Applications and performance of monoclonal antibodies to human tartrate resistant acid phosphatase.

BACKGROUND: Tartrate-resistant acid phosphatase (TRACP) is an enzyme common to cells of the mononuclear phagocyte system and a clinically relevant biomarker for osteoclasts and inflammatory macrophages. The purpose was to assess applications and performance of six anti-TRACP monoclonal antibodies. METHODS: Mab9C5, 14G6, 162, 203, 220, and 89 were used as capture and detection antibodies in quantitative immunoassay, and for western blot (WB), immunoprecipitation, and immunohistochemistry of paraffin sections containing chronic inflammatory infiltrates. The clinical performance of mab14G6 for immunoassay of serum TRACP5b activity was compared to two commercial kit methods. RESULTS: Mab9C5 is useful for WB and immunohistochemistry methods only. Mab14G6, 162, and 203 are useful for quantitative immunoassay and immunoprecipitation, however, mab203 causes inactivation of enzymatic activity. Mab220 and 89 are specific for TRACP5a and useful in all applications. Mab14G6 has similar clinical sensitivity and specificity as two commercial methods. CONCLUSIONS: TRACP is an important marker in osteoimmunology. Specific antibodies with unique specificity for TRACP isoforms and defined applications will be valuable for clinical evaluation of bone metabolic, inflammatory and autoimmune diseases and will aid in basic research of TRACP biochemistry and biology.

Serum tartrate-resistant acid phosphatase isoform 5a (TRACP5a) as a potential risk marker in cardiovascular disease.

OBJECTIVE: This study was undertaken to determine the association between serum tartrate-resistant acid phosphatase 5a (TRACP5a) and cardiovascular disease (CVD) risk. METHODS: Four hundred patients were enrolled including, 291 asymptomatic subjects grouped by the number of traditional risk factors, 36 patients undergoing cardiac arteriography, 34 undergoing percutaneous cardiac intervention, and 39 with acute myocardial infarction. Serum was collected at baseline and, in arteriograpy and intervention groups, periodically for 1 week afterward. In addition to laboratory and clinical evaluation for risk assessment, serum TRACP5a, C-reactive protein (CRP) and interleukin-6 (IL-6) were determined. RESULTS: All biomarkers rose with increasing CVD risk. Only serum TRACP5a, logCRP and cholesterol were elevated in symptomatic patients. Serum TRACP5a was higher in men and correlated with age, logCRP, logIL-6 and log-triglycerides, and in symptomatic patients, with the number of diseased coronary arteries. IL-6 and CRP showed acute phase responses, whereas TRACP5a did not change over 1 week after arteriography or intervention. After adjustment for all other variables and risk factors, TRACP5a and logCRP were the only biomarkers to associate with symptomatic disease. TRACP5a was more specific than CRP to predict myocardial infarction among all subjects. CONCLUSIONS: Serum TRACP5a is a macrophage-derived inflammation marker associated with CVD risk, and with coronary vessel disease and its severity and may be a useful marker for screening and assessment of CVD risk.

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

BACKGROUND: Serum 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. METHODS: We 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. RESULTS: Estrogen 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). CONCLUSIONS: We 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.

Biology and clinical significance of tartrate-resistant acid phosphatases: new perspectives on an old enzyme.

Type 5 tartrate-resistant acid phosphatase (TRAP) has been a clinically relevant biomarker for about 50 years. It has always been a reliable and specific cytochemical marker for hairy cell leukemia and for differentiated cells of monocytic lineage. Only recently has the test for serum TRAP activity been accepted as sensitive and specific enough for clinical use as a marker of osteoclasts and bone resorption. This has come about through steady advances in knowledge about TRAP enzymology, structure, function, and molecular regulation and a consequent appreciation that TRAP isoforms 5a and 5b have very different clinical significance. As a measure of osteoclast number and bone resorption, TRAP 5b has diagnostic and prognostic applications in osteoporosis, cancers with bone metastasis, chronic renal failure, and perhaps other metabolic and pathologic bone diseases. Serum TRAP 5a, on the other hand, has no relationship to bone metabolism but seems instead to be a measure of activated macrophages and chronic inflammation. Exploration of the real clinical usefulness of serum TRAP 5a for diagnosis and disease management in a wide variety of chronic inflammatory diseases is only now beginning. This perspective traces the important basic scientific developments that have led up to the refinement of serum TRAP isoform immunoassays and their validation as biomarkers of disease. Many unanswered questions remain, providing a wealth of opportunity for continued research of this multifaceted enzyme.

