Differential effects of sex hormones on peri- and endocortical bone surfaces in pubertal girls.

CONTEXT: The role of sex steroids in bone growth in pubertal girls is not yet clear. Bone biomarkers are indicators of bone metabolic activity, but their value in predicting bone quality has not been studied in growing girls. OBJECTIVE: This study examines the association of sex hormones and bone markers with bone geometry and density in pubertal girls. DESIGN: The study was designed as a 2-yr longitudinal study in pubertal girls. Measurements were performed at baseline and at 1- and 2-yr follow-ups. SETTING: The study was conducted in a university laboratory. PARTICIPANTS: A total of 258 10- to 13-yr-old healthy girls at the baseline participated. METHODS: Peripheral quantitative computed tomography was used to scan the left tibial shaft. Serum 17beta-estradiol (E2), testosterone (T), SHBG, osteocalcin (OC), bone-specific alkaline phosphatase, and tartrate-resistant acid phosphatase isoform 5b were assessed. Data were analyzed using hierarchical linear models with random effect. RESULTS: E2 was a positive predictor for total bone mineral density (BMD), cortical thickness, and a negative predictor for endocortical circumference but had no predictive value for total bone cross-sectional area or periosteal circumference. T was a positive predictor for total cross-sectional area and periosteal circumference as well as endocortical circumference, and a negative predictor for total BMD. OC was negatively correlated with cortical BMD (R2 = 0.325; P < 0.001). CONCLUSIONS: In pubertal girls, E2 and T have different influences on bone properties at the long bone shaft. The results suggest that, at the endocortical surface, E2 inhibits bone resorption during rapid growth, and later, after menarche, acts at higher concentrations to promote bone formation. At the periosteal surface, T promotes bone formation, whereas E2 does not affect it. In addition, OC might be used as a predictor of cortical BMD.

Properties and expression of human tartrate-resistant acid phosphatase isoform 5a by monocyte-derived cells.

Human serum tartrate-resistant acid phosphatase exists as two enzyme isoforms (TRACP 5a and 5b), derived by differential, post-translational processing of a common gene product. Serum TRACP 5b is from bone-resorbing osteoclasts (OC) and becomes elevated in diseases of increased bone resorption. TRACP 5a is secreted by macrophages (MPhi) and dendritic cells (DC) and is increased in many patients with rheumatoid arthritis. Our purpose was to fully characterize the properties of human TRACP isoforms and to produce an antibody specific to TRACP 5a for use as a biomarker in chronic inflammatory diseases. Partially purified, natural serum TRACP isoforms and recombinant TRACP 5a (rTRACP 5a) were compared with respect to specific activity and subunit structure and presence of sialic acid. Mice were immunized with rTRACP 5a, and resulting hybridomas were screened for monoclonal antibody to serum TRACP 5a. One antibody, 220, was tested for its epitope specificity and use in various immunological techniques. rTRACP 5a had properties identical to serum TRACP 5a. Antibody 220 was specific for the trypsin-sensitive epitope in the loop peptide, present only in TRACP 5a. Antibody 220 was effective for specific immunoprecipitation, immunoassay, and immunoblot of TRACP 5a. Intact TRACP was present in MPhi, DC, and OC. TRACP 5a was the predominant isoform secreted by MPhi and DC, whereas TRACP 5b was the predominant isoform secreted by OC. TRACP isoforms 5a and 5b may have different functions inside and outside of monocyte-derived cells. Antibody 220 is an important resource for studies of the biosynthetic relationship among TRACP isoforms and of the significance of serum TRACP 5a as a marker in diseases of bone metabolism and inflammation.

Serum TRACP 5b is a useful marker for monitoring alendronate treatment: comparison with other markers of bone turnover.

