The effect of oral glucose tolerance test on serum osteocalcin and bone turnover markers in young adults.

Osteocalcin (OC) is an osteoblast-derived protein implicated in the regulation of glucose tolerance and energy metabolism. This endocrine function has been suggested to be exerted via its undercarboxylated form, which has been shown to induce expression of adiponectin, insulin, and islet cell proliferation in mice. Furthermore, insulin has recently been shown to regulate the biological activity of OC in bone. Our aim was to explore the association between glucose and bone metabolism by evaluating the effect of a standard 75 g oral glucose tolerance test (OGTT) on serum OC, carboxylated OC (cOC) and bone-turnover markers (BTMs) C terminal telopeptide (ßCTX-I) and N terminal propeptide (PINP) of type I collagen and tartrate-resistant acid phosphatase 5b (TRACP5b). Serum samples collected at 0 and at 120 min were analyzed in a cohort of normoglycemic young adults (n = 23, mean age 23.6 years). During OGTT a significant decrease was observed in all BTMs (P < 0.001 for all variables). The median decreases from 0 to 120 min for OC, cOC, ßCTX-I, PINP, and TRACP5b were -32.1% (-37.9 to -19.6), -34.4% (-39.8 to -22.2), -61.4% (-68.5 to -53.0), -26.8% (-33.2 to -19.2), and -44.5% (-48.3 to -40.2), respectively. A strong association between the changes in OC and cOC was observed (r = 0.83, P < 0.001). The decrease in PINP was associated with changes in OC, whereas the changes in ßCTX-I and TRACP5b were not associated with decreases in OC or cOC. The observed OGTT-induced changes in bone-derived proteins were partially independent of each other and potentially mediated by different mechanisms.

Effect of impact exercise on bone metabolism.

SUMMARY: Regular impact exercise in premenopausal women caused positive osteogenic effects associated to low basal serum parathormone (PTH) but had no effects on bone turnover markers PINP or TRACP5b. The low serum basal PTH levels during impact exercise may be a sign of increased incorporation of calcium to bone. INTRODUCTION: This study aimed to determine the long-term effects of high-impact exercise on bone turnover and calciotropic hormones. METHODS: We performed a 12-month population-based, randomized, controlled exercise trial in 120 women (age 35-40 years) randomly assigned to an exercise group (EG; n = 60) or a control group (CG; n = 60). The exercise regimen consisted of supervised high-impact exercises three times per week. Daily impact loading was assessed by using an accelerometer. Bone turnover markers and calciotropic hormones were analyzed at 0, 6, and 12 months. RESULTS: Twelve months of impact exercise did not reveal any treatment effects in bone turnover markers PINP or TRAPC5b, whereas serum basal PTH decreased significantly more in the EG than in the CG (-11.2 vs. -2.2 pg/mL; p = 0.03). The change in PTH was dose dependent and most clearly seen in subjects with 96 to 130 daily impacts at 2.5 to 5.3 g (e.g., running or jumping). CONCLUSIONS: Regular impact exercise does not cause persistent alterations in bone turnover emphasizing necessity of continuous training to achieve bone benefits. Impact exercise training lowers the serum basal PTH levels and possibly enables greater difference between the basal PTH and transient exercise-induced PTH peaks leading to osteogenic effects.

Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface.

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.

Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ruffled borders of osteoclasts.

Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H(+)-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60- and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatment of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K(+)-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through vacuolar H(+)-ATPase.

Removal of osteoclast bone resorption products by transcytosis.

Osteoclasts are multinucleated cells responsible for bone resorption. During the resorption cycle, osteoclasts undergo dramatic changes in their polarity, and resorbing cells reveal four functionally and structurally different membrane domains. Bone degradation products, both organic and inorganic, were endocytosed from the ruffled border membrane. They were then found to be transported in vesicles through the cell to the plasma membrane domain, located in the middle of the basal membrane, where they were liberated into the extracellular space. These results explain how resorbing osteoclasts can simultaneously remove large amounts of matrix degradation products and penetrate into bone.

Can elevated serum TRACP-5b levels predict stress fractures? A cohort study.

