staining proteins under 100 kD that are secreted from three different human osteoblastic cell cultures. The major proteins secreted by normal human osteoblasts from adult trabecular bone were identified by N-terminal sequencing to be gelatinase, osteonectin, the C-terminal propeptides of the α1 and α2 chains of type I collagen, tissue inhibitor of metalloproteinase 1 (TIMP-1), and β2-microglobulin. The amounts of each of these proteins secreted into medium over a 24 h interval did not change over the 7 consecutive days of culture under serum-free conditions, which indicates that this pattern of protein secretion is not significantly affected by the serum-free conditions needed for protein identification by this method. In addition, radioimmunoassay for bone gla protein (BGP), a marker for osteoblast phenotype, revealed that BGP secretion remained high over 7 days of culture under serum-free conditions and was comparable to the rate of BGP secretion in control cultures with 10% serum. The major proteins secreted by MG-63 cells were identified by N-terminal sequencing to be gelatinase, a novel 40 kD human bone protein we termed YKL-40, TIMP-1, the recently discovered TIMP-2, and β2-microglobulin. Further studies revealed that YKL-40 is the only protein detectable by Coomassie staining of SDS gels of MG-63 media proteins that is induced by extended time at confluence or by treatment with 1,25-(OH)2D3. The apparent absence of detectable Coomassie-stained bands corresponding to the C-terminal propeptides of collagen in the medium of MG-63 cells suggests that these transformed cells may not be a good model for bone matrix formation. The major proteins secreted by normal fetal osteoblastic cells were identified by N-terminal sequencing to be osteonectin and the C-terminal propeptides of the α1 and α2 chains of type I collagen. Gelatinase and TIMP could not be detected among the conditioned medium proteins by these methods. These observations indicate that fetal osteoblasts primarily express proteins that are matrix constituents and adult human osteoblasts secrete, in addition to these, proteins that could function in matrix turnover.
Risk of fractures in patients with pernicious anemia
Authors: Goerss Jean B. , Kim Chung H. , Atkinson Elizabeth J. , Eastell Richard , O'Fallon W. Michael , Dr. Melton L. Joseph
Published: February 2010

In a series of postmenopausal women previously studied by our group, pernicious anemia was associated with reduced bone mineral density of the spine and with vertebral fractures. To determine whether osteoporotic fractures represent an important clinical problem for such patients, we conducted a population-based retrospective cohort study of all Rochester, Minnesota residents newly diagnosed with pernicious anemia from 1950 through 1979. These 131 individuals were followed through the inpatient and outpatient records of all medical care providers in the community for 1550 person-years. In comparison with fracture rates from the general community, patients with pernicious anemia had a 1.9-fold increase in proximal femur fractures, a 1.8-fold increase in vertebral fractures, and a 2.9-fold increase in distal forearm fractures (all p < 0.05). Increases in the risk of proximal humerus and pelvis fractures were not statistically significant. Clinicians should be aware that pernicious anemia is associated with an increased risk of osteoporotic fractures, but the mechanism is unknown.
cAMP promotion of osteoclast-like cell development from mouse bone marrow cells requires a permissive action of 1,25-(OH)2D3
Authors: Rubin Janet , Titus Louisa , Nanes Mark S.
Published: February 2010

