RESUMO
UNLABELLED: Sagopilone, a fully synthetic epothilone and very potent anti-tumor agent, has proved to be efficient in inhibiting bone destruction and tumor burden in a mouse model of breast cancer bone metastasis. In addition to its antiproliferative effects, this study shows direct effects of sagopilone on bone resorption and osteoclast activity. INTRODUCTION: Sagopilone, a novel fully synthetic third-generation epothilone, has proved to be efficient in inhibiting bone destruction and tumor burden in a mouse model of breast cancer bone metastasis. The aim of this study was to investigate whether the effect was primarily due to sagopilone's antiproliferative effect and consequent inhibition of tumor cell growth, or if sagopilone exerts direct effects on bone resorption and osteoclast activity. METHODS: Sagopilone was studied and compared to paclitaxel in vitro in human osteoclast differentiation and activity cultures. For studying the potential of sagopilone for inhibiting bone resorption in vivo, a mouse model of ovariectomy (ovx)-induced osteoporosis was utilized. RESULTS: Sagopilone inhibited osteoclast differentiation and activity more efficiently than paclitaxel and showed less cytotoxicity. Whereas sagopilone showed inhibitory effects on human osteoclast differentiation and activity already at 5 and 15 nM, respectively, paclitaxel started to show effects only at 20 and 100 nM concentrations, respectively. Sagopilone treatment increased BMD In the mouse ovx model even though a non-optimized dose was used which is effective in tumor-bearing mice. CONCLUSION: This is the first study to evaluate sagopilone's effects on bone resorption in non-cancerous situation. The evidence that sagopilone is beneficial for bone will strengthen the status of sagopilone as an anti-cancer compound compared to other microtubule stabilizing agents.
Assuntos
Benzotiazóis/farmacologia , Reabsorção Óssea/tratamento farmacológico , Epotilonas/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Paclitaxel/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Densidade Óssea , Reabsorção Óssea/etiologia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Osteoporose/etiologia , Ovariectomia/efeitos adversosRESUMO
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.
Assuntos
Fosfatase Ácida/sangue , Biomarcadores Tumorais/sangue , Neoplasias Ósseas/sangue , Neoplasias Ósseas/secundário , Neoplasias da Mama/sangue , Isoenzimas/sangue , Fragmentos de Peptídeos/sangue , Pró-Colágeno/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/sangue , Neoplasias Ósseas/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Colágeno Tipo I , Estudos Transversais , Feminino , Humanos , Pessoa de Meia-Idade , Peptídeos , Sensibilidade e Especificidade , Fosfatase Ácida Resistente a TartaratoRESUMO
Skeletal metastases are a significant problem in prostate cancer (PC). The patients are also exposed to treatment-related skeletal changes. This cross-sectional study evaluated a marker of bone resorption, TRACP 5b in relation to the standard analyte total alkaline phosphatase (tALP) as a marker of skeletal changes. Serum levels of TRACP 5b, tALP and PSA were measured in 130 prostate cancer patients. Comparison was made between patients with (BM+, n = 25) and without (BM-, n = 105) skeletal metastases, and between those treated with (n = 64) or without (n = 66) androgen deprivation (AD). Sensitivities and specificities were calculated for each marker and diagnostic accuracy was evaluated by ROC curve analysis. ROC curves indicated the superior accuracy of tALP, whereas TRACP 5b and PSA were comparable. With tALP the best combination of sensitivity (96%) and specificity of (91%) was reached at a cut-off point 224 U/L, the corresponding values were for TRACP 5b sensitivity (76%), specificity (89%) with a cut-off point 4.89 U/L, and for PSA sensitivity (65%), specificity (81%) at 23 ng/L for skeletal metastases. Patients treated with AD showed with increasing duration an increase in TRACP 5b values. TRACP 5b was less specific than tALP as a marker of skeletal metastases. TRACP 5b may have a role in the diagnostics of skeletal changes in PC with a focus on treatment-related skeletal changes.