BACKGROUND AND AIMS: Stress fracture is a common overuse injury in athletes and military conscripts. The reliable diagnosis of stress fractures is often difficult, however, because it is usually based solely on radiographic findings. Biochemical markers of bone resorption reflect bone degradation and may also reflect the rate of bone loss. The aim of the study was to examine whether elevated serum tartrate-resistant acid phosphatase isoform 5b (TRACP-5b) levels reflect enhanced bone remodeling and predict the occurrence of stress fractures in military conscripts. MATERIAL AND METHODS: Randomly selected military conscripts [mean age, 19.8 (range 18-28) years; n = 820] were followed for 3 months. Baseline blood samples were drawn upon arrival to the service. Four subsequent samples were obtained from subjects that developed stress fractures and one sample each was obtained from two asymptomatic control subjects for each fracture case. RESULTS: Plain radiography was used to diagnose stress fractures in 20 of the 820 conscripts (2.4%). Follow-up data were available for 14 subjects with 21 stress fractures and 28 control subjects. Subjects with proportionally increasing serum TRACP-5b levels had an 8-fold greater probability of stress fracture than controls. No statistically significant difference was detected. CONCLUSIONS: Although assessing serum TRACP-5b levels appears to be a promising method to predict bone stress injuries, the present study failed to give a conclusive statement of its usefulness as a diagnostic tool.

Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage.

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption.

Differential effects of selective oestrogen receptor modulators (SERMs) tamoxifen, ospemifene and raloxifene on human osteoclasts in vitro.

BACKGROUND AND PURPOSE: Several selective oestrogen receptor modulators (SERMs) with oestrogen agonist effects in bone cells and without increased risk of breast and endometrial cancer have been developed. Here, we have investigated the effects of different types of SERMs on osteoclast differentiation, bone resorption and apoptosis in vitro. EXPERIMENTAL APPROACH: Human peripheral blood-derived CD14+ monocytes were cultured on bovine bone slices in the presence of RANKL, M-CSF, TNF-alpha and dexamethasone for seven days. Also, CD14+ monocytes were co-cultured either with human SaOS-2 or MG-63 osteosarcoma cells, in the presence of parathyroid hormone. Osteoclast cultures were treated with different SERMs. TRACP+ multinucleated cells and C-terminal telopeptide of type I collagen were used as markers for osteoclast formation and bone resorption, respectively. KEY RESULTS: In CD14+ monocyte cultures, tamoxifen directly inhibited human osteoclast formation and bone resorption, while raloxifene and ospemifene had no inhibitory effect. In the co-cultures either with SaOS-2 or MG-63 cells, ospemifene and raloxifene as well as tamoxifen inhibited osteoclast formation in a concentration-dependent manner. The inhibitory effect was associated with an increased production of osteoprotegerin. The anti-oestrogen ICI 182 780 completely reversed the effects of these SERMs. CONCLUSION AND IMPLICATIONS: Tamoxifen had an oestrogen receptor dependent, direct, inhibitory effect on human osteoclast differentiation and bone resorption, whereas ospemifene and raloxifene required osteoblastic cells to achieve a similar inhibition. The effects of ospemifene and raloxifene were mediated by oestrogen receptors by a mechanism involving paracrine induction of osteoprotegerin in cultures with osteoblast derived osteosarcoma cells.

Resorption-cycle-dependent polarization of mRNAs for different subunits of V-ATPase in bone-resorbing osteoclasts.

Protein sorting in eukaryotic cells is mainly done by specific targeting of polypeptides. The present evidence from oocytes, neurons, and some other polarized cells suggests that protein sorting can be further facilitated by concentrating mRNAs to their corresponding subcellular areas. However, very little is known about the mechanism(s) involved in mRNA targeting, or how widespread and dynamic such mRNA sorting might be. In this study, we have used an in vitro cell culture system, where large multinucleated osteoclasts undergo continuous structural and functional changes from polarized (resorbing) to a nonpolarized (resting) stage. We demonstrate here, using a nonradioactive in situ hybridization technique and confocal microscopy, that mRNAs for several vacuolar H(+)-ATPase subunits change their localization and polarity in osteoclasts according to the resorption cycle, whereas mRNA for cytoplasmic carbonic anhydrase II is found diffusely located throughout the osteoclast during the whole resorption cycle. Antisense RNA against the 16-kDa or 60-kDa V-ATPase subunit inhibits polarization of the osteoclasts, as determined by cytoskeleton staining. Antisense RNA against carbonic anhydrase II, however, has no such effect.

Serum TRACP 5b and ICTP as markers of bone metastases in breast cancer.