Recruitment of osteoclasts from monocytic precursors is modulated by local signals. We previously showed that monoblastic differentiation in U937 cells is stimulated by 1,25-(OH)2D3 and cAMP in series. We investigate here the combined effects of these agents to stimulate differentiation of osteoclast-like cells from mouse marrow. Cells from mouse marrow were harvested and cultured in α-MEM with 10% fetal bovine serum. The appearance of tartrate-resistant acid phosphatase-containing multinuclear cells was measured after 8 days in culture by cytochemical staining. Continuous exposure of cultures to 10 nM 1,25-(OH)2D3 positively stimulated development of these cells after 8 days (101 + 3 cells per well, n = 74). No osteoclast-like cells were found when 1,25-(OH)2D3 was added for the first 4 days followed by 4 days more with no treatment. PGE2 (1 μM) as a single agent added during the last 4 days of culture was not able to recruit osteoclast-like cells. However, cultures exposed to 1,25-(OH)2D3 during the first 4 days and 1 μM PGE2 during the second 4 days developed osteoclast-like cells at 8 days [66 + 8% of the formation seen with 1,25-(OH)2D3 alone, p < 0.05]. Dibutyryl cAMP (1 μM to 3 mM) was also not effective used as a single agent, but was able to stimulate formation of TRAP-positive multinuclear cells when 1,25-(OH)2D3 preceded its addition to culture medium. cAMP analogs therefore mimicked the effect of 1 μM PGE2, but these experiments do not allow us to assign the PGE2 action entirely to activation of cAMP second messenger. We postulate that continuous 1,25-(OH)2D3 stimulation is alone sufficient to recruit osteoclasts from precursors but that the effect of cAMP to stimulate osteoclast formation occurs only when combined with a permissive action of 1,25-(OH)2D3.
Influence of oophorectomy on serum levels of sex steroids and bone metabolism and assessment of bone mineral density in lumbar trabecular bone by QCT-C value
Authors: Ohta Hiroaki , Makita Kazuya , Suda Yoshio , Ikeda Toshiyuki , Masuzawa Toshihide , Nozawa Shiro
Published: February 2010

The present cross-sectional study was carried out to determine the influence of oophorectomy (OPX) on serum levels of sex steroids and bone metabolism, as well as bone mineral density (BMD), in OPX subjects in comparison with age- and body size-matched controls. Quantitative computed tomography (QCT) and dual-photon absorptiometry (DPA) demonstrated a remarkable reduction in BMD in OPX subjects. In particular, the QCT of the centrum of vertebral bone (QCT-C) in these subjects was no more than 69.33 + 3.40% (X + SEM) of the control value, and this parameter was much lower than the QCT integral (QCT-I) value of total lumbar vertebrae. This means that BMD decreases specifically in spongy portions after OPX. The serum level of estrone (E1) was significantly lower in OPX subjects than in controls. The hormonal action of E1 on target organs has been thought to be only one-third of that of estradiol (E2), but the marked reduction in serum E1 level seemed to be a significant cause of the reduction in BMD. The serum level of androstenedione (Δ4) significantly decreased in OPX subjects and appeared to affect bone metabolism negatively. Both bone formation and bone resorption were found to be stimulated following OPX, but the rate of bone resorption was found to be higher than that of bone formation: there was an imbalance between bone formation and bone resorption in OPX subjects. However, it was not possible to prove a relationship between Ca regulating hormone and this phenomenon. In conclusion, the QCT-C value reflects the changes in spongy vertebral BMD more sensitively than the QCT-I value or DPA. It is also necessary to limit the region of interest strictly to the spongy portion to grasp the changes in BMD.
Relationship between bone mass and rates of bone change at appendicular measurement sites
Authors: Davis James W. , Grove John S. , Ross Philip D. , Vogel John M. , Wasnich Richard D.
Published: February 2010

The rate of bone change among postmenopausal women may vary depending upon the initial bone mass. Examining this possibility is difficult, however, because of a negative statistical bias that occurs when change is regressed against the initial value of the same variable. In this article, four statistical methods were applied to measure the association between bone mass and the rate of bone change. The study population was Japanese-American women, who were monitored for approximately 5 years. Bone changes were determined for the calcaneus and the distal and proximal radius. The results were consistent across the bone sites but differed between statistical methods. Three of the four methods indicated that the women with the greater bone mass had the greater loss rates. The fourth method did not support this association. Possible reasons for the discordant results are discussed. Using the “best” estimate of the relationship, a gradual convergence of bone mass was projected over time toward the population mean. The convergence occurred because women with higher bone mass had a somewhat faster loss rate than women with lower bone mass. Overall, however, the variation in bone mass between individuals was large compared to the rate of convergence.
Effects of heparin on osteoclast activity
Authors: Chowdhury Majeedul H. , Hamada Chieko , Dempster David W.
Published: February 2010

