Serum Tartrate-resistant Acid Phosphatase 5b Is a Specific and Sensitive Marker of Bone Resorption

Bone-resorbing osteoclasts and activated macrophages express high amounts of type 5 tartrate-resistant acid phosphatase (TRACP; EC 3.1.3.2) (1). Osteoclasts secrete TRACP into the circulation, and serum TRACP has been considered a potentially useful marker of bone resorption. Several technical problems have prevented the use of serum TRACP as a specific marker of bone resorption. Early enzymatic assays lacked specificity because of the presence of interfering acid phosphatases derived from platelets and erythrocytes (2)(3). Serum TRACP can be determined by use of fluoride (4) or by immunoassays with antibodies specific for TRACP (5)(6)(7)(8)(9). However, there are two forms of TRACP in the serum, namely TRACP 5a and TRACP 5b. Of these, TRACP 5b is derived from osteoclasts, whereas TRACP 5a originates from other, as yet unidentified sources (10)(11)(12). Two diagnostic assays have been developed recently for serum TRACP 5b: a kinetic assay based on the use of specific inhibitors (13), and an immunoassay based on a highly characterized specific monoclonal antibody (12). The TRACP 5b-specific immunoassay has been shown to be a useful method for monitoring antiresorptive therapy (12). We have now further characterized the immunoassay by studying the clinical specificity and clinical sensitivity of serum TRACP 5b in various bone diseases and nonskeletal diseases. We obtained serum samples from 303 individuals after written informed consent. At the time of sampling, none of the subjects was receiving antiosteoporotic treatment. Healthy premenopausal women, healthy postmenopausal women, and breast cancer (BC) patients without evidence of bone metastases (BC−) were included as control populations. Patients with bone diseases included subjects with primary vertebral osteoporosis (OPO), osteopenia, active Paget disease of bone, and BC patients with overt bone metastases (BC+). All patients with OPO had at least one vertebral compression fracture. Patients with chronic hepatic dysfunction (HF; n = 17), chronic renal failure (RF; n = 49), and rheumatoid arthritis (RA; n = 40) were included as reference populations. All patients with RF had endstage renal disease, and they had been on chronic hemodialysis for several years (mean duration, 5 years) at the time of sample collection. The study was approved by the local ethics committees and performed in accordance with the Declaration of Helsinki. Serum TRACP 5b activity was measured with a solid-phase immunofixed enzyme activity assay as described (12), using TRACP 5b purified from human osteoclasts as a calibrator (7). The distribution of TRACP 5b activity was within reference values in the control groups, and statistical analysis was performed using one-way ANOVA. The interassay CV was 2.2%, and the intraassay CV was 1.8%. The reference interval (mean ± 2 SD of healthy premenopausal women) of TRACP 5b activity in serum was 0.50–3.80 U/L. Compared with healthy premenopausal women, serum TRACP 5b activity was significantly higher (P <0.001) in healthy postmenopausal women and in all groups of patients with bone diseases (Table 1 and Fig. 1A ). The clinical specificity of TRACP 5b (percentage of healthy premenopausal women with TRACP 5b activity within reference values) was 95.0%, and the clinical sensitivity (percentage of individuals in a disease group with TRACP 5b activity above reference values) was 48.3% in osteopenia, 81.3% in OPO, 71.4% in Paget disease, and 80.0% in BC+ subjects (Fig. 1A ). Serum TRACP 5b activity in healthy women and in patients with metabolic bone diseases (mean ± SD). Serum TRACP 5b activity in healthy women and in patients with metabolic bone diseases (mean ± SD). Serum TRACP 5b in bone diseases and nonskeletal diseases. (A), serum TRACP 5b activity in healthy women and patients with metabolic bone diseases. Pre, premenopausal; Post, postmenopausal; OPE, osteopenia; PD, Paget disease. (B), serum TRACP 5b, bone sialoprotein (BSP), CTX, and NTX in BC patients. (C), serum TRACP 5b activity (5b), total TRACP mass (Total), and NTX in patients with nonskeletal diseases. The results in B and C are given as relative amounts, where the upper limit of normal