Tartrate-resistant Acid Phosphatase 5b Activity Research Articles

Impact of leptin or melatonin on Sema4D overexpression-related bone metabolism

PurposeThe current study aims to investigate the regulatory impact of leptin or melatonin on bone metabolism as well as the underlying mechanism in conjunction with Sema4D (monoclonal antibody to semaphorin 4D).MethodsRats were used to create the osteoporosis model utilizing the OVX (OVariectomize) technique. Rat tibial specimens from each side were collected for three-dimensional reconstruction and Micro-CT scanning examination. The Hematoxylin-osinstaining (HE) staining technique was used to determine the pathological condition of bone tissues. The ELISA (Enzyme-Linked Immunosorbent Assay) assay was used to measure the amount of estradiol present in the serum. In the current study, there were six groups: control, OVX, OVX + NL (no load group), OVX + Sema4D, OVX + Sema4D + leptin, and OVX + Sema4D + MT (melatonin). Rats were given injections of the Sema4D or leptin overexpressing vectors via the tail vein in accordance with the aforementioned classification. By using a high-resolution micro-CT technology, 3D bone structure was discovered. The activity of tartrate-resistant acid phosphatase-5b (TRAP-5b) and bone-derived alkaline phosphatase (BALP) in serum was assessed using an ELISA. The number of osteoclasts in the metaphysis of the upper tibia was determined using TRAP (tartrate-resistant acid phosphatase) staining. Immunohistochemistry was used to find leptin and bone morphogenetic protein-2 (BMP-2) expressions in bone tissue.ResultsThe BV/TV (Bone volume/Tissue volume), Tb.N (Trabecular number), BMD (Bone Mineral Density), and BMC (Bone Mineral Content) levels were significantly higher in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups compared to OVX + NL, while Tb.Sp (Trabecular separation) levels were significantly lower. In contrast to the OVX group, the bone trabeculae in the OVX + Sema4D + leptin and OVX + Sema4D + MT groups had a relatively complete structure and tended to be organized closely. The amount of bone trabeculae grew drastically, whereas the proportion of TRAP-positive osteoclasts declined dramatically. BMP-2 and leptin were also elevated, while BALP and TRAP-5b activity was reduced.ConclusionLeptin or melatonin improved Sema4d's role in trabecular bone microstructure, bone production, and repairment of trabecular bone loss in osteoporosis rats.

Effects of I-125 seeds combined with anlotinib on tumor growth and bone metabolism in A549 tumor-bearing mice

Purpose This study aimed to investigate the therapeutic potential of tumor suppression and mechanism for different implantation modes of iodine-125 (I-125) seeds irradiation in a mice xenograft model, and its skeletal complications. Materials and methods A total of 24 mice carrying A549 lung tumor-derived xenografts were randomly assigned to four groups, including non-radioactive (sham) seeds implantation, I-125 seeds fractional implantation, I-125 seeds single implantation and I-125 seeds single implantation combined with anlotinib. Ki67 immunohistochemistry, TUNEL immunofluorescence and CD31 morphometric analysis were used to determine the proliferation index, rate of apoptotic cells and microvessel density, respectively. Additionally, the side effects on the skeletal system in mice treated with I-125 seeds implantation were evaluated by histomorphometric staining with tartrate-resistant acid phosphate (TRAP) and alkaline phosphatase (ALP) expression in femur, tartrate-resistant acid phosphatase 5b (TRACP-5b) and procollagen type I N-terminal propeptide (PINP) levels in serum were evaluated by enzyme-linked immunosorbent assay (ELISA). Results The I-125 seeds single and fractionated implantation had similar therapeutic effects and complications when the total number of I-125 seeds was the same. A single implantation of I-125 seeds with or without anlotinib could analogously inhibit the tumor growth in xenografts mice, while the single implantation combined with anlotinib had more effective in tumor inhibition. The results of Ki67, TUNEL and CD31 staining confirmed an evident reduction in tumor cell proliferation and angiogenesis, as well as an increase in apoptosis. A relatively integrated bone metabolism was indicated after I-125 seeds single implantation with or without anlotinib, and the results were similar in I-125 seeds fractional implantation, including a reduction in the number of TRAP-positive cells and an increase in ALP expression level. Additionally, the serum TRACP-5b activity was decreased and the serum PINP concentration was increased following I-125 seeds implantation. Conclusions Single and fractionated implantation pattern of I-125 radioactive seeds had similar therapeutic efficacy against tumor growth, while brachytherapy with I-125 seeds implantation may be an effective and safe treatment strategy for its potential protection against cancer treatment-induced bone loss.

