Tartrate-resistant Acid Phosphatase Isoform 5b Research Articles

Zoledronic Acid Is an Effective Treatment for Osteoporosis in Patients with Thalassemia Major: Results of a Randomized, Placebo-Controlled, Trial.

Osteoporosis in thalassemia major (TM) is due to several factors, including bone marrow expansion, iron overload, hormonal deficiency, and increased osteoclast activity. Potent inhibitors of osteoclast function, such as bisphosphonates, have been recently used in the management of TM-induced osteoporosis. The aim of this trial was to determine the efficacy and safety of a third-generation aminobisphosphonate, zoledronic acid, on osteoporosis and bone remodeling in patients with TM. Sixty-six patients with TM-induced osteoporosis (21M/45F; median age 35.5 years) have been enrolled in this study. The majority of patients had pathological fractures and/or bone pain due to osteoporosis at baseline. Patients were randomized to receive zoledronic acid at a dose of 4 mg, iv, in 15 min infusion, every 6 months (23 patients; group A) or every 3 months (21 patients; group B), or to receive placebo every 3 months (22 patients; group C), for a period of one year. All patients were under oral calcium (500 mg) administration during the treatment period. Effects were monitored by measuring bone mineral density (BMD) in comparison with a series of serum bone remodeling indices: i) bone resorption markers [C-telopeptide of type-I collagen (CTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)], ii) bone formation markers [bone-alkaline phosphatase (bALP), osteocalcin (OC), and C-terminal propeptide of collagen type-I (CICP)], and iii) osteoclast stimulating factors [receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG)]. BMD of the lumbar spine, femoral neck and forearm was determined using DEXA, before and 12 months after treatment. The above biochemical parameters were measured at baseline and before the administration of zoledronic acid or placebo in each cycle. We also evaluated these markers in 30 healthy, age and gender-matched, controls. All patients had increased values of CTX, TRACP-5b, bALP, CICP, sRANKL, OPG, and sRANKL/OPG ratio, compared with controls (p<0.02). Patients of group A showed no differences in terms of BMD of all three evaluated sites after 12 months of treatment, while they showed a reduction in serum levels of CTX (p=0.02), bALP (p=0.02) and OC (p=0.01). Conversely patients of group B achieved a significant increase in their lumbar spine BMD after 12 months of therapy (p=0.007), which was accompanied by a dramatic decrease in serum CTX (p=0.004), bALP (p<0.0001), CICP (p<0.0001), and OC (p=0.005). Patients of group C showed an aggravation of BMD of the lumbar spine (p=0.041) at 12 months, while there was no alteration in BMD of other sites as well as in markers of bone remodeling. There was no effect of zoledronic acid on sRANKL and OPG in all patients. Zoledronic acid reduced bone pain in patients of groups A and B, while there was no bone pain relief in placebo group. No severe side-effects were observed in this study. We conclude that zoledronic acid, at a dose of 4 mg, iv, every 3 months is an effective treatment in increasing BMD and reducing osteoclast activity and bone resorption in TM-induced osteoporosis. Longer follow-up studies are required to clarify the exact role of zoledronic acid in the management of these patients.

Differential Effects of Sex Hormones on Peri- and Endocortical Bone Surfaces in Pubertal Girls

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. This study examines the association of sex hormones and bone markers with bone geometry and density in pubertal girls. 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. The study was conducted in a university laboratory. A total of 258 10- to 13-yr-old healthy girls at the baseline participated. 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. 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). 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.

Evaluation of the Activity of Tartrate-resistant Acid Phosphatase Isoform 5b in Normal Chinese Children - A Novel Marker for Bone Growth