The effect of heparin on osteoclastic bone resorption was studied in vitro using the disaggregated osteoclast resorption assay. Bone resorption was assessed by counting the resorption lacunae on bone slices by light microscopy. Low concentrations of heparin (5 μg/ml) increased bone resorption by isolated chick and rat osteoclasts. Among other glycosaminoglycans tested at 5 μg/ml, only dextran sulfate showed a small but significant stimulation of resorption. Chondroitin sulfates A, B, and C were without effect at 25 and 100 μg/ml, whereas resorption was increased by 100 μg/ml of heparan sulfate. With chick osteoclasts, which could be maintained in serum-free conditions, a stimulatory effect of heparin was found both in the presence of 5% fetal calf serum and in serum-free media containing insulin, transferrin, and selenium. The magnitude of the heparin-induced increase in resorption was similar in the presence or absence of serum. The stimulation of resorption was associated with an increase in the number of osteoclasts on bone slices. Pretreatment of the bone slices with heparin also enhanced resorption. In time course experiments, 5 μg/ml of heparin caused a doubling of chick osteoclast activity index (number of resorption pits per number of osteoclasts) at 12 and 24 h. In 24 h cultures, treatment with 10 μg/ml of the arginine-rich basic protein, protamine, 1 μg/ml of the immunosuppressant, cyclosporine A, or 5 μg/ml of the cysteine-proteinase inhibitor, leupeptin, negated the heparin effect on bone resorption. Leupeptin also inhibited basal resorption. We conclude that heparin causes an increase in bone resorption in vitro both by increasing the number of differentiated osteoclasts and by enhancing the activity of individual osteoclasts.
Quantitation of skeletal alkaline phosphatase isoenzyme activity in canine serum
Authors: Farley John R. , Hall Susan L. , Ritchie Candace , Herring Sandra , Orcutt Christopher , Miller Barbara E.
Published: February 2010

Pursuing the hypothesis that quantitation of skeletal alkaline phosphatase (ALP) activity in canine serum would provide an index of the rate of bone formation, we compared three methods for isoenzyme-specific identification of skeletal ALP activity in canine serum: heat inactivation, wheat germ agglutinin (WGA) precipitation, and concanavalin A (ConA) precipitation. ALP isoenzyme activities were extracted from canine bone, intestine, and liver, diluted into heat-inactivated canine serum (i.e., serum without ALP activity), and used as calibrators of ALP isoenzyme activities. Differential sensitivity to inhibition by 10 mM L-homoarginine was used to distinguish intestinal ALP activity from hepatic and skeletal ALP activities (i.e., 9, 80, and 72% inhibition, respectively). To allow resolution of skeletal ALP activity from hepatic ALP activity, we tested two established methods (heat inactivation and WGA precipitation) and a novel method, ConA precipitation. The organ-derived skeletal and hepatic ALP isoenzyme activities were used to compare these three methods with respect to linearity, isoenzyme separation, and precision. All three methods were linear, but the WGA and ConA methods afforded greater isoenzyme separation and precision. The relative extent of isoenzyme separation (i.e., the difference in percentage remaining skeletal and hepatic ALP isoenzyme activities) averaged 23, 40, and 47% remaining ALP activity for the heat, WGA, and ConA methods, respectively. However, when these methods were applied to the quantitation of skeletal ALP activity in sera from 10 young and 10 adult beagles, the WGA method was found to be unacceptable because most of the results fell outside the range of the WGA assay calibrators (i.e., >100% skeletal ALP activity). The heat and ConA methods showed that the amount of skeletal ALP activity in the beagle sera decreased with age, both as ALP activity per liter and as percentage of total serum ALP activity (p < 0.001 for each). Skeletal ALP activity levels determined by ConA were correlated with values determined by heat inactivation (r = 0.87, p < 0.001) but not with WGA-determined levels (r = 0.26). Intestinal ALP activity was detected in only 1 of these 20 sera. We conclude that ConA precipitation can be used for quantitation of skeletal ALP activity in beagle serum.
Dual-energy X-ray absorptiometry of the forearm: Reproducibility and correlation with single-photon absorptiometry
Authors: Leboff Meryl S. , Fuleihan Ghada El-Hajj , Angell Jennifer E. , Chung Susan , Curtis Kate
Published: February 2010