for each marker is 1. The upper limit of normal is the mean + 2 SD of premenopausal women in A and C and the mean + 2 SD of BC− in B. The lines show the upper limits of normal. The clinical sensitivity in BC+ was substantially higher for TRACP 5b than for other serum markers of bone resorption (Fig. 1B ), including cross-linked N- and C-terminal telopeptides of type I collagen (NTX and CTX, respectively) and bone sialoprotein. Concentrations of all markers in the BC− group were within the reference interval. Reference values for all markers in this study were obtained from measurements of healthy premenopausal women and were comparable to published reference values. Two enzymatic pathways are involved in osteoclastic bone resorption, one involving cathepsin K and releasing NTX and CTX from bone collagen, and the other involving matrix metalloproteinases without releasing NTX or CTX. The substantially lower clinical sensitivity of NTX and CTX compared with TRACP 5b in BC+ patients suggests that the increased bone resorption in BC+ patients is primarily mediated by matrix metalloproteinases. Serum markers of bone resorption are accumulated in the circulation of patients with HF and RF (14). Mean TRACP 5b activity was not increased in patients with HF or RF, whereas mean NTX was significantly increased in both diseases (P <0.001) and were above the upper limit of normal in 58.8% of patients with HF and 93.2% of patients with RF. Total TRACP mass [measured as described previously (15)] was significantly increased (P <0.001) and above the upper limit of normal in 51.0% of the RF subjects (Fig. 1C ). These results suggest that the function of the liver and the kidneys has no effect on circulating TRACP 5b activity. Although the mean TRACP 5b activity was within the reference interval in RF patients, a subpopulation (12.5%) had TRACP 5b activity above the upper limit of normal, suggesting that they have increased bone resorption. This is not surprising because RF often is associated with secondary hyperparathyroidism and renal osteodystrophy. Greatly increased mean total serum TRACP mass in patients with RF suggests that enzymatically active TRACP 5b molecules are inactivated and degraded into fragments before they are removed from the circulation. Thus, inactive TRACP fragments may be accumulated in a manner similar to other markers. TRACP released into the uterine fluid of pigs is rapidly inactivated and degraded into fragments by proteases (16). Approximately 90% of serum TRACP circulates as fragments (7)(8), suggesting that a similar rapid inactivation and degradation may occur for osteoclast-derived TRACP 5b in serum, as has been suggested previously (17). In addition to osteoclasts, activated macrophages express high amounts of TRACP (1), suggesting that TRACP released from macrophages may affect the specificity of our immunoassay. We show here that in patients with RA, a disease associated with increased activity of macrophages, total serum TRACP was substantially increased and above the upper limit of normal in 35% of the subjects (Fig. 1C ). Instead, neither the resorption marker NTX nor TRACP 5b was increased, suggesting that bone resorption was not increased. These findings suggest that the observed high total serum TRACP in RA patients may be attributable to increased secretion of TRACP from cells other than the osteoclasts, possibly activated macrophages, that are not detected by the immunoassay measuring TRACP 5b. We conclude that serum TRACP 5b is greatly increased in patients with bone diseases. The most pronounced changes were observed in patients with BC+ and OPO. Data from this study and from a previous study (12) suggest that serum TRACP 5b is a sensitive and specific marker of bone resorption that can be used in the diagnosis and follow-up of skeletal pathologies. Serum TRACP 5b may be derived exclusively from osteoclasts, and it may not accumulate in the circulation in RF or HF patients. Larger studies are needed to confirm the clinical relevance of serum TRACP 5b measurements and their predictive power regarding future bone loss and fractures. This work was supported by the Academy of Finland, the State Technology Development Center of Finland, and the Graduate School of Musculo-Skeletal Diseases.