Peripartum changes in serum activities of three major alkaline phosphatase isoenzymes in Holstein dairy cows.

This study investigated changes in serum levels of hepatic, bone, and intestinal alkaline phosphatase (ALP) isoenzymes (ALP2, ALP3, and ALP5, respectively) in Holstein cows around parturition. Tartrate-resistant acid phosphatase 5b (TRAP5b) activity and calcium (Ca) concen-trations were also measured. We analyzed blood samples from 11 late-pregnant heifers (primipa-rous group) and 13 multiparous (2-4 lactations; multiparous group) cows at 3 weeks (18-24 days prepartum; -3 weeks), 2 weeks (17-11 days prepartum; -2 weeks), and 1 week (10-4 days prepar-tum; -1 weeks) before parturition; the day of calving (within 12 h post-calving; day 0); and 5 days postpartum (5 days). ALP3 activity was significantly higher in the primiparous group than in the multiparous group, whereas the activities decreased significantly in both groups after 5 days. ALP2 and ALP5 activities did not change, whereas ALP2 activity was significantly higher in the primiparous group than in the multiparous group. TRAP5b activity was significantly higher in the primiparous group than in the multiparous group and showed a transient significant increase at day 0. Ca concentration significantly decreased at day 0 in both groups; the Ca level at day 0 was significantly higher in the primiparous group than in the multiparous group. These data show that ALP3 activity in serum may indicate a change in osteoblastic bone forma-tion after calving, but further study is needed to determine the clinical application for measuring ALP isoenzymes in bovine medicine.

Sodium alendronate loaded poly(l-lactide- co-glycolide) microparticles immobilized on ceramic scaffolds for local treatment of bone defects

Bone tissue regeneration in critical-size defects is possible after implantation of a 3D scaffold and can be additionally enhanced once the scaffold is enriched with drugs or other factors supporting bone remodelling and healing. Sodium alendronate (Aln), a widely used anti-osteoporosis drug, exhibits strong inhibitory effect on bone resorption performed by osteoclasts. Thus, we propose a new approach for the treatment of bone defects in craniofacial region combining biocompatible titanium dioxide scaffolds and poly(l-lactide-co-glycolide) microparticles (MPs) loaded with Aln. The MPs were effectively attached to the surface of the scaffolds’ pore walls by human recombinant collagen. Drug release from the scaffolds was characterized by initial burst (24 ± 6% of the drug released within first 24 h) followed by a sustained release phase (on average 5 µg of Aln released per day from Day 3 to Day 18). In vitro tests evidenced that Aln at concentrations of 5 and 2.5 µg/ml was not cytotoxic for MG-63 osteoblast-like cells (viability between 81 ± 6% and 98 ± 3% of control), but it prevented RANKL-induced formation of osteoclast-like cells from macrophages derived from peripheral blood mononuclear cells, as shown by reduced fusion capability and decreased tartrate-resistant acid phosphatase 5b activity (56 ± 5% reduction in comparison to control after 8 days of culture). Results show that it is feasible to design the scaffolds providing required doses of Aln inhibiting osteoclastogenesis, reducing osteoclast activity, but not affecting osteoblast functions, which may be beneficial in the treatment of critical-size bone tissue defects.

Ginsenoside Rg3 Attenuates Aluminum-Induced Osteoporosis Through Regulation of Oxidative Stress and Bone Metabolism in Rats.