Although single-photon absorptiometry (SPA) has been the predominant tool used to assess bone mineral density (BMD) in the forearm, the development of dual-energy x-ray absorptiometry (DEXA) provides the benefits of greater source stability, reduced scanning time, and improved image resolution compared to SPA. In the present study we used the DEXA bone densitometer (Hologic, Inc., Waltham, MA) to (1) measure BMD in the one-third radius and ultradistal radius; (2) examine the reproducibility of these BMD measurements; and (3) compare the BMD at the one-third radius with SPA (SP2, Lunar Corp., Madison, WI). In 65 normal women (ages 22–74 years) we examined changes in the forearm DEXA BMD with age, revealing significant quadratic regression equations. The reproducibility of DEXA BMD (mean ± SEM) in 7 normal subjects aged 22–50 years is 0.85 ± 0.16% for the predominantly cortical one-third radius site and 0.97 ± 0.15% for the more trabecular ultradistal site. The regression relationship between DEXA and SPA of the one-third radius in 26 subjects (ages 22–68 years) is DEXA BMD = 0.105 + 0.826 (SPA BMD); R = 0.97, R2 = 0.94, p < 0.0001. Bone densitometry of the forearm using DEXA may be performed relatively rapidly, providing reproducibility and image resolution that are generally superior to those observed with SPA.
Change in bone mass immediately before menopause
Authors: Recker Robert R. , Lappe Joan M. , Davies K. Michael , Kimmel Donald B.
Published: February 2010

A group of 75 healthy women nearing menopause were entered into a longitudinal study of bone mass and bone cell function. All were at least 46 years, were still menstruating, and had premenopausal levels of follicle stimulating hormone (FSH) and estradiol (E2). Bone density measurements of the spine (dual-photon absorptiometry, DPA) and total-body (DPA) and forearm (single-photon absorptiometry, SPA) were made at 6 month intervals along with measurements of FSH and E2 over an average of 2.04 + 0.45 years. The annual rate of change in bone mass was determined for each individual by regressing the bone mass measurement at each site at the time of observation. The slopes were pooled, and the percentage annual change was calculated as the mean slope divided by the initial mean bone mass value. The mean rates of change in bone mass were calculated with and without weighting by length of observation. No meaningful differences in the results were seen in the weighted values compared to the unweighted values, and none of the sites showed a slope significantly different from zero. Demonstrated reproducibility and sample size provided sufficient power to detect annual weighted rates of change of 0.81% for spine BMC, 0.69% for spine BMD, 1.31% for forearm BMC, 1.55% for forearm BMC/BW, and 1.09% for total body bone mineral. We conclude that if changes in bone mineral are occurring in women at this age, they are substantially less than 1%/ year for spine and not much more for the forearm or total skeleton.
Advantages of peripheral radiogrametry over dual-photon absorptiometry of the spine in the assessment of prevalence of osteoporotic vertebral fractures in women
Authors: Meema Erik H. , Meindok Harry
Published: February 2010