UNLABELLED: We studied clinical performance of serum TRACP 5b and other bone turnover markers, including S-CTX, U-DPD, S-PINP, S-BALP, and S-OC, for monitoring alendronate treatment. TRACP 5b had higher clinical sensitivity, area under the ROC curve, and signal-to-noise ratio than the other markers. INTRODUCTION: The purpose of this study was to compare the clinical performance of serum TRACP 5b (S-TRACP5b) with that of other markers of bone turnover in the monitoring of alendronate treatment. MATERIALS AND METHODS: This double-blinded study included 148 healthy postmenopausal women that were randomly assigned into two groups: one receiving 5 mg alendronate daily (n=75) and the other receiving placebo (n=73) for 12 months. All individuals in both groups received calcium and vitamin D daily. The bone resorption markers S-TRACP5b, serum C-terminal cross-linked telopeptides of type I collagen (S-CTX), and total urinary deoxypyridinoline (U-DPD), and the serum markers of bone formation procollagen I N-terminal propeptide (S-PINP), bone-specific alkaline phosphatase (S-BALP), and total osteocalcin (S-OC) were assessed at baseline and at 3, 6, and 12 months after initiation of treatment. Lumbar spine BMD (LBMD) was measured at baseline and 12 months. RESULTS: Compared with the placebo group, LBMD increased, and all bone markers decreased significantly more in the alendronate group (p<0.001 for each parameter). The decrease of S-TRACP5b after first 3 months of alendronate treatment correlated significantly with the changes of all other markers except S-OC, the best correlation being with S-CTX (r=0.60, p<0.0001). The changes of LBMD at 12 months only correlated significantly with the changes of S-TRACP5b (r=-0.32, p=0.005) and S-CTX (r=-0.24, p=0.037) at 3 months. Based on clinical sensitivity, receiver operating characteristic (ROC) curves, and signal-to-noise ratio, S-TRACP5b, S-CTX, and S-PINP were the best markers for monitoring alendronate treatment. Clinical sensitivity, area under the ROC curve, and signal-to-noise ratio were higher for S-TRACP5b than for the other markers. CONCLUSION: These results show that S-TRACP5b, S-CTX, and S-PINP are useful markers for monitoring alendronate treatment.

Impaired bone resorption in cathepsin K-deficient mice is partially compensated for by enhanced osteoclastogenesis and increased expression of other proteases via an increased RANKL/OPG ratio.

Previous reports indicate that mice deficient for cathepsin K (Ctsk), a key protease in osteoclastic bone resorption, develop osteopetrosis due to their inability to properly degrade organic bone matrix. Some features of the phenotype of Ctsk knockout mice, however, suggest the presence of mechanisms by which Ctsk-deficient mice compensate for the lack of cathepsin K. To study these mechanisms in detail, we generated Ctsk-deficient (Ctsk-/-) mice and analyzed them at the age of 2, 7, and 12 months using peripheral quantitative computed tomography, histomorphometry, resorption marker measurements, osteoclast and osteoblast differentiation cultures, and gene expression analyses. The present study verified the previously published osteopetrotic features of Ctsk-deficient mice. However, these changes did not exacerbate during aging indicating the absence of Ctsk to have its most severe effects during the rapid growth period. Resorption markers ICTP and CTX were decreased in the media of Ctsk-/- osteoclasts cultured on bone slices indicating impaired bone resorption. Ctsk-/- mice exhibited several mechanisms attempting to compensate for Ctsk deficiency. The number of osteoclasts in trabecular bone was significantly increased in Ctsk-/- mice compared to controls, as was the number of osteoclast precursors in bone marrow. The mRNA levels for receptor activator of nuclear factor (kappa)B ligand (RANKL) in Ctsk-/- bones were increased resulting in increased RANKL/OPG ratio favoring osteoclastogenesis. In addition, expression of mRNAs of osteoclastic enzymes (MMP-9, TRACP) and for osteoblastic proteases (MMP-13, MMP-14) were increased in Ctsk-/- mice compared to controls. Impaired osteoclastic bone resorption in Ctsk-/- mice results in activation of osteoblastic cells to produce increased amounts of other proteolytic enzymes and RANKL in vivo. We suggest that increased RANKL expression mediates enhanced osteoclastogenesis and increased protease expression by osteoclasts. These observations underline the important role of osteoblastic cells in regulation of osteoclast activity and bone turnover.

Biochemical markers of bone turnover are influenced by recently sustained fracture.