Aluminum (Al)-induced bone metabolism disorder is a primary cause of osteoporosis. Ginsenoside Rg3 (Rg3) has demonstrated therapeutic properties in the treatment of osteoporosis. The present study aimed to identify potential bone protection mechanisms of Rg3 against Al-induced osteoporosis in rats. In this study, forty healthy male Sprague-Dawley rats were randomly allocated into groups in which they were treated with AlCl3 (64mg/kg/day) and/or Rg3 (20mg/kg/day). AlCl3 was given orally to rats for 120days, and from the 91st day, treated orally with Rg3 for 30days. Rg3 attenuated AlCl3-induced accumulation of Al by decreasing the bone mineral density in the lumbar spines, femoral metaphysis, and tibia, and inhibited AlCl3-induced oxidative stress in rat bone by decreasing the levels of reactive oxygen species and malondialdehyde, while increasing glutathione peroxidase and superoxide dismutase activity. Rg3 facilitated bone formation by increasing the concentration of calcium, phosphorus, amino-terminal propeptide of type I procollagen, and carboxyl-terminal propeptide of type I procollagen, bone alkaline phosphatase activity in serum, and type I collagen, osteocalcin, and osteopontin protein expressions. Rg3 inhibited bone resorption by decreasing the content of N-terminal cross-linking telopeptide of type I collagen, C-terminal cross-linking telopeptide of type I collagen, and tartrate-resistant acid phosphatase 5b activity in serum. Rg3 promoted the mRNA expression of growth regulation factors by increasing transforming growth factor-β1, bone morphogenetic protein-2, insulin-like growth factor I, and core-binding factor α1. The results demonstrate that Rg3 can significantly attenuate Al accumulation, facilitate bone formation, inhibit bone resorption, resist oxidative stress, and promote the expression of factors that regulate growth. The results indicate that Rg3 is effective in alleviating AlCl3-induced osteoporosis.

Manipulation of osteoclastogenesis: Bioactive multiphasic silica/collagen composites and their effects of surface and degradation products

The intent of the present study was to demonstrate that multiphasic silica/collagen xerogels are able to manipulate cellular processes. These xerogels were prepared by a sol-gel approach allowing the incorporation of mineral phases. The resulting nanocomposites are designed as biomaterial for bone regeneration. Human osteoclasts derived from peripheral blood mononuclear cells were cultured both indirectly and directly, either in presence of different xerogel types or on their surface, to investigate the factor with the main influence on osteoclastogenesis. To this end, the incorporation of a third phase to silica/collagen xerogels was used to affect osteoclastogenesis. In cell culture, ambient ion conditions controlled by both the degradation products of the xerogel and the bioactivity-dependent ion release and reprecipitation were shown to have the main effect on osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP) 5b. Late stage of osteoclastogenesis characterized by resorption was strongly dependent on the xerogels composition. Surface chemistry of the xerogels was displayed to play an important role in osteoclast resorption. Biphasic silica/collagen xerogels and triphasic xerogels with calcium carbonate offered widespread resorbed areas, whereas hydroxyapatite containing xerogels showed distinctly reduced resorption. The incorporation of strontium carbonate and phosphate, respectively, as third phase changed TRAP 5b activity dose-dependently and inhibited resorption within 21 days. Quantitative evaluation on osteoclast differentiation was carried out using biochemical methods (TRAP 5b, cathepsin K) and was supported by confocal laser scanning microscopy and scanning electron microscopy (SEM). Qualitative estimation of resorption was carried out by SEM.

Pain prediction by serum biomarkers of bone turnover in people with knee osteoarthritis: an observational study of TRAcP5b and cathepsin K in OA

To investigate serum biomarkers, tartrate resistant acid phosphatase 5b (TRAcP5b) and cathepsin K (cath-K), indicative of osteoclastic bone resorption, and their relationship to pain and pain change in knee osteoarthritis (OA). Sera and clinical data were collected from 129 people (97 with 3-year follow-up) with knee OA from the Prediction of Osteoarthritis Progression (POP) cohort. Knee OA-related outcomes in POP included: WOMAC pain, National Health and Nutrition Examination Survey (NHANES) I (pain, aching and stiffness), subchondral sclerosis, and radiographically determined tibiofemoral and patellofemoral OA. Two putative osteoclast biomarkers were measured in sera: TRAcP5b and cath-K. Medial tibia plateaux were donated at knee arthroplasty for symptomatic OA (n=84) or from 16 post mortem (PM) controls from the Arthritis Research UK (ARUK) Pain Centre joint tissue repository. Osteoclasts were stained for tartrate resistant acid phosphatase (TRAcP) within the subchondral bone of the medial tibia plateaux. Serum TRAcP5b activity, but not cath-K-immunoreactivity, was associated with density of TRAcP-positive osteoclasts in the subchondral bone of medial tibia plateaux. TRAcP-positive osteoclasts were more abundant in people with symptomatic OA compared to controls. Serum TRAcP5b activity was associated with baseline pain and pain change. Our observations support a role for subchondral osteoclast activity in the generation of OA pain. Serum TRAcP5b might be a clinically relevant biomarker of disease activity in OA.