Since osteoporosis develops in most postmenopausal women and is probably the most important single factor in the pathogenesis of osteoporotic fractures of the spine, hip, and wrist (and at other sites), methods suitable for mass screening should be developed. In this study of 97 women aged 24–79, measurements of the lumbar spine mineral content by dual-photon absorptiometry (DPA) were compared with the summed combined cortical thickness measurements from radiographs of the radius and metacarpal II (MR). There was good correlation between the two methods (r = 0.90). The correlation of age with MR was higher than with DPA. The correlation of years postmenopause was significant with MR but not with DPA. Taking the -2 SD level of the premenopausal means to be previously established vertebral fracture thresholds, 24% of the DPA measurements, but no MR measurements in patients with vertebral compressions, were above the fracture threshold. Since MR measurement requires taking only two small plain radiographs using ordinary x-ray equipment, it is concluded that this less expensive method is better suited to screening for osteoporotic vertebral fracture risk in postmenopausal women than DPA.
Relationship between the location of osteoblastic alkaline phosphatase activity and bone formation in human iliac crest bone
Authors: Bradbeer Jeremy N. , Zanelli Joan M. , Lindsay Peter C. , Pearson Josephine , Reeve Jonathan
Published: February 2010

It is not feasible to use in vivo tetracycline double labeling to study bone formation in biopsies taken during the emergency fixation of fractures. We therefore compared the trabecular localization and extent of osteoblastic alkaline phosphatase (AP) perimeters with tetracycline and osteoid perimeters in iliac crest biopsies from 7 women with postmenopausal osteoporosis and 13 women without metabolic bone disease. Fresh biopsies were chilled to -70°C, and triplicate serial unfixed undecalcified cryostat sections were cut and reacted for AP, stained for osteoid, or mounted unstained. At individual remodeling sites, the mineralizing perimeter (M.Pm) was measured as the extent of a double or single label accompanied by ≥ 1 lamella of osteoid and ≥ 1 lamella of mineralized matrix between the mineralization front and the adjacent label. Osteoid perimeters (O.Pm) and AP perimeters (AP.Pm) were also measured. In each biopsy there was good agreement between the location of AP and bone formation (x statistic, range 0.71–1.0). The overall sensitivity and specificity of AP as an indicator of the location of bone formation were 0.963 and 0.902, respectively. At the level of the basic multicellular unit, in those samples in which >3 active BMUs were found, there was (1) significant positive correlation between the M.Pm and both AP.Pm and AP-positive O.Pm (except 1 patient) and (2) no significant difference between the M.Pm and AP-positive O.Pm (17 of 18 patients and 18 of 18 patients at the tissue level). At the tissue level, the line of regression of AP-positive O.Pm on M.Pm (calculated either as label 2 extent or conventionally as dL + 1/2sL extent) was not significantly different from the line of identity. The presence of osteoblastic AP activity in biopsies from normal and osteoporotic patients distinguishes osteoid currently contributing to bone formation and should be useful for the study of bone formation in unlabeled bone samples from such individuals. Further studies are necessary to establish the broader applicability of this technique, particularly when a mineralization defect is involved, as in osteomalacia.
Extracellular matrix formation by osteoblasts from patients with osteogenesis imperfecta
Authors: Fedarko Neal S. , Moerike Martin , Brenner Rolf , Robey Pamela Gehron , Vetter Ulrich
Published: February 2010

Extracellular matrix proteins synthesized by bone cells isolated from 16 patients with different forms of osteogenesis imperfecta (OI) were analyzed in vitro. Specific components of the extracellular matrix by OI and age-matched cultures were investigated by steady-state radiolabeling followed by quantitation of label into specific proteins and comparison of OI cultures to those of age-matched controls. The in vitro proliferation of OI bone cells was found to be lower than that of control cells. In seven patients, abnormalities of the α1(I) and/or α2(I) chains of type I collagen were detected by gel electrophoresis. In two of these patients, the mutations in the COLIA1 and COLIA2 genes have been previously identified. Although the amount of total protein synthesized by the cells in culture was the same for OI bone cells and age-matched control cells, OI bone cells showed a significantly reduced synthesis of not only collagen but also other bone matrix glycoproteins. The synthesis of osteonectin (SPARC/BM40) and three proteoglycans [a large chondroitin sulfate proteoglycan, biglycan (PGI), and decorin (PGII)] was found to be decreased in OI cells. The reduction was most pronounced at the developmental age at which these macromolecules reach maximal levels during normal development.
Effect of fluoride on bone and bone cells in ovariectomized rats
Authors: Modrowski Dominique , Miravet Livia , Feuga Michel , Bannié Florence , Marie Pierre J.
Published: February 2010