BACKGROUND: The purpose of this cross-sectional study was to evaluate the value of serum tartrate-resistant acid phosphatase 5b (TRACP 5b) and carboxyterminal telopeptide of type I collagen (ICTP) separately and in combination as markers of bone metastases compared to total alkaline phosphatase (tALP) in breast cancer. MATERIALS AND METHODS: Two groups of patients were studied, one with verfied bone metastases (N=46) and one without bone metastases (N=141). Bone marker levels were correlated with the presence or absence of bone metastases. RESULTS: Serum TRACP 5b concentrations exhibited the largest area under the receiver-operating characteristics (ROC) curve (AUC=0.845), followed by ICTP (0.818) and tALP (0.814) when all patients were included in the analysis. With the combination of TRACP 5b and ICTP, the AUC increased to 0.881. In multivariate regression analysis, all three markers were significant predictors of bone metastases. CONCLUSION: Serum TRACP 5b, ICTP and tALP exhibited equal performances in the detection of bone metastases. The combination of TRACP with ICTP did not significantly improve the detection of bone metastases over tALP.

Synthesis and secretion of a 38-kDa glycopolypeptide coincides with L6 myoblast fusion.

Rat L6 myoblastic cell line fused rapidly after two day cultivation in a medium containing horse serum and insulin. We analyzed whether the induction of plasma membrane or secreted proteins occurred simultaneously with ongoing fusion. Thus the cells were metabolically labeled with [35S]methionine followed by biotinylation of the cell surface proteins. Detergent-solubilized proteins derivatized with biotin were isolated with streptavidin-agarose and subjected to SDS polyacrylamide gel electrophoresis. This analysis did not show fusion-associated induction of any surface proteins. However, analysis of the microsomal fraction revealed a fusion-associated 38-kDa glycopolypeptide. This polypeptide appeared simultaneously with the formation of the multinucleated cells and then declined with decreasing fusion activity. Pulse-chase labeling experiments showed that the 38-kDa component was secreted into the medium. These results indicate that a secreted protein component is induced during the fusion of L6 myoblasts.

Kinetics of the osteoclast cytoskeleton during the resorption cycle in vitro.

Resorption and migration phases alternate in the life of the osteoclast. We have previously described a specific microfilament structure at the attachment sites in resorbing osteoclasts. In the present study we have examined microfilaments and microtubules in both resorbing and migrating rat osteoclasts cultured on bone slices. In migrating osteoclasts microfilaments form so-called podosome structures containing vinculin, talin, and F-actin at the paramarginal area of the cell. When the osteoclast prepares itself for resorption, the podosomes gather to a certain area and form a broad ring around the area, which is then resorbed. In the resorbing osteoclast, vinculin and talin form a continuous double circle, which may be partially formed by podosomes, and between these double circles a broad zone is formed by F-actin. Narrow vinculin and F-actin rings were found in osteoclasts at the end of the resorption phase. The different configurations of microfilaments in 1 and 2 day cultures were correlated in terms of their relationship to the resorption lacunae. The vitamin A derivative isotretinoin significantly stimulated resorption and increased the number of microfilament configurations associated with the resorption pits. On the other hand, Bt2cAMP abolished resorption and prevented the formation of a specific ring structure of microfilaments. Based on these data, a kinetic model of the whole migration-resorption cycle of the osteoclast cultured on the bone slice is presented. With alpha-tubulin stainings of microtubules two different cytoskeletal organizations were observed. In migrating osteoclasts, microtubules were evenly distributed over the whole cell. In the resorbing osteoclast, there was a noticeable concentration of these cytoskeletal structures at cytoplasmic sites closest to the resorption lacuna. This orientation of microtubules may reflect the active secretory function of the resorbing osteoclast.

Exercise can provide protection against bone loss and prevent the decrease in mechanical strength of femoral neck in ovariectomized rats.

The effect of treadmill exercise on bone loss in ovariectomized (OVX) rats was studied in two different sets of experiments. In the first experiment rats were either ovariectomized (n = 38) or sham operated (n = 18) at the age of 12 weeks. Half the OVX rats were trained twice a day for 30 minutes by running at 10 m/minute for 7 or 17 weeks. In the second experiment 40 female rats, aged 12 weeks, were divided into five groups (n = 8). One group of rats was sacrificed on day 0 for the baseline data. Other rats were sham operated or ovariectomized for 9 weeks. Half of both groups were trained using the same training program as in the first experiment. OVX reduced trabecular bone volume (TBV) in the distal femur to 42.7 and 48.3% in 8 and 18 weeks, respectively. Exercise opposed this effect significantly but could not prevent it totally. Exercise did not have any significant effect on sham-operated animals. OVX induced a 17.7 and 30.7% decrease in maximal failure load of femoral neck in 8 and 18 weeks, respectively. A corresponding decrease was also observed in the torque capacity of tibia. Exercise was able to prevent almost totally the decrease in bone strength of femoral neck, tibia, and humerus. In conclusion, our results suggest that the measurement of bone strength in aging female rat femoral neck can be used as a useful indicator of the deleterious effect of OVX in bone. These results further indicate that exercise can overcome a significant part of the decrease in trabecular bone volume and maintain the mechanical strength of femoral neck and tibial shaft in the OVX rats.