Assuntos
Fosfatase Ácida/sangue , Biomarcadores Tumorais/sangue , Neoplasias Ósseas/enzimologia , Isoenzimas/sangue , Neoplasias da Próstata/enzimologia , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/sangue , Antagonistas de Androgênios/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Reabsorção Óssea , Estudos Transversais , Humanos , Masculino , Pessoa de Meia-Idade , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Curva ROC , Sensibilidade e Especificidade , Fosfatase Ácida Resistente a TartaratoRESUMO
Tartrate-resistant acid phosphatase (TRACP) is produced by macrophages and other cells of the monohistiocytic lineage. In particular, osteoclasts are characterized for a high expression of this enzyme. Yet, several data suggest that other bone cell types, such as osteocytes and osteoblasts, may also express activity of this enzyme. This is particularly obvious at sites were osteoclasts resorb bone, suggesting that osteoclasts (or their precursors) somehow induce TRACP activity in osteoblasts. In the present study, we investigated this by culturing human osteoblast-like cells with and without conditioned medium (MCM) from human blood monocytes (as a source of osteoclast precursors). High levels of TRACP activity were found in osteoblast-like cells cultured with MCM. Depletion of TRACP from this medium resulted in the absence of its activity in osteoblast-like cells, thus suggesting that the TRACP activity in these cells was the result of endocytosed TRACP that was released by the monocytes in the MCM. Osteoblast-like cells cultured in control (non-conditioned) medium contained very low levels of TRACP-like activity. However, the cells expressed TRACP mRNA and incubation of extracts of these cells with active cathepsin B did induce activity of a TRACP-like enzyme. Inhibition of the activity of cysteine proteinases in general and of cathepsin B in particular, completely blocked TRACP activity of the osteoblast-like cells. This TRACP-like enzyme but not the alleged endocytosed fraction of TRACP was inhibited by fluoride, suggesting that the fractions may be different isoenzymes. Our data seem to indicate that osteoblast-like cells may contain two different fractions of TRACP, one that is released by monocytes and subsequently endocytosed by osteoblast-like cells and a second endogenous fraction that is present in an inactive proform. We hypothesize that the capacity of osteoblast-like cells to endocytose TRACP is important for the removal of this enzyme during or following the bone resorptive activity of the osteoclast.
Assuntos
Fosfatase Ácida/metabolismo , Endocitose/fisiologia , Expressão Gênica/genética , Isoenzimas/metabolismo , Osteoblastos/enzimologia , Fosfatase Ácida/efeitos dos fármacos , Fosfatase Ácida/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Reabsorção Óssea/metabolismo , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsina B/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/farmacologia , Meios de Cultivo Condicionados/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Vesículas Citoplasmáticas/química , Dipeptídeos/farmacologia , Ativação Enzimática , Humanos , Isoenzimas/efeitos dos fármacos , Isoenzimas/genética , Modelos Biológicos , Monócitos/citologia , Monócitos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Fluoreto de Sódio/farmacologia , Fosfatase Ácida Resistente a TartaratoRESUMO
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.
Assuntos
Fosfatase Ácida/sangue , Densidade Óssea/fisiologia , Osso e Ossos/metabolismo , Isoenzimas/sangue , Fosfatase Ácida/antagonistas & inibidores , Biomarcadores/sangue , Feminino , Heparina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/antagonistas & inibidores , Cinética , Pessoa de Meia-Idade , Pós-Menopausa , Análise de Regressão , Fosfatase Ácida Resistente a TartaratoRESUMO
Estrogens are important for the male skeleton. Osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), interleukin-6 (IL-6), IL-1 and tumor necrosis factor alpha (TNFalpha) have been suggested to be involved in the skeletal effects of estrogen. We treated orchidectomized mice with estradiol for 2 weeks and observed a 143% increase in the trabecular bone mineral density of the distal metaphysis of femur that was associated with a decreased OPG/RANKL mRNA ratio in vertebral bone. A similar decreased OPG/RANKL ratio was also seen after estrogen treatment of ovariectomized female mice. The effect of estrogen receptor (ER) inactivation on the OPG/RANKL ratio was dissected by using intact male mice lacking ER alpha (ERKO), ER beta (BERKO) or both receptors (DERKO). The expression of OPG was increased in ERKO and DERKO but not in BERKO male mice, resulting in an increased OPG/RANKL ratio. Furthermore, serum levels of IL-6 and tartrate-resistant acid phosphatase 5b (TRAP 5b) were decreased in ERKO and DERKO, but not in BERKO male mice. These results demonstrate that ER alpha, but not ER beta, is involved in the regulation of the vertebral OPG/RANKL ratio, serum levels of IL-6 and TRAP 5b in male mice.