Most parents are very concerned about the height of their children. Biochemical markers of bone formation and resorption may provide useful clinical predictors for bone growth. Tartrate-resistant acid phosphatase 5b (TRAcP 5b) has been advocated as a biomarker of osteoclast activity and bone resorption. However, the TRAcP 5b levels of children at different ages are still unknown. It is necessary to accumulate and analyze the data for healthy children at different ages. We use an improved immunoassay for bone TRAcP 5b to examine sera from children to see whether it is a significant marker of bone growth. Serum, including cord blood, was collected from 404 normal Chinese children (age range 0-17 years; 225 male, 179 female). The venous blood was withdrawn from the peripheral vein and stored at 4 degrees C before centrifugation for serum collection. All sera were stored at -70 degrees C and thawed at 37 degrees C immediately before TRAcP 5b levels were measured. Bone-specific alkaline phosphatase (BAP) was also used for comparison with TRAcP 5b levels at different ages. TRAcP 5b levels were extremely high in infants of both genders, gradually decreasing with age (p <0.001). A second peak in TRAcP 5b values occurred at 12-13 and 10-11 years in males and females, respectively (p <0.001). Age alone, as well as age-related changes between the male and female groups, were independent predictors of TRAcP 5b levels (p <0.001). There was no significant between-gender difference in serum TRAcP 5b levels for any age group (p = 0.682). BAP values did not show a significant second peak in females. Age and gender alone, as well as the age-related changes between male and female groups, were independent predictors of BAP values (p <0.001). Preliminary results were established for serum TRAcP 5b and BAP values of normal Chinese children of different ages. Elevated serum TRAcP 5b values were observed during infancy and puberty for both genders. The pattern of this age-related change in serum TRAcP 5b levels is similar to the shape of the standard height velocity curve for healthy children. Values of BAP were less specific than TRAcP 5b. These data may prove valuable as a normal reference in future research about bone markers.

Long Term Treatment with Hydroxyurea Does Not Prevent Development of Renal Dysfunction and Osteodystrophy in Patients with Sickle Cell Disease.

Hydroxyurea (HU) is presently considered as the main treatment for the reduction of sickle-cell crises; however, information regarding the potential of HU to inhibit progressive organ failure is scarce. The gradually failing renal function and renal osteodystrophy are well known complications of sickle cell disease (SCD). The pending question is whether administration of hydroxyurea over very long period of time may delay or prevent the appearance of these abnormalities. To this effect we evaluated the renal function and bone metabolism in 57 patients with HbS/β-thal (31M/26F; median age 32 years, range: 19–67 years) receiving hydroxyurea (usually 1 g/daily) continuously for 1 to 14 years (median 10.2 years). In addition to conventional renal biochemistry we measured the levels of serum and urinary β2-microglobulin (β2M), serum cystatin C (specific and sensitive index of glomerular filtration rate), and urine N-acetyl-b-D-glucosaminidase (NAG; reflecting the distal tubular cells function). The extent of renal osteodystrophy was evaluated by DEXA scans assessing bone mineral density (BMD), and by assaying various markers of (a) osteoclast function [soluble receptor activator of nuclear factor κB ligand (sRANKL), osteoprotegerin (OPG), and tartrate resistant acid phosphatase isoform 5b (TRACP-5b)], (b) bone resorption [C-telopeptide of collagen type I (CTX)], and (c) bone formation [bone-alkaline phosphatase (bALP) and osteocalcin (OC)]. The above parameters were also evaluated in 16 age- and gender-matched controls. Three patients (5.2%) had increased serum creatinine levels; 16 patients (28%) had more than 300 mg/day protein excretion in the urine, and 13 patients (22.8%) had microalbuminuria. Moreover, serum cystatin C was elevated in 16 patients (28%), NAG in 21 (36.8%), serum β2M in 34 (59.6%) and urinary β2M in one patient. In addition, 17 patients (29.8%) had osteoporosis/osteopenia in DEXA scans (comparable to HbS/β-thal patients who did not receive HU). Furthermore, all patients displayed significantly elevated levels of OPG (p=.001), sRANKL (p<.01), sRANKL/OPG ratio (p=.022), and CTX (p=.02), while significant correlations were found between serum cystatin C vs. both serum OPG and β2M levels as well as between cystatin C and the sRANKL/OPG ratio. Not only do these results suggest that HU does not prevent renal dysfunction in this cohort of patients but also highlight the role of RANKL/OPG pathway in the renal-induced bone disease of sickle cell syndromes. Furthermore, NAG, cystatin C and OPG may be useful as early biochemical markers for the assessment of renal impairment in SCD patients.

Role of receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin and macrophage protein 1-alpha (MIP-1a) in monoclonal gammopathy of undetermined significance (MGUS).