In striving to refine the clinical utility of different markers of bone metabolism, we should take into account numerous confounders, many of which are well known, such as sampling time, fasting status, and bone density. One further confounder may be ongoing fracture healing and/or post-fracture immobilization, which at least theoretically should impose an increased bone formation and resorption. Since both recent fracture and high bone turnover are independent predictors for new fracture, we thought it of importance to define the potential influence of such fracture on markers of bone turnover. From a population-based cohort of 1604 women, all 75 years old (the OPRA-study), 1024 women attended a clinical examination. The bone metabolism was assessed in serum, by three markers of bone formation [bone-specific alkaline phosphatase (S-Bone ALP), intact and N-Mid osteocalcin (S-Total OC), and total carboxylated osteocalcin (S-cOC)], two markers of bone resorption [C-terminal cross-linked telopeptides of type I collagen (S-CTX) and tartrate-resistant acid phosphatase type 5b (S-TRACP5b)], and in urine by one marker of bone resorption [deoxypyridinoline/creatinine (U-DPD/crea)] and two putative markers of bone resorption [urinary osteocalcins (U-OC/crea)]. Current physical activity and retrospective fracture data were recorded by questionnaires. The fracture data, for the entire cohort of 1604 women, were validated with radiographic referrals and reports, saved since the beginning of the last century. All data provided, except date of occurrence of retrospectively sustained fracture, were thus obtained cross-sectionally and in all women at the age of 75. Fracture had ever been sustained by 727 of the entire cohort (n = 1604), and by 523 of the attending women (n = 1024). All markers were marginally higher (significant only for U-DPD/crea, P = 0.027) in women who had ever sustained fracture, compared to women without fracture. In women with recent retrospective fracture (since 2 years) (n = 100), the levels of all markers, except the two S-OCs, were significantly higher (r = 0.20-0.33, P = 0.049-0.001) the more recently the fracture had been sustained. Women with low current physical activity had elevated levels of U-DPD/crea (P < 0.001) and one U-OC (P = 0.014), while the other markers were unaffected.

Potential function for the ROS-generating activity of TRACP.

TRACP is an enzyme with unknown biological function. It is expressed primarily in bone-resorbing osteoclasts and activated macrophages. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). In resorbing osteoclasts, TRACP is localized in transcytotic vesicles transporting bone matrix degradation products from the resorption lacuna to a functional secretory domain in the basolateral membrane. ROS generated by TRACP are capable of destroying organic bone matrix components, suggesting that they may be targeted to further destroy initial matrix degradation products in the transcytotic vesicles. The transcytotic route of osteoclasts is analogous with the antigen presentation route of macrophages transporting endocytosed foreign material into cell surface for presentation to other cells of the immune system. Macrophages overexpressing TRACP have elevated levels of intracellular ROS. In alveolar macrophages, TRACP is colocalized with endocytosed Staphylococcus aureus, a pathogen whose clearance is reduced in TRACP-deficient mice, suggesting that ROS generated by TRACP may be targeted to destroy foreign material in the antigen presentation route of macrophages. These data suggest that the ROS generating activity of TRACP may have an important role both in bone resorption and in the immune defense system.

Biochemical markers of bone metabolism and prediction of fracture in elderly women.