Assuntos
Proteínas de Transporte , Glicoproteínas/metabolismo , Interleucina-6/sangue , Glicoproteínas de Membrana , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Estrogênio/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fosfatase Ácida/sangue , Animais , Biomarcadores/sangue , Densidade Óssea/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Isoenzimas/sangue , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Orquiectomia , Osteoprotegerina , Ovariectomia , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Fosfatase Ácida Resistente a TartaratoRESUMO
There are two known estrogen receptors, estrogen receptor-alpha (ER alpha) and estrogen receptor-beta (ER beta), which may mediate the actions of estrogen. The aim of the present study was to compare fat content, skeletal growth and adult bone metabolism in female mice lacking ER alpha (ERKO), ER beta (BERKO) or both ERs (DERKO). We demonstrate that endogenous estrogens decrease the fat content in female mice via ER alpha and not ER beta. Interestingly, the longitudinal bone growth was decreased in ERKO, increased in BERKO, but was intermediate in DERKO females, demonstrating that ER alpha and ER beta exert opposing effects in the regulation of longitudinal bone growth. The effects on longitudinal bone growth were correlated with similar effects on serum levels of IGF-I. A complex regulation of the trabecular bone mineral density (BMD), probably caused by a disturbed feedback regulation of estrogen and testosterone, was observed in female ER-inactivated mice. Nevertheless, a partial functional redundancy for ER alpha and ER beta in the maintenance of the trabecular BMD was observed in the female mice at 60 days of age. Thus, ER alpha and ER beta may have separate effects (regulation of fat), opposing effects (longitudinal bone growth) or partial redundant effects (trabecular BMD at 60 days of age), depending on which parameter is studied.
Assuntos
Fêmur/metabolismo , Receptores de Estrogênio/metabolismo , Absorciometria de Fóton/métodos , Animais , Constituição Corporal , Densidade Óssea/fisiologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Fêmur/diagnóstico por imagem , Fêmur/crescimento & desenvolvimento , Fator de Crescimento Insulin-Like I/metabolismo , Modelos Lineares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Estrogênio/genética , Tomografia Computadorizada por Raios XAssuntos
Fosfatase Ácida/sangue , Reabsorção Óssea/diagnóstico , Isoenzimas/sangue , Adulto , Idoso , Biomarcadores/sangue , Doenças Ósseas Metabólicas/sangue , Reabsorção Óssea/sangue , Neoplasias da Mama/sangue , Feminino , Humanos , Imunoensaio , Pessoa de Meia-Idade , Osteíte Deformante/sangue , Osteoporose Pós-Menopausa/sangue , Sensibilidade e Especificidade , Fosfatase Ácida Resistente a TartaratoRESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
Assuntos
Fosfatase Ácida/metabolismo , Isoenzimas/metabolismo , Osteoclastos/metabolismo , Fosfatase Ácida/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Soros Imunes , Imunoensaio , Isoenzimas/imunologia , Camundongos , Osteoclastos/citologia , Osteoclastos/enzimologia , Coelhos , Fosfatase Ácida Resistente a TartaratoRESUMO
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.
Assuntos
Fosfatase Ácida/sangue , Reabsorção Óssea/diagnóstico , Terapia de Reposição de Estrogênios , Isoenzimas/sangue , Anticorpos Monoclonais , Biomarcadores/sangue , Reabsorção Óssea/sangue , Reabsorção Óssea/enzimologia , Método Duplo-Cego , Estabilidade Enzimática , Estradiol/uso terapêutico , Feminino , Humanos , Pessoa de Meia-Idade , Neuraminidase , Noretindrona/uso terapêutico , Placebos , Pós-Menopausa , Valores de Referência , Fosfatase Ácida Resistente a TartaratoRESUMO
Osteoclasts are multinucleated cells responsible for bone resorption. They have developed an efficient machinery for dissolving crystalline hydroxyapatite and degrading organic bone matrix rich in collagen fibers. When initiating bone resorption, osteoclasts become polarized, and three distinct membrane domains appear: a ruffled border, a sealing zone and a functional secretory domain. Simultaneously, the cytoskeleton undergoes extensive re-organisation. During this process, the actin cytoskeleton forms an attachment ring at the sealing zone, the membrane domain that anchors the resorbing cell to bone matrix. The ruffled border appears inside the sealing zone, and has several characteristics of late endosomal membrane. Extensive vesicle transport to the ruffled border delivers hydrochloric acid and proteases to an area between the ruffled border and the bone surface called the resorption lacuna. In this extracellular compartment, crystalline hydroxyapatite is dissolved by acid, and a mixture of proteases degrades the organic matrix. The degradation products of collagen and other matrix components are endocytosed, transported through the cell and exocytosed through a functional secretory domain. This transcytotic route allows osteoclasts to remove large amounts of matrix-degradation products without losing their tight attachment to underlying bone. It also facilitates further processing of the degradation products intracellularly during the passage through the cell.