Clinical performance of six different serum tartrate-resistant acid phosphatase assays for monitoring alendronate treatment.

Two forms of tartrate-resistant acid phosphatase (TRACP) circulate in human blood, TRACP 5a derived from inflammatory macrophages and TRACP 5b derived from osteoclasts. We compared the clinical performance of the following TRACP immunoassays for monitoring alendronate treatment in postmenopausal women: 1) TRACP 5b activity using a selective pH; 2) TRACP 5b activity using a selective substrate; 3) Total TRACP activity; 4) Total TRACP protein amount; 5) TRACP 5a activity; 6) TRACP 5a protein amount. TRACP and other bone turnover markers were measured before the start of treatment and at 3 months. Alendronate treatment decreased TRACP values determined with assays 1, 2 and 3, and had no effect on the values determined with assays 4, 5 and 6. Clinical performance of assays 1, 2 and 3 was good, and these assays correlated with each other and with the other bone markers. This study showed that TRACP 5b specific methods are useful for monitoring changes in bone resorption during alendronate treatment, and alendronate treatment does not affect serum TRACP 5a levels.

Significance of serum TRACP in rheumatoid arthritis.

Human serum contains two related isoforms of TRACP: TRACP 5a and TRACP 5b. Serum TRACP 5a protein is increased in about one third of rheumatoid arthritis (RA) sera. This study was undertaken to examine the significance of serum TRACP isoforms 5a and 5b as disease markers of inflammation and bone destruction in RA. One hundred eighteen patients were recruited including 50 with RA (25 with nodules), 26 with osteoarthritis (OA), and 42 with other rheumatic diseases. Twenty-six healthy adults served as controls. Serum TRACP 5a activity, TRACP 5a protein, and TRACP 5b activity were determined by in-house immunoassays. C-reactive protein (CRP) was determined by in-house immunoassay using commercial antibodies and CRP. Other commercial markers included bone-specific alkaline phosphatase (BALP), C-telopeptides of type-I collagen (ICTP), cartilage glycoprotein-39 (YKL-40), and IgM rheumatoid factors (IgM-RF). Mean TRACP 5a protein was significantly elevated only in RA compared with healthy controls and other disease groups. TRACP 5a protein correlated significantly only with IgM-RF in RA. Among RA patients, mean TRACP 5a protein and IgM RF were significantly higher in nodule formers. In contrast, TRACP 5b activity was slightly elevated in RA and correlated with BALP, ICTP, and YKL-40 but not with IgM-RF or CRP. Mean TRACP 5b activity was no different in RA patients with or without nodules. TRACP isoforms could be useful disease markers in RA; TRACP 5a protein may be a measure of systemic inflammatory macrophage burden and disease severity. TRACP 5b activity is a marker for osteoclast number and perhaps local or systemic bone destruction.

The semiquantitative bone scintigraphy index correlates with serum tartrate-resistant acid phosphatase activity in breast cancer patients with bone metastasis.

OBJECTIVE: 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. PARTICIPANTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Tartrate-resistant acid phosphatase as an immunohistochemical marker for inflammatory macrophages.