The aim of this study was to evaluate the role of markers of bone remodelling, and osteoclast activation/function in patients with monoclonal gammopathy of undetermined significance (MGUS). We have measured serum levels of soluble RANKL (sRANKL), osteoprotegerin (OPG), macrophage inflammatory protein-1alpha (MIP-1alpha), markers of bone resorption [N-telopeptide of collagen type-I (NTX), and tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)] and bone formation [bone-alkaline phosphatase (bALP)] in 40 MGUS patients. These parameters were compared with those of 42 newly diagnosed myeloma patients, and 45 healthy, gender- and age-matched controls. MGUS patients had elevated levels of NTX, sRANKL, and sRANKL/OPG ratio compared with controls (P < 0.0001). Furthermore, TRACP-5b, MIP-1alpha and NTX were decreased in patients with MGUS compared with myeloma patients (P < 0.001), while OPG and bALP were increased (P < 0.001). Serum levels of MIP-1alpha, as well as TRACP-5b, and sRANKL/OPG ratio were reduced, while bALP was increased in MGUS patients, even when compared with myeloma patients who had stage I/II disease. These results demonstrate that increased osteoclastogenesis leading to increased bone resorption is present in MGUS but seems to be compensated for by normal bone formation, which is absent in MM. Furthermore MIP-1alpha, bALP, and sRANKL/OPG may be useful tools for distinguishing between cases of MGUS and early myeloma.

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.

Clinical performance of immunoreactive tartrate-resistant acid phosphatase isoform 5b as a marker of bone resorption

Previous immunoassays developed for the measurement of serum tartrate-resistant acid phosphatase (TRACP) have lacked specificity for osteoclastic TRACP, TRACP 5b, or have not shown satisfactory clinical performance. The aim of this study was to evaluate the clinical performance of a novel immunocapture activity assay for TRACP 5b, in comparison to telopeptide fragments of type I collagen. Within-subject variability and the effect of feeding on TRACP 5b and telopeptides of type I collagen were assessed in 20 healthy premenopausal women. Diurnal variation of TRACP 5b and serum β C-terminal cross-linked telopeptide of type I collagen (sβCTX) was assessed in 12 healthy postmenopausal women. Renal clearance was assessed in 19 end stage renal failure patients undergoing routine haemodialysis. Response to antiresorptive treatment and calcium supplementation was assessed in osteoporotic postmenopausal women treated with alendronate and calcium ( n = 16) or with calcium alone ( n = 7) for 24 weeks. Within-subject variability (CV i) of TRACP 5b was 6.6%, lower than CV i of urinary and serum telopeptides. TRACP 5b decreased by 2.4 ± 0.8%, in response to feeding ( P < 0.05) compared to 7.0 ± 2.6% to 7.9 ± 3.7% for urinary telopeptides ( P < 0.05 to < 0.01) and 8.5 ± 1.7% to 17.8 ± 2.6% for serum telopeptides ( P < 0.0001). The amplitude of the diurnal rhythm for TRACP 5b was small compared to that of sβCTX, 14 ± 4% vs. 137 ± 14%. Haemodialysis did not have a significant effect on TRACP 5b but reduced sβCTX by 46 ± 4% ( P < 0.0001). In response to alendronate, TRACP 5b decreased by 39 ± 4% compared to 49 ± 4% to 69 ± 5% for urinary telopeptides and 75 ± 8% for sβCTX. We conclude that TRACP 5b shows an attenuated response to antiresorptive therapy in comparison with other markers of bone resorption, but that this may be offset by lower biological variability. TRACP 5b may provide useful additional information about bone resorption.

Pamidronate is an effective treatment for osteoporosis in patients with beta-thalassaemia.

Osteoporosis in beta-thalassaemia is multifactorial; increased osteoclast function seems to play an important role in its pathogenesis. The aim of this study was to evaluate the effect of pamidronate on the osteoporosis of thalassaemia. To this effect we studied 26 patients who received this drug in doses of 30 or 60 mg i.v. once a month over 12 months. The effects were monitored by measuring bone mineral density (BMD) in association with markers of osteoclast function [soluble receptor activator of nuclear factor-kappa B ligand (sRANKL), osteoprotegerin (OPG)] and of bone remodelling [N-telopeptide of collagen type-I (NTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b), bone-alkaline phosphatase (bALP), and osteocalcin (OC)]. Thirty healthy individuals were also studied, as controls. NTX, TRACP-5b, bALP and OC levels were significantly higher in thalassaemic patients compared with controls; in contrast, OPG levels were significantly lower, while the levels of sRANKL varied within normal limits. Administration of pamidronate was followed by a clear decrease of NTX, TRACP-5b, OPG, and OC, and by a significant increase in the BMD of the lumbar spine, which was similar in patients of both treatment groups. These data suggest that pamidronate, at a monthly dose of 30 mg, is an effective treatment for thalassaemic osteoporosis.