Assuntos
Reabsorção Óssea/patologia , Osteoclastos/citologia , Animais , Transporte Biológico , Matriz Óssea/metabolismo , Movimento Celular , Polaridade Celular , Células Cultivadas , Colágeno/metabolismo , Citoesqueleto/ultraestrutura , Durapatita/metabolismo , Endopeptidases/metabolismo , Humanos , Ácido Clorídrico/metabolismo , Camundongos , Camundongos Knockout , Organelas/fisiologia , Osteoclastos/fisiologia , Bombas de Próton/metabolismo , Espécies Reativas de OxigênioRESUMO
BACKGROUND: Tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) is a product of osteoclasts and a biochemical marker of bone resorption rate. However, erythrocytes and platelets contribute to total TRAP activity in serum, reducing the specificity of direct biochemical assays in serum. Osteoclast TRAP is also known as type-5 TRAP and is antigenically unique. Immunoassays are sought to improve the specificity and sensitivity of TRAP as a bone marker. METHODS: We developed two colorimetric microplate assays for type-5 TRAP: an enzyme capture immunoassay to measure antibody-bound enzymatic activity, and a two-site immunoassay to measure bound enzyme protein. Both use the same monoclonal antibody (14G6) to capture type-5 TRAP, which permits determination of specific activity of serum TRAP in health and disease. RESULTS: Both TRAP assays were linear from one-tenth to fivefold the mean value in 18 healthy subjects. In these subjects, the mean (SD) TRAP activity was 3.2 (0.54) U/L for the enzyme capture assay and 37 (13) microg/L for the two-site assay. Mean TRAP activity was not significantly increased in 64 patients with endstage renal disease requiring hemodialysis (HD) or 99 unselected patients with rheumatic diseases. By contrast, TRAP protein was increased in both the HD and rheumatic disease groups. The specific activity of TRAP in the 17 of 64 HD sera that had increased TRAP activity (0.088 U/microg) was similar to that in healthy subjects (0.091 U/microg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/microg) was significantly decreased. CONCLUSIONS: Wide sample distributions for TRAP activity in HD patients and TRAP protein in rheumatic disease patients suggest the presence of subpopulations of HD patients with increased TRAP activity and of rheumatic patients with increased TRAP protein. Each assay for TRAP activity and protein may have its own biological significance and clinical applications in specific groups of patients.
Assuntos
Fosfatase Ácida/análise , Imunoensaio/métodos , Isoenzimas/análise , Fosfatase Ácida/sangue , Fosfatase Ácida/imunologia , Adulto , Especificidade de Anticorpos , Reabsorção Óssea/sangue , Feminino , Peroxidase do Rábano Silvestre , Humanos , Isoenzimas/sangue , Isoenzimas/imunologia , Falência Renal Crônica/sangue , Masculino , Diálise Renal , Doenças Reumáticas/sangue , Sensibilidade e Especificidade , Fosfatase Ácida Resistente a TartaratoRESUMO
Tartrate-resistant acid phosphatase (TRAP) is highly expressed in bone-resorbing osteoclasts and activated macrophages. It has been suggested that a redox-active iron in the binuclear iron center of TRAP could have the capacity to react with hydrogen peroxide to produce highly destructive reactive oxygen species (ROS). Here we show that TRAP can generate ROS in vitro and that cells over-expressing TRAP produce higher amounts of intracellular ROS than their parent cells. We further demonstrate that these ROS can be targeted to destroy collagen and other proteins. In resorbing osteoclasts, TRAP was found in transcytotic vesicles transporting matrix degradation products through the cell, suggesting that TRAP-facilitated fragmentation of endocytosed material takes place in a specific cellular compartment. These results suggest that bone matrix degradation occurs not only extracellularly in the resorption lacunae but also intracellularly in the transcytotic vesicles. We propose that proteins containing redox-active iron could represent a novel mechanism of physiological fragmentation of organic molecules. This mechanism could be important in tissue remodeling and as a defense mechanism of phagocytosing cells.