UNLABELLED: We studied the ability of various markers of bone turnover to predict fracture in 1040 randomly recruited 75-year-old women. A total of 178 of the women sustained at least one fracture during follow-up (mean, 4.6 years). In elderly women, TRACP5b and urinary fragments of osteocalcin are promising new markers for prediction of fracture, in particular, vertebral fracture. INTRODUCTION: Biochemical markers reflecting bone turnover may improve the prediction of fractures. MATERIALS AND METHODS: The ability of 10 markers of bone turnover to predict fracture in 1040 elderly women in the Malmö OPRA study was studied. Serum bone-specific alkaline phosphatase and four different forms of serum osteocalcin (S-OC) were analyzed as markers of bone formation and serum C-terminal cross-linking telopeptides of type I collagen (S-CTX), serum TRACP isoform 5b (S-TRACP5b) and urinary free deoxypyridinoline (U-DPD) as markers of bone resorption. Two novel assays for osteocalcin fragments in urine (U-OC) were analyzed. Areal BMD (aBMD) was measured by DXA in the femoral neck and lumbar spine. RESULTS: In total, 231 fractures were sustained by 178 of the women during a 3- to 6.5-year (mean, 4.6 years) follow-up period. When women with prospective fractures were compared with women without fractures, S-TRACP5b, S-CTX, one S-OC, and one U-OC were higher in women with a fracture of any type (all p < 0.05), and all bone markers were higher in women with clinical vertebral fracture (all p < 0.05). Markers were not significantly elevated in women with hip fracture. When women within the highest quartile of a bone marker were compared with all others, S-TRACP5b and one U-OC predicted the occurrence of a fracture of any type (odds ratio [OR]), 1.55 and 1.53; p < 0.05). S-TRACP5b, the two U-OCs, and S-CTX predicted vertebral fracture (OR, 2.28, 2.75, 2.71, and 1.94, respectively; all p < 0.05), and the predictive value remained significant for S-TRACP5b and the two U-OCs after adjusting for aBMD (OR, 2.02-2.25; p < 0.05). Bone markers were not able to predict hip fracture. CONCLUSION: These results show that biochemical markers of bone turnover can predict fracture, and in particular, fractures that engage trabecular bone. S-TRACP5b and U-OC are promising new markers for prediction of fracture.

Intracellular machinery for matrix degradation in bone-resorbing osteoclasts.

UNLABELLED: In osteoclasts, TRACP co-localized with cathepsin K in transcytotic vesicles and was activated by cathepsin K in vitro, suggesting that TRACP may degrade organic matrix components in transcytotic vesicles in an event regulated by cathepsin K. INTRODUCTION: TRACP is an enzyme with unknown biological function. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). Bone-resorbing osteoclasts contain large amounts of TRACP, and transgenic animal models suggest that TRACP has a role in bone resorption. Osteoclasts resorb bone by secreting acid and lysosomal enzymes such as cathepsin K into an extracellular resorption lacuna between the cell membrane and bone surface. Matrix degradation products are then endocytosed, transcytosed, and secreted through a functional secretory domain in the basolateral membrane facing bone marrow. MATERIALS AND METHODS: We have studied intracellular localization of TRACP in osteoclasts with antibodies against various known endosomal and lysosomal proteins using confocal microscopy. We also studied co-localization of TRACP with cathepsin K and endocytosed bone matrix components and the effect of cathepsin K digestion on the ROS generating activity of TRACP in vitro. RESULTS: Double-staining experiments of TRACP with endosomal and lysosomal markers showed that, although some endosomal staining was detected, TRACP was not present in lysosomes. However, TRACP was present in transcytotic vesicles, where it co-localized with cathepsin K. Cathepsin K digestion of TRACP in vitro increased the phosphatase activity by 5.6-fold and the ROS generating activity by 2.0-fold. CONCLUSIONS: These results suggest that cathepsin K may activate the ROS-generating activity of TRACP in transcytotic vesicles of resorbing osteoclasts, the ROS being targeted to finalize degradation of organic bone matrix components during their transcytosis.

A novel immunoassay for the determination of tartrate-resistant acid phosphatase 5b from rat serum.

Osteoclasts secrete tartrate-resistant acid phosphatase 5b (TRACP 5b) into the circulation. We have developed an immunoassay for the determination of rat TRACP 5b activity. Intra-assay variation of the immunoassay was 4.5%, interassay variation was 3.8%, dilution linearity was 104.6 +/- 7.6%, and recovery of recombinant rat TRACP was 99.1 +/- 5.8%. We studied serum TRACP 5b as a marker of bone resorption using orchidectomized (ORC) rats as a model for osteoporosis and age-matched sham-operated rats as controls in a 6-month study. After the operation, trabecular bone mineral density decreased significantly more in the ORC group than in the sham group, whereas cortical bone mineral density increased similarly in both groups. Serum TRACP 5b activity was significantly elevated within the first week after ORC, returned to the control level in the third week, and was not increased above the sham level at any of the later time points. At 6 months, trabecular bone volume was 80% lower in ORC rats than in controls. Osteoclast number per trabecular bone perimeter was slightly increased, but the absolute number of osteoclasts in trabecular bone was significantly decreased. These results suggest that absolute bone resorption is increased within the first week after ORC. Later, it is decreased because there is less bone to be resorbed. However, relative bone resorption (compared with the amount of remaining bone) is still increased, leading to further bone loss. We conclude that serum TRACP 5b is a useful marker for monitoring changes in the bone resorption rate in rat ORC model.