Manipulation of In Vitro Osteoclastogenesis: Bioactive Multiphasic Silica/Collagen Composites and their Effects of Surface and Degradation Products

The intent of the present study was to demonstrate that multiphasic silica/collagen xerogels are able to manipulate cellular processes. These xerogels were prepared by a sol-gel approach allowing the incorporation of mineral phases. The resulting nanocomposites are designed as biomaterial for bone regeneration. Human osteoclasts derived from peripheral blood mononuclear cells were cultured both indirectly and directly, either in presence of different xerogel types or on their surface, to investigate the factor with the main influence on osteoclastogenesis. To this end, the incorporation of a third phase to silica/collagen xerogels was used to affect osteoclastogenesis. In cell culture, ambient ion conditions controlled by both the degradation products of the xerogel and the bioactivity-dependent ion release and reprecipitation were shown to have the main effect on osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP) 5b. Late stage of osteoclastogenesis characterized by resorption was strongly dependent on the xerogels composition. Surface chemistry of the xerogels was displayed to play an important role in osteoclast resorption. Biphasic silica/collagen xerogels and triphasic xerogels with calcium carbonate offered widespread resorbed areas, whereas hydroxyapatite containing xerogels showed distinctly reduced resorption. The incorporation of strontium carbonate and phosphate, respectively, as third phase changed TRAP 5b activity dose-dependently and inhibited resorption within 21 days. Quantitative evaluation on osteoclast differentiation was carried out using biochemical methods (TRAP 5b, cathepsin K) and was supported by confocal laser scanning microscopy and scanning electron microscopy (SEM). Qualitative estimation of resorption was carried out by SEM.

Serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity as a biomarker for bone metastasis in non-small cell lung cancer patients

Background Diagnosis and follow-up of bone metastasis (BMet) in non-small cell lung cancer (NSCLC) patients usually rely on symptoms and image studies. A serum marker of bone resorption may improve the quality of treatment in such patients. Tartrate-resistant acid phosphatase 5b (TRACP5b) is a specific marker for osteoclasts and we proposed it can be used as a marker of BMet in NSCLC patients. Methods In November 2002 till August 2008 serum samples were obtained from 141 newly diagnosed stage IIIA, IIIB or IV NSCLC patients and 41 normal subjects. All patients received baseline bone scintinography examination and evaluation of clinical symptoms as a standard of BMet diagnosis. Patients were divided into 2 groups by having BMet (Group I, n = 72) or not (Group II, n = 69). An in-house immunoassay using a TRACP-specific monoclonal antibody, 14G6, was used to measure the serum TRACP5b activity at pH 6.1. Results The mean serum TRACP5b activities of Group I, Group II and normal subjects were 3.50 ± 2.23 U/l, 2.09 ± 0.72 U/l and 2.33 ± 0.52 U/l, respectively. After adjusting for age, stage, gender, and histology in a generalized linear model, Group I has significantly higher TRACP5b activity than Group II ( p < 0.001). The receiver operating characteristic analysis established a cutoff value of 2.551 U/l to identify BMet in NSCLC patients with a sensitivity of 63.9% and a specificity of 76.8%. TRACP5b activity declined in patients who responded to treatment ( p = 0.047), and elevated in patients who developed new BMet ( p = 0.05). Conclusions Serum TRACP5b activity test is a potentially useful adjunct in diagnosing and monitoring BMet in NSCLC. Further study is warranted to establish its real value in diagnosis and monitoring of BMet in NSCLC patients.

Serum tartrate-resistant acid phosphatase 5b activity as a prognostic marker of survival in breast cancer with bone metastasis

BackgroundSerum tartrate-resistant acid phosphatase 5b (TRACP 5b) activity is a marker of osteoclast number and is elevated in breast cancer (BC) patients with extensive bone metastasis, which might in turn reflect the tumour burden. We tested the hypothesis that baseline serum TRACP 5b activity and its interval change are potential prognostic markers of survival in BC patients with bone metastasis.MethodsWe analyzed the data from previous prospective studies. A total of 100 patients with newly diagnosed bone metastasis were included. Cox proportional regression model was used to evaluate the correlation between the overall survival time (OS) and baseline serum TRACP 5b activity and its interval changes. The least significant change (LSC) of TRACP 5b was calculated from data obtained from 15 patients with early BC.ResultsEstrogen receptor status (Hazard Ratio (HR) = 0.397; p = 0.003) and visceral metastasis (HR = 0.492; p = 0.0045) were significantly correlated with OS. The OS was significantly shorter in those patients with higher baseline TRACP 5b activity based on a cut-off value to delineate the highest tertile (HR = 3.524; p < 0.0001). Further analysis demonstrated that among patients in the highest tertile, OS was significantly longer in those patients who had achieved a decrease of serum TRACP 5b activity greater than the LSC (38.59%) (p = 0.0015).ConclusionsWe found that TRACP 5b activity and its interval change after treatment bore a prognostic role in BC patients with bone metastasis and a high baseline serum TRACP 5b activity. Further prospective phase II study is necessary to confirm these results.