Assuntos
Fosfatase Ácida/metabolismo , Reabsorção Óssea , Isoenzimas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Células Cultivadas , Colágeno/metabolismo , Ratos , Fosfatase Ácida Resistente a TartaratoRESUMO
Tartrate-resistant acid phosphatase (TRAP), an enzyme expressed in bone-resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two-site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4 degreesC and -20 degreesC or five thawing-freezing cycles, did not change the TRAP concentration detected using the two-site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1-5C1 and O1A-J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1-5C1 and O1A-J1B may be useful in determining the bone resorption rate.
Assuntos
Fosfatase Ácida/análise , Fosfatase Ácida/imunologia , Anticorpos Monoclonais , Imunoensaio/métodos , Isoenzimas/análise , Isoenzimas/imunologia , Osteoclastos/enzimologia , Fosfatase Ácida/sangue , Adulto , Animais , Reabsorção Óssea/enzimologia , Estabilidade Enzimática , Mapeamento de Epitopos , Feminino , Humanos , Imuno-Histoquímica , Isoenzimas/sangue , Menopausa/metabolismo , Camundongos , Pessoa de Meia-Idade , Fosfatase Ácida Resistente a TartaratoRESUMO
Tartrate-resistant acid phosphatase (TRAP) is an enzyme with unknown biological function. In human tissues, its expression is restricted to bone-resorbing osteoclasts and activated macrophages. Osteoclasts secrete TRAP to the circulation during bone resorption. Reduction of the enzyme's binuclear iron center is important in regulating its activity. The purple form of the enzyme is inactive and contains two ferric ions. Mild reduction activates it to a pink form containing one ferric and one ferrous ion. Instead, strong reduction removes the iron content, resulting in a colorless, inactive enzyme. We describe spontaneous activation of the purple form to the pink form upon incubation at +37 degrees C. Further incubation results in slow inactivation of the enzyme and color change to yellowish. The enzyme purified from osteoclasts is a mixture of the purple and pink forms, but the enzyme purified from serum represents the yellowish form. We suggest that the newly synthesized enzyme is purple and reduced in the cell to the functionally active pink form. After fulfilling its biological function in the cell, the enzyme is further reduced to the yellowish form and secreted into the circulation. In the serum, further reduction would dissociate the iron content. The enzymes from osteoclasts and macrophages had similar catalytic properties, both being active as a protein tyrosine phosphatase (PTPase). The acid phosphatase (AcP) and PTPase activities were similar, and the preferred AcP substrate, pNPP, was processed in the same active site as phosphotyrosine. Our results suggest that redox-regulated PTPase activity may be a major function of TRAP in vivo.
Assuntos
Fosfatase Ácida/metabolismo , Isoenzimas/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Fosfatase Ácida/sangue , Fosfatase Ácida/isolamento & purificação , Animais , Sítios de Ligação , Cor , Ativação Enzimática , Estabilidade Enzimática , Humanos , Isoenzimas/sangue , Isoenzimas/isolamento & purificação , Macrófagos Alveolares/enzimologia , Osteoclastos/enzimologia , Proteínas Tirosina Fosfatases/sangue , Proteínas Tirosina Fosfatases/isolamento & purificação , Ratos , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fosfatase Ácida Resistente a TartaratoRESUMO
Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP) to the circulation, where the amount of TRAP is expected to correlate with the bone resorption rate. We have developed two monoclonal antibodies, O1A and J1B, using purified human bone TRAP as antigen. The antibodies recognized different epitopes, allowing us to develop a two-site fluoroimmunoassay. The immunoreactivity in fresh serum specimens was less than 10% of the concentrations measured from the same specimens after 24 h of storage at 4 degrees C, or after addition of 5 mM EDTA or EGTA to them. When fresh serum was gel filtrated using Sephacryl S-200 column, all of the enzyme eluted in the void volume as a complex with a molecular weight of more than 250 kDa. If the serum was treated with EDTA before the gel filtration, the complex was destroyed and the enzyme eluted in fractions corresponding to a molecular weight of 30 kDa, the size of monomeric purified human bone TRAP. The immunoassay was used to measure TRAP concentrations from serum samples that had been stored at 4 degrees C for 24 h. According to the assay, premenopausal women had 13.1 +/- 3.1, postmenopausal women 17.6 +/- 4.2, and children 32.6 +/- 12.2 microg TRAP/l of serum. We conclude that TRAP circulates in the serum as part of a complex, which also contains Ca2+, and that TRAP-immunoassay is a potentially useful method for determining bone resorption rates, as long as the complex is destroyed before the assay.