Macrophages overexpressing tartrate-resistant acid phosphatase show altered profile of free radical production and enhanced capacity of bacterial killing.

Activated macrophages and osteoclasts express high amounts of tartrate-resistant acid phosphatase (TRACP, acp5). TRACP has a binuclear iron center with a redox-active iron that has been shown to catalyze the formation of reactive oxygen species (ROS) by Fenton's reaction. Previous studies suggest that ROS generated by TRACP may participate in degradation of endocytosed bone matrix products in resorbing osteoclasts and degradation of foreign compounds during antigen presentation in activated macrophages. Here we have compared free radical production in macrophages of TRACP overexpressing (TRACP+) and wild-type (WT) mice. TRACP overexpression increased both ROS levels and superoxide production. Nitric oxide production was increased in activated macrophages of WT mice, but not in TRACP+ mice. Macrophages from TRACP+ mice showed increased capacity of bacterial killing. Recombinant TRACP enzyme was capable of bacterial killing in the presence of hydrogen peroxide. These results suggest that TRACP has an important biological function in immune defense system.

Phosphatase and oxygen radical-generating activities of mammalian purple acid phosphatase are functionally independent.

Bone-resorbing osteoclasts and activated macrophages express large amounts of tartrate-resistant acid phosphatase (TRAP), an iron-containing enzyme with unknown biological function. We studied acid phosphatase (AcP) and reactive oxygen species (ROS)-generating activities of recombinant rat TRAP. pH optimum was 4.5 for AcP activity and 6.5 for ROS-generating activity. Replacement of His113 and His216 by site-directed mutagenesis severely inhibited AcP activity, but had no significant effects on ROS-generating activity. Substrate specificity was not affected by the mutations. These results suggest that AcP and ROS-generating activities of TRAP are functionally independent.

Osteoclast-derived serum tartrate-resistant acid phosphatase 5b in Albers-Schonberg disease (type II autosomal dominant osteopetrosis).

BACKGROUND: Albers-Schönberg disease, or autosomal dominant osteopetrosis type II (ADO2), is caused by ineffective osteoclastic bone resorption resulting from mutations in the chloride channel 7 (ClCN7) gene. Individuals with ADO2 have increased numbers of large ineffective osteoclasts in addition to increased serum total tartrate-resistant acid phosphatase (TRACP) activity. METHODS: We investigated the serum activity of the osteoclast-derived 5b isoform of TRACP (TRACP 5b) and concentrations of the bone formation marker osteocalcin in clinically affected individuals, unaffected gene carriers, and healthy controls from 10 ADO2 families with known ClCN7 gene mutations. Bone fracture prevalence was studied in association with the serum markers. RESULTS: Similar to total TRACP, TRACP 5b was significantly increased in clinically affected individuals compared with age-matched controls. TRACP 5b correlated significantly with total TRACP (r = 0.833; P <0.001), suggesting that most of the TRACP in the serum of ADO2 patients is osteoclast-derived TRACP 5b. Osteocalcin was significantly increased in affected adults and slightly decreased in affected children. TRACP 5b and total TRACP were significantly increased in clinically affected individuals with severe fractures (P <0.05). CONCLUSIONS: The results indicate that in ADO2, serum TRACP 5b reflects the number of osteoclasts and that the extremely high serum TRACP 5b activity is a specific indicator of the disease. Similar to total TRACP, TRACP 5b appears to be a potentially useful marker to stratify individuals with ClCN7 gene mutations into clinically affected and unaffected gene carriers. It may also have a prognostic value in the prediction of fractures in patients with a ClCN7 gene mutation.