Tartrate-resistant acid phosphatase isoform 5a as an inflammation marker in end-stage renal disease

End-stage renal disease (ESRD) is often complicated by chronic inflammation and malnutrition. We tested whether serum tartrate-resistant acid phosphatase (TRACP) isoform 5a relates to other markers of inflammation in ESRD. Predialysis serum was collected from 99 ESRD patients (51 male, 48 female) aged 55 +/- 15 years and a control group of 36 healthy subjects (8 male, 28 female) aged 43.2 +/- 10.5 years. Serum TRACP 5a activity and protein, TRACP 5b activity and C-reactive protein (CRP) were estimated by in-house immunoassays. Commercial kits were used for serum bone-specific alkaline phosphatase, Ntelopeptides of Type I collagen, interleukin-6 (IL-6) and fetuin-A. Intact parathyroid hormone was determined by chemiluminescent assay. Albumin, cholesterol, triglycerides, ferritin and hemoglobin were compared to the hospital reference ranges. Bone mineral density (BMD) was measured at the heel in 69 patients and all control subjects and expressed as g/cm2 and age-corrected T-score. Mean (median) levels of all serum markers were significantly elevated in ESRD except fetuin-A, which was significantly reduced. Mean BMD (g/cm2) was not different than control, but mean T-score was significantly reduced. TRACP 5a protein correlated with CRP, triglycerides and ferritin, but not with IL-6 or any other nutritional or bone markers or BMD. TRACP 5b activity correlated with all bone markers and BMD, but not with inflammation or nutritional markers. Our findings suggest that TRACP 5a may be a useful marker to estimate the degree of inflammation in ESRD patients on chronic hemodialysis.

The Semiquantitative Bone Scintigraphy Index Correlates With Serum Tartrate-Resistant Acid Phosphatase Activity in Breast Cancer Patients With Bone Metastasis

To determine if a correlation exists between the semiquantitative bone scintigraphy index (SQBSI) and serum tartrateresistant acid phosphatase 5b (TRACP5b) activity, a novel osteoclast marker that has been shown to be useful for monitoring bone metastasis in breast cancer (BC) patients. Among patients enrolled in 2 prospective studies conducted at Tri-Service General Hospital, Taipei, Taiwan, between December 2000 and July 2002, we identified post hoc 52 patients with both BC and bone metastasis who had detailed records of clinical condition, bone scintigraphy, and concordant serum TRACP5b levels. Between January 1, 2003, and December 31, 2005, we performed bone scintigraphy and serum TRACP5b activity assays to monitor these patients, while they were treated according to clinical need. To assess clinical condition, we obtained information from patient records, such as performance status and visual analogue pain score, as well as from selected laboratory tests for tumor markers and serum TRACP5b activity. Those patients with BC and bone metastasis who had undergone whole-body bone scintigraphy and serum TRACP5b activity determination before any therapeutic intervention were designated the pretreated group (n=30). We developed our own formula for calculating SQBSI on the basis of bone scintigraphy findings. A significant correlation was observed between SQBSI and serum TRACP5b activity in pretreated BC patients with bone metastasis, but the strength of the correlation lessened after treatment. No significant correlation was noted between the change in serum TRACP5b activity and the change in SQBSI in treated patients. Compared with the change in SQBSI, the change in TRACP5b activity had higher sensitivity, specificity, and positive predictive value as well as a greater likelihood ratio for reflecting the clinical scenarios of bone morbidity over time. As monitors of the response of bone metastasis in BC to treatment, serial determinations of serum TRACP5b activity and SQBSI were both shown to be useful by our preliminary findings. However, serum TRACP5b activity proved the better monitoring tool. If follow-up studies were conducted within 6 months, the combined use of SQBSI and TRACP5b would allow distinction of genuine disease progression from the "flare" phenomenon, in which bone metastasis can appear to progress in bone scintigraphic images although clinical symptoms improve. Larger prospective studies are needed to confirm these findings.