Tartrate-resistant acid phosphatase from human bone: purification and development of an immunoassay.

Tartrate-resistant acid phosphatase (TRAP) was purified 20,000-fold to apparent homogeneity from human bone. The purified enzyme consisted of one 32 kd subunit, which was cleaved by beta-mercaptoethanol into two subunits of 15 kd and 20 kd, as shown by sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The purified enzyme was identified by N-terminal amino acid sequencing, and it was shown to be homologous with previously purified TRAPs from other sources. We developed a polyclonal antiserum against the purified enzyme in mice. In immunohistochemistry, the antiserum recognized osteoclasts from human bone and alveolar macrophages from human lung tissue, but no cells from human spleen tissue. It also stained osteoclasts from rat bone cells cultured on bovine bone slices. Purified TRAP could be inhibited by vanadate and molybdate, but not by tartrate, and it was activated 2-fold by beta-mercaptoethanol. The glycoprotein structure of human bone TRAP was analyzed, and it was shown to contain only high-mannose type carbohydrates. We used the polyclonal antibody to develop a competitive fluorescence immunoassay for measuring serum TRAP concentrations. According to the assay, children have higher serum TRAP concentrations than adults, and postmenopausal women have higher concentrations than premenopausal women. Postmenopausal women also have higher serum TRAP concentrations than postmenopausal women on estrogen replacement therapy.

Alteration in the mechanical competence and structural properties in the femoral neck and vertebrae of ovariectomized rats.

The structural and mechanical properties of bone in the femoral neck and various other sites were investigated in intact (INT), sham-operated (Sham), and ovariectomized (OVX) rats. Six weeks after operation, the maximal load and energy absorption of the femoral neck were significantly lower in the OVX than in the INT or Sham groups, being 73.2 +/- 1.4 (SE) N, 86.3 +/- 4.1 N, and 87.1 +/- 3.2 N, respectively (p < 0.01) for load. The total cross-sectional area of the femoral neck did not change after OVX, but the marrow cavity area was enlarged, leading to a reduced bone area (including both cortical and trabecular bone) (p < 0.01). Histomorphometric analysis showed that new bone formation could not be detected at the periosteum of the femoral neck below the femoral head, but at the endocortical surfaces the double tetracycline labeling revealed an increased mineral apposition rate (MAR) and bone formation rate (BFR) in OVX animals (p < 0.001). In contrast, MAR and BFR were significantly increased in both periosteal and endocortical surfaces of the tibia, humerus, and femoral shaft, thus preventing a decrease in cortical bone area. The maximal bending loads of the tibia and humerus were not different in the various groups of animals. The correlation coefficient between maximal load and bone area revealed positive relationships in the femoral neck (r = 0.54, p < 0.01), tibia (r = 0.46, p < 0.01), and humerus (r = 0.51, p < 0.01). Ovariectomy resulted in a decreased trabecular bone volume of lumbar vertebra VI (L6) decreased compressive loads of lumbar vertebrae I, III, and IV. These lumbar bone loads were positively related to their L6 bone area (L4/L6: r = 0.66, p < 0.001). Element analyses (energy dispersion spectrometer) from trabecular and cortical areas of bone showed some changes related to aging but not to OVX. These results indicate that ovariectomy influences the biomechanical properties of rat bone by changing structural properties rather than material ones.

Inhibition of bone resorption by a monoclonal antibody that reacts with a 150 kD membrane protein in chicken osteoclasts.

Bone resorption is a multistep process that includes the maturation of osteoclast precursors, the special attachment of fully differentiated osteoclasts to mineralized bone surface, and the dissolution of inorganic mineral, as well as the breakdown of organic matrix. We have produced a large panel of monoclonal antibodies directed against chicken osteoclasts to obtain specific probes for studying the function of osteoclasts. One of our antibodies, K20, inhibited bone resorption of isolated osteoclasts almost completely. Several pieces of evidence suggested that the antigen detected by this antibody was located in the plasma membrane of the osteoclast. In western blot analysis K20 antibody specifically recognized a 150 kD protein in the medullary bone microsome fraction under reducing and nonreducing conditions. In addition to osteoclasts and some bone and bone marrow mononuclear cells, a positive immunoreaction was seen in the kidney tubules. These data suggest that monoclonal antibody K20 reacts with an osteoclast surface antigen that is functionally important in bone resorption.

Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption.

Human serum contains two forms of tartrate-resistant acid phosphatase (TRAP), 5a and 5b. Of these, 5a contains sialic acid and 5b does not. We show here that antigenic properties and pH optimum of TRAP purified from human osteoclasts are identical to those of serum TRAP 5b and completely different from those of serum TRAP 5a, suggesting that 5b would be derived from osteoclasts and 5a from some other source. We developed a novel immunoassay specific for 5b using a monoclonal antibody O1A as capture antibody. O1A did not bind acid phosphatase derived from platelets and erythrocytes. Western analysis showed that O1A was specific for TRAP in both human bone and serum. We measured bound TRAP activity at pH 6.1, where 5b is highly active and 5a almost completely inactive. The immunoassay detected more than 90% of the initial TRAP 5b activity after 8-h incubation of serum samples at 25 degrees C and after 3 days incubation at 4 degrees C. Serum TRAP 5b activity decreased significantly after 6 months of hormone replacement therapy (HRT) of postmenopausal women compared with the change observed in postmenopausal women receiving placebo (p < 0.0001). Instead, no significant differences were observed between the changes in the placebo and HRT groups in total serum TRAP amount. These results show that serum TRAP 5b is a specific and sensitive marker for monitoring antiresorptive treatment. Instead, total serum TRAP cannot be used for that purpose. These findings may turn out to be a significant improvement in using serum TRAP as a resorption marker.

Short-term intravenous bisphosphonates in prevention of postmenopausal bone loss.

This study was performed to test the efficacy of short-term intravenous clodronate and etidronate in the prevention of postmenopausal bone loss. Healthy postmenopausal women, exhibiting a decreasing trend in bone mineral density, were randomized to five groups (clodronate at doses of 150, 300, and 600 mg; etidronate at a dose of 300 mg; and a placebo group) of 21-22 subjects. The drugs were administered intravenously three times with 1-week intervals, followed by regular evaluation for up to 24 months. During the first year, 300 mg of clodronate retarded bone loss significantly in the lumbar spine and femoral neck, where significant protection still persisted after 24 months. Other doses of clodronate (150 and 600 mg) were not bone protective. Etidronate (300 mg) retarded bone loss significantly in the lumbar spine up to 24 months, relative to placebo. Serum concentrations of procollagen I carboxy-terminal propeptide and urinary Ca2+ and hydroxyproline excretion decreased in all bisphosphonate groups during the first month after treatment, but the values returned later toward baseline. In the etidronate-group, serum osteocalcin concentrations also decreased significantly during the first 3 months of the study. Otherwise, no uniform serum responses to bisphosphonate-treatment were detected in circulating markers of bone formation, alkaline phosphatase, or osteocalcin. No significant differences in the serum concentrations of cross-linked carboxy-terminal telopeptide of type I collagen were detected between the groups. Patient acceptance of both bisphosphonates was excellent, and no drug-related adverse side effects were detected. These results suggest that infrequently repeated intravenous treatment with bisphosphonates may effectively counteract postmenopausal bone loss.

Demonstration of the predominant urine osteocalcin fragments detectable by two-site immunoassays.

We have isolated and characterized human osteocalcin (OC) fragments from pubertal urine. The fragments were isolated by immunoaffinity chromatography based on monoclonal antibody 6F9 and further purified by reverse phase chromatography. The major isolated forms, which were detectable with two-site immunofluorometric assays for serum OC, span residues 6-30 and 7-30 as determined by mass spectrometry and N-terminal amino acid sequencing. Full-length OC was not detectable in the supernatant fraction of urine but could be extracted with guanidinium hydrochloride from the sediment of urine samples. Urine samples from subjects with different menopausal status were measured by two different two-site assays. Urine OC (uOC) concentrations were 12- to 16-fold higher in the pubertal group than in the adult group. Also, the uOC concentration in a postmenopausal group was significantly higher than in a premenopausal group. The difference was 125% and 75% (values for p < 0.0001), respectively, when measured with the two assays. uOC concentrations in postmenopausal subjects on hormone replacement therapy were indistinguishable from the premenopausal subjects. The fact that uOC can be measured by a noncompetetive two-site assay design offers improved analytical sensitivity. Urine as the sample matrix is also especially interesting because the predominant markers of bone resorption, collagen type I peptides or cross-links, are performed on urine samples. Our results from the technical validation of two-site assays for uOC and from applying these to human pubertal and pre- and postmenopausal samples calls for more extensive clinical validation.