To evaluate whether treatment with a mitogenic agent may increase bone formation and bone mass in osteopenia induced by estrogen deficiency, we determined the effect of oral fluoride treatment on bone and bone cells in ovariectomized rats. Sodium fluoride (NaF) was administered to 3-month-old ovariectomized rats 1 day after ovariectomy (OVX) for 1, 3, and 6 months. NaF was given in drinking water at the dose of 1 mg/kg body weight per day. Fluoride administration led to a partial prevention of the bone loss induced by OVX as shown by histologic analysis of tibial metaphysis and by evaluation of femoral calcium content. These beneficial effects of fluoride were more striking at early time points (1 and 3 months postovariectomy) than after 6 months of treatment. The increase in trabecular bone volume in OVX rats treated with fluoride was associated with a rise in the osteoblast surface, which was increased by 60, 72, and 235% at 1, 3, and 6 months postovariectomy compared to untreated OVX rats. In OVX rats and in sham-operated rats plasma osteocalcin was increased in correlation with the osteoblast surface. However, these two parameters were not correlated in OVX rats treated with fluoride. The heat-labile bone-specific alkaline phosphatase in plasma was decreased in OVX rats treated with fluoride compared to OVX rats, suggesting that both the number and the activity of osteoblasts were affected by NaF treatment. To examine the effect of fluoride on the osteocalcin production and the proliferative capacity of bone cells, osteoblastic cells were isolated by collagenase digestion from the bone surface of tibia in treated and untreated OVX rats. In OVX rats DNA synthesis by cultured bone cells was markedly increased compared to sham rats. In OVX rats treated with fluoride DNA synthesis tended to be further increased compared to untreated OVX rats as evaluated by thymidine incorporation into DNA. Osteocalcin production by osteoblastic cells in vitro was comparable in the different groups. The results of this study show that oral treatment with fluoride partially prevents the bone loss induced by estrogen deficiency in OVX rats. This beneficial effect of fluoride results from a further stimulation of bone formation as shown in vivo by an increased extent of bone-forming cells and in vitro by an enhancement of the proliferative capacity of osteoblastic cells isolated from the bone surface.
The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin
Authors: Worcester Elaine M. , Blumenthal Samuel S. , Beshensky Ann M. , Lewand Donna L.
Published: February 2010

Urine contains proteins that inhibit the growth of calcium oxalate (CaOx) crystals and may prevent the formation of kidney stones. We have identified a potent crystal growth inhibitor in the conditioned media from primary cultures of mouse kidney cortical cells. Conditioned media, incubated with the kidney cells for 6–72 h, was assayed for crystal growth inhibition; inhibitory activity increased 15-fold by 24 h. Inhibitory activity was purified from serum-free media containing proteinase inhibitors using anion-exchange and gel-filtration chromatography. A single band of molecular weight 80,000 daltons was seen after SDS-polyacrylamide gel electrophoresis. The sequence of the N-terminal 21 amino acids of this protein matched that of osteopontin (OP), a phosphoprotein initially isolated from bone matrix. Antisera raised to fusion proteins produced by plasmids containing the N-terminal or C-terminal portions of OP cDNA also cross-reacted with the protein purified from cell culture media on western blots. The effect of the purified protein on the growth of CaOx crystals was measured using a constant composition assay. A 50% inhibition of growth occurred at a protein concentration of 0.85 μg/ml, and the dissociation constant of the protein with respect to CaOx crystal was 3.7 × 10−8 M. The concentration of OP in mouse urine, measured using antibodies raised to the purified protein, was approximately 8 μg/ml. We conclude that OP is synthesized by kidney cortical tubule cells and functions as a crystal growth inhibitory protein in urine.
Genetic expression of extracellular matrix proteins correlates with histologic changes during fracture repair
Authors: Jingushi Seiya , Joyce Michael E. , Bolander Mark E.
Published: February 2010