Unusual association between increased bone resorption and presence of paroxysmal nocturnal hemoglobinuria phenotype in multiple myeloma.

Paroxysmal nocturnal hemoglobinuria (PNH) clones deficient in glycosylphosphatidylinositol-anchored molecules, including CD55 and CD59, have been previously described in patients with multiple myeloma (MM). The aim of this study was to investigate the possible association between existence of the PNH phenotype and myeloma bone disease. Forty-three patients with newly diagnosed MM were the subjects of the study. Radiographic evaluation of the skeleton was performed in all patients at diagnosis. The following biochemical markers were measured: bone resorption markers (tartrate-resistant acid phosphatase isoform 5b [TRACP-5b]and N-terminal cross-linking telopeptide of type-I collagen [NTX]), bone formation markers (bone alkaline phosphatase [bALP] and osteocalcin [OC]), osteoprotegerin (OPG), soluble receptor activator of nuclear factor KB ligand (sRANKL), and interleukin 6 (IL-6). Detection of CD55- and/or CD59-deficient red cell populations was performed after diagnosis. Patients with MM had elevated mean baseline NTX, TRACP-5b, sRANKL, and IL-6 levels compared with controls, whereas the mean values of bALP, OC, and OPG were significantly decreased. Four patients had no osteolytic lesions, whereas 8 patients had 1 to 3 lytic lesions, and 31 patients had more than 3 lytic lesions and/or pathologic fractures in the skeletal survey. CD55- and/or CD59-deficient red cell populations were observed in 56% of patients with MM. There was a strong correlation between the presence of PNH-like erythrocytes and increased bone resorption, as measured by NTX, TRACP-5b, and sRANKL/OPG ratio (P < .03, P < .02, and P < .02, respectively). There was also a significant correlation between PNH phenotype and severe bone disease (P < .02). These results suggest that there is a possible link between PNH phenotype and increased osteoclastic activity in MM owing to a potential effect of myeloma microenvironment on a preexisting PNH clone. Further studies are required for clarifying this phenomenon and investigating possible mechanisms of this unusual association.

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.

Tartrate-resistant Acid Phosphatase Isoform 5b as Serum Marker for Osteoclastic Activity

Tartrate-resistant acid phosphatase (AcP) 5b is a marker of osteoclastic activity and bone resorption. Immunoassays for serum TRAcP may lack sensitivity and specificity because of the presence of non-bone isoform 5a. The purpose of this study was to isolate the serum isoforms, quantify their disease-related expressions, and test an improved immunoassay for TRAcP 5b. We separated TRAcP isoforms chromatographically from pooled sera of healthy, rheumatoid arthritis (RA) and endstage renal disease (ESRD) subjects. TRAcP isoforms were identified by electrophoresis and quantified by biochemical and immunochemical assays. Serum TRAcP activity in healthy, RA, and ESRD cohorts was assessed at pH 5.5 and 6.1, and compared with bone alkaline phosphatase (BAP) and N-telopeptides of type I collagen (NTx). TRAcP isoforms 5a and 5b were present in all sera; 5b was identical to osteoclastic TRAcP. In serum from healthy subjects, 5a accounted for 87% of the enzyme protein but only 55% of the activity. In RA, both isoforms were increased two- to threefold in protein, but their specific activities were subnormal. In ESRD, only 5b was abnormal, being increased fivefold in protein and threefold in activity. In RA sera, TRAcP activity did not correlate with either BAP or NTx. In ESRD sera, TRAcP activity correlated with BAP and NTx only when measured at pH 6.1. All sera contained both TRAcP isoforms 5a and 5b, but only 5b was present in bone. TRAcP isoform expression was variable in different diseases. Measurement of TRAcP activity at pH 6.1 improves the specificity of immunoassay for isoform 5b.