Effects of cis‐9, trans‐11, CLA in rats at intake levels reported for breast‐fed infants

CLA intake in exclusively breast-fed infants is close to levels found to have physiological effects in animals. However, in the majority of studies mixtures of CLA isomers have been used and the independent effects of the major CLA isomer in human milk, cis-9,trans-11 CLA, at the intake level in exclusively breast-fed infants have hardly been studied. We therefore studied the effects of cis-9,trans-11 CLA on plasma lipids and glucose, immune function, and bone metabolism in growing rats. Thirty male Sprague-Dawley rats (n = 10/group) were fed either 20 mg/kg/d cis-9,trans-11 CLA and 20 mg/kg/d sunflower oil (CLA20), 40 mg/kg/d cis-9,trans-11 CLA (CLA40), or 40 mg/kg/d sunflower oil (placebo) for 8 wk. No significant differences between groups were found in plasma lipids, glucose, insulin, C-reactive protein, or lipid peroxidation. Liver fat content was lowest in the CLA20 group. In vitro interleukin 2 (IL-2) production increased, and tumor necrosis factor alpha, IL-1beta, prostaglandin E2, and leukotriene B4 production decreased in the CLA20 group. No differences between groups were detected in IL-4, IL-6, or interferon gamma production, plasma osteocalcin, insulin-like growth factor, or urinary deoxypyridinoline crosslinks. Plasma tartrate-resistant acid phosphatase 5b activity was significantly increased in the CLA40 group. The results indicate anti-inflammatory effects and enhanced T-cell function for the CLA20 group. No adverse effects were seen in the CLA20 group, whereas indications of increased bone resorption rate were observed in the CLA40 group.

Tartrate-Resistant Acid Phosphatase 5b Activity Is a Useful Bone Marker for Monitoring Bone Metastases in Breast Cancer Patients after Treatment

Metabolic markers of bone metabolism may be useful for the diagnosis and monitoring of bone metastasis in breast cancer patients. Serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity is a novel bone resorption marker. The treatment response of serum TRACP5b activity, bone alkaline phosphatase (BAP) activity, and concentrations of NH(2)-terminal telopeptide of type 1 collagen (NTX) in 68 breast cancer patients with bone metastasis were determined. These patients were treated and followed up as clinically indicated. Fifty-four healthy women were recruited as control. Serum TRACP5b activity, BAP activity, and NTX level of breast cancer patients with bone metastasis were significantly higher than those of normal controls. In normal subjects, serum TRACP5b activity and NTX level are significantly correlated (P < 0.0001). Neither was correlated with BAP activity. In breast cancer patients with bone metastasis, all marker pairs correlated to each other significantly (P < 0.0001). Biomarkers were examined repeatedly in 38 patients who were evaluable for treatment response. Based on clinical criteria, 20 patients were responders and 18 were nonresponders. In the 20 responders, serum TRACP5b activity and NTX level decreased significantly (P < 0.0001 and 0.0107, respectively) after treatment. In the 18 nonresponders, only NTX level showed significant increase (P = 0.0342) after treatment; TRACP5b and BAP were unchanged. By means of multiple logistic regression with stepwise selection, we determined that TRACP5b activity has a higher probability than NTX level to indicate treatment response as a function of percent change after treatment (18 times versus 12 times). Our data support the use of either TRACP5b activity or NTX level to follow up breast cancer patients with bone metastasis after treatment instead of the prevailing BAP activity.

Tartrate-Resistant Acid Phosphatase 5b is a Useful Serum Marker for Extensive Bone Metastasis in Breast Cancer Patients

Previous studies showed that serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity was increased in 70% to 94% of breast cancer (BC) patients with bone metastasis (BM). This study aims to determine whether serum TRACP5b is useful for identifying limited or extensive BM in BC patients. Serum TRACP5b activity was measured in 168 BC patients, including 81 who were newly diagnosed with early BC, 20 with extraosseous metastasis, 24 with limited BM, and 43 with extensive BM. Serum TRACP5b activity was also measured monthly in 151 patients with early BC until they developed BM. Four hundred and twenty-seven (427) healthy women ages 18 to 90 served as control. One-way ANOVA was used to compare serum TRACP5b among groups. The sensitivity and specificity of serum TRACP5b as a marker for BM were estimated by receiver operator characteristic (ROC) curves. Serum TRACP5b increased with age in healthy women ( P < 0.0001). It was significantly elevated in patients with extensive BM compared with all other groups ( P < 0.0001). ROC analysis established a cutoff value of 4.026 units/L to identify patients with extensive BM with a specificity of 98% and a sensitivity of 93% (area under the curve = 0.9807; 95% CI = 0.9698-0.9915). Among the 151 patients with early BC, 6 developed limited BM and 2 developed extensive BM during the follow-up period. Serum TRACP5b remained below the cutoff value in patients with limited BM, but became significantly increased in those whose BM became extensive. Serum TRACP5b activity is a useful diagnostic marker for extensive BM in patients with BC.