We characterized gene expression in the reparative callus that formed after fracture of the rat femur. The callus was divided into regions of bone formation (hard callus) and cartilage formation (soft callus), and gene expression was examined separately in each region. Expression of extracellular matrix protein genes varied with the progression of repair and differed between hard and soft calluses. Messenger ribonucleic acids (mRNAs) for osteonectin, alkaline phosphatase, and type I procollagen were detected in the hard callus at maximal levels during endochondral ossification and bone remodeling (day 15) and at 50% maximal levels during intramembranous bone formation (day 7). Messenger RNAs for these proteins in the soft callus were detected at low levels during chondrogenesis (day 9) but increased to 80% of maximal levels with chondrocyte hypertrophy and mineralization of the cartilage matrix (day 13). Messenger RNAs for type II procollagen and proteoglycan core protein were detected at maximal levels in the soft callus during chondrogenesis (day 9). Osteocalcin gene expression was detected in the hard callus during endochondral ossification and remodeling but not during intramembranous bone formation or at any time in the soft callus. Osteonectin mRNA was detected in both the hard and soft callus throughout the entire course of fracture repair. Expression of cartilage and bone-related genes correlated with the temporal sequence of histologic changes, suggesting transcriptional regulation of gene expression during repair. Differences in gene expression between hard and soft callus and in each of these regions as repair progressed suggest local regulation of gene expression during cell differentiation and matrix synthesis.
Estrogen and progesterone replacement therapy reduces glucocorticoid-induced bone loss
Authors: Lukert Barbara P. , Johnson Bruce E. , Robinson Ralph G.
Published: February 2010

This is a retrospective study of 15 postmenopausal or amenorrheic women aged 34–78 years who had taken prednisone for 6–108 months and were followed for 1 year while continuing to take doses of 5–15 mg/day. A total of 8 patients were treated with 0.6256 mg Premarin daily for 25 days and 5 mg/day of medroxyprogesterone on days 15–25 (ERT, group 2); 7 were followed without ERT (group 1). A group of 17 women, matched for age, were randomly selected from our computerized data base to serve as a control group (group 3), and 10 women of similar age who were taking ERT only (group 4) were selected to compare the response to ERT to that of group 2. Bone density (BD) was measured in the lumbar spine baseline and at 1 year using dual-photon or dual-energy x-ray absorptiometry. Spine density did not change significantly during the year of observation in group 1. Although BD decreased in 5 of 7 patients, the change was not significant (-0.034 ± 0.018 g/cm2, p = 0.10). In group 2 BD increased significantly, with 7 of 8 patients showing an increase (0.037 ± 0.011 g/cm2, p = 0.008). BD did not change significantly in the control group (0.013 ± 0.008 g/cm2, p = 0.16). Loss of bone from the spine was significantly greater in group 1 than in controls (p = 0.02), but changes in group 2 were similar to those in the control group (p = 0.66). Changes in BD in group 2 were similar to those in group 4; that is, the response to ERT of patients taking prednisone was comparable to that of women taking ERT only. The change in spine density correlated with the cumulative dose of prednisone taken during the year of observation in the group not taking ERT (r = 0.759, p = 0.05) but not in the group taking ERT (r = 0.134, p = 0.73). This suggests that ERT protects against bone loss for at least 1 year throughout the dosage range studied. We concluded that ERT prevents vertebral bone loss for at least 1 year in the majority of women taking prednisone chronically in doses of 5–15 mg/day.