The association of bone metabolism with bone mineral density, serum sex hormone concentrations, and regular exercise in middle-aged men

Physical activity is an important factor in attaining bone mass. Our aim was to investigate if low to moderate intensity exercise affects bone resorption [serum tartrate-resistant acid phosphatase (TRAP) 5b activity] and formation (serum osteocalcin concentration) in a randomized controlled exercise intervention trial in Finnish middle-aged men. In addition, the relations of these bone turnover markers with bone mineral density (BMD) and serum sex hormone concentrations [circulating testosterone (T), estradiol (E 2), and sex hormone-binding globulin (SHBG) concentrations] were evaluated. Serum TRAP 5b activity and osteocalcin concentration were measured at randomization and after 1 and 4 years of the exercise intervention. BMDs of the lumbar spine (L2–L4), femoral neck, and total proximal femur were measured with a dual-energy X-ray absorptiometry (DXA). At randomization, TRAP 5b activity was strongly correlated with the osteocalcin concentration (Spearman r = 0.541, P < 0.0001). In addition, TRAP 5b activity was significantly correlated with proximal femur BMD values ( r = −0.201, P = 0.018) and osteocalcin concentration with femoral neck and proximal femur BMD values ( r = −0.187, P = 0.028; r = −0.240, P = 0.005, respectively). Serum E 2, free E 2, and free T concentrations were inversely correlated with both bone turnover markers. After 1 year of exercise intervention, TRAP 5b activity was significantly lower in the exercise than reference group ( P = 0.006). However, after 4 years of exercise intervention, the difference was no longer statistically significant. There were no differences in the osteocalcin concentrations between the study groups during the intervention. Our results show a connection between serum TRAP 5b activity and osteocalcin concentration. Furthermore, our results suggest that low to moderate exercise intervention and serum sex hormone concentrations may induce changes in bone metabolism in middle-aged men. However, exercise-induced effects on bone metabolism should be confirmed in other randomized controlled exercise trials taking into account exercise intensity and dose–response issues.

Alternative immunoassay for tartrate-resistant acid phosphatase isoform 5b using the fluorogenic substrate naphthol ASBI-phosphate and heparin

Objective: Our purpose was to develop a specific immunoassay for tartrate-resistant acid phosphatase (TRACP) 5b using naphthol ASBI phosphate (N-ASBI-P) as a selective substrate for isoform 5b and heparin as a selective inhibitor of isoform 5a. Methods: Serum TRACP 5a and 5b and recombinant TRACP 5a were used to optimize and standardize the immunoassay for specificity, linearity, analytical recovery, sensitivity and reproducibility. Serum N-telopeptide cross-links (NTX) and bone alkaline phosphatase (bone ALP) were also measured. The clinical sensitivity and specificity were assessed in healthy control subjects and patients with osteoarthritis (OA), rheumatoid arthritis (RA) and endstage renal disease (ESRD). Results: TRACP 5b specificity was achieved at pH 6.3 with 2.5 mmol/l substrate and 25 U/ml heparin. Isoform 5b specificity was increased over our original immunoassay using 4-nitrophenyl phosphate (4-NPP) without heparin. The alternative immunoassay was linear with 110% analytical recovery and no serum matrix effects. The average intra-assay error was 10.65%; the average inter-assay error was 10.11% for values of 1–3 U/l and 6.5% for values of 7–11 U/l. Mean serum TRACP 5b in OA and RA were not significantly different from control using either immunoassay. Mean serum TRACP 5b was significantly increased in ESRD with both immunoassays. Serum TRACP 5b levels correlated significantly with NTX and bone ALP in the disease groups, but not always in the control group. Conclusion: This alternative immunoassay for TRACP 5b activity is highly specific. It should have applications in evaluating patients with bone disease and will improve our understanding of the biological significance of TRACP 5b expression in health and disease.

Serum tartrate-resistant acid phosphatase 5b, but not 5a, correlates with other markers of bone turnover and bone mineral density.

Human serum contains two isoforms of tartrate-resistant acid phosphatase (TRACP) known as TRACP 5a and TRACP 5b with pH optima of 5.0 and 5.8, respectively. Preliminary data suggest that serum TRACP 5b is derived from osteoclasts and serum TRACP 5a from some other cells. It has been reported that heparin inhibits TRACP 5a but has no effect on the activity of TRACP 5b. Here we show that heparin has no effect on serum TRACP activity, as determined using our previously published immunoassay, suggesting that the immunoassay does not detect TRACP 5a. The change of serum TRACP 5b activity after 6 months HRT, determined by this immunoassay, correlated significantly with the changes of all markers of bone turnover determined, including serum N- and C-terminal propeptides of type I collagen and urinary-free deoxypyridinoline. Serum TRACP 5b activity was significantly elevated in patients with osteoporosis and had a significant negative correlation with bone mineral density (BMD). Serum TRACP 5a activity, determined by an immunoassay, showed no correlation with serum TRACP 5b activity, with BMD, or with any of the markers of bone turnover. These results show that serum TRACP 5b, but not 5a, reflects the bone resorption rate, and that our TRACP 5b immunoassay may be a specific method for the determination of the bone resorption rate from serum samples.

Naphthol-ASBI Phosphate as a Preferred Substrate for Tartrate-Resistant Acid Phosphatase Isoform 5b

Tartrate-resistant acid phosphatase (TRAP) isoform 5b is a potential serum marker for osteoclastic activity. Biochemical assays for serum TRAP activity with para-nitrophenylphosphate (pNPP) have low specificity for bone because of hydrolysis by unrelated nontype 5 TRAPs of blood cells and by related isoform 5a. Our purpose was to increase the specificity of TRAP assay for osteoclastic activity by using naphthol-ASBI phosphate (N-ASBI-P) as a substrate for serum type 5 TRAP activity and heparin as an inhibitor of isoform 5a. TRAP activity in individual and pooled sera of normal subjects and patients with endstage renal disease (ESRD) and rheumatologic diseases was quantitated using pNPP and N-ASBI-P as substrate at pH 5.5 and 6.1. For some experiments, heparin (23U/ml) was added as a specific inhibitor of isoform 5a activity. Isoforms 5a and 5b were separated from serum pools by cation exchange chromatography and identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). N-ASBI-P was selectively hydrolyzed by TRAP isoform 5b. TRAP assays with pNPP and N-ASBI-P correlated only in ESRD sera, which contained primarily isoform 5b. The two assays did not correlate in normal or rheumatic sera with significant amounts of 5a. Heparin inhibited isoform 5a activity approximately 50% but had little effect on isoform 5b activity. Biochemical assay of serum TRAP activity can be made specific for isoform 5b by using N-ASBI-P and heparin. This method can be adapted to simple microplate biochemical or immunochemical assays. This simplified method for assessment of osteoclastic TRAP 5b activity warrants a detailed investigation in diseases of bone metabolism.

Rapid Screening Method for Osteoclast Differentiation in Vitro That Measures Tartrate-resistant Acid Phosphatase 5b Activity Secreted into the Culture Medium

Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) 5b into the circulation. We studied the release of TRAP 5b from osteoclasts using a mouse in vitro osteoclast differentiation assay. We developed and characterized a polyclonal antiserum in rabbits, using purified human osteoclastic TRAP 5b as antigen. The antiserum was specific for TRAP in Western analysis of mouse osteoclast culture medium and was used to develop an immunoassay. We cultured mouse bone marrow-derived osteoclast precursor cells for 3-7 days with or without clodronate in the presence of vitamin D and analyzed the number of osteoclasts formed and the amount of TRAP 5b activity released into the culture medium. TRAP 5b activity was not secreted from osteoclast precursor cells. Addition of clodronate-containing liposomes decreased in a dose-dependent manner the number of osteoclasts and TRAP 5b activity released in 6-day cultures. The amount of TRAP 5b activity in the medium detected by the immunoassay correlated significantly with the number of osteoclasts formed (r = 0.94; P<0.0001; n = 120). The TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts in the osteoclast differentiation assay and to test the effects of potential therapeutic agents on osteoclast differentiation, enabling fast screening of large amounts of potential therapeutic agents.