Human serum contains 2 isoforms of type-5 tartrate-resistant acid phosphatase (TRACP): 5a and 5b. TRACP-5b is osteoclastic. Our goal was to determine if serum TRACP-5a could originate from inflammatory macrophages (MPhi). We stained 246 paraffin-embedded tissue samples for TRACP using monoclonal antibody 9C5 (mab9C5) to isoforms 5a and 5b and a novel mab220 specific to isoform 5a. CD68 and lysozyme were also stained. MPhi of chronic and granulomatous inflammation and in tissues that undergo strong antigenic stimulation were strongly positive for TRACP, more so with mab220 than with mab9C5. Noninflammatory MPhi in lymph node sinuses or germinal centers and red pulp MPhi of spleen were weak or negative for TRACP. Marginal zone lymphocytes and sebaceous glands of skin were weakly positive for TRACP. Tissue mast cells displayed strong TRACP staining. Neuroendocrine cells of gastrointestinal tissues were strongly immunoreactive with mab9C5 but negative with mab220. Restricted expression of TRACP primarily in inflammatory MPhi supports our hypothesis that circulating TRACP-5a could be a biomarker of chronic inflammatory disease activity.

Effects of calcitriol on type 5b tartrate-resistant acid phosphatase and interleukin-6 in secondary hyperparathyroidism.

BACKGROUND/AIMS: Secondary hyperparathyroidism (SHP) is characterized by high bone turnover and elevated serum bone remodeling markers. Elevation of serum interleukin-6 (IL-6) levels is also characteristic of end-stage renal disease. This study investigates the effects of intravenous calcitriol on serum bone resorptive markers, namely, type 5b tartrate-resistant acid phosphatase (TRACP5b) and IL-6 in patients with SHP. METHODS: Intravenous calcitriol therapy was given for 16 weeks to 24 patients on maintenance hemodialysis with plasma intact parathyroid hormone (iPTH) levels >300 pg/ml. Blood was drawn at baseline and every 4 weeks for 16 weeks for determination of the levels of biochemical parameters, iPTH, IL-6 and bone remodeling markers, including bone-specific alkaline phosphatase (bAP) and TRACP5b. RESULTS: Only 21 patients responded to the calcitriol therapy, with significant decrements in serum iPTH after 4 weeks of therapy and thereafter. After 16 weeks of calcitriol therapy, 21 patients had significant decrements in serum iPTH (707.9 +/- 317.8 vs. 205.0 +/- 63.1 pg/ml, p < 0.01). Prior to treatment, a significant correlation was found between increased levels of serum iPTH and IL-6 levels (r = 0.45, p < 0.05). After treatment, there was also a significant and parallel lowering of levels of serum iPTH, IL-6 (8.52 +/- 3.59 vs. 7.24 +/- 2.81 pg/ml, p < 0.01), bAP (54.68 +/- 36.17 vs. 24.55 +/- 13.84 U/l, p < 0.01) and TRACP5b (3.41 +/- 1.89 vs. 1.80 +/- 0.55 U/l, p < 0.01). Our results additionally showed significant positive correlationsbetween baseline levels of serum IL-6 and those of iPTH, bAP and TRACP5b. After 16 weeks of calcitriol treatment, the correlation between IL-6 and iPTH levels lost significance but levels of serum IL-6, bAP and TRACP5b remained significantly correlated. CONCLUSIONS: Elevated levels of serum IL-6 and bone remodeling markers, namely, bAP and TRACP5b which are common features of SHP, are effectively suppressed by calcitriol therapy. This indicates that hyperparathyroidism not only accelerates bone remodeling but may also aggravate inflammation in patients on maintenance hemodialysis.

Development of immunoassays for serum tartrate-resistant acid phosphatase isoform 5a.

BACKGROUND: Serum tartrate-resistant acid phosphatase (TRACP) consists of 2 structurally related isoforms, TRACP 5a and 5b. TRACP 5b is from bone-resorbing osteoclasts. TRACP 5a may be a macrophage product of inflammation. We used a novel antibody to TRACP 5a to standardize immunoassays for serum TRACP 5a activity and protein. METHODS: Biotinylated anti-TRACP antibodies were used to immobilize serum TRACP isoforms. TRACP activity was measured using 4-nitrophenyl phosphate as substrate. TRACP 5a protein was measured with an independent peroxidase-conjugated anti-TRACP antibody. Immunoassays were standardized for linearity of serum dose response, sensitivity and precision. Reference ranges for TRACP 5a were established from serum of 50 healthy males and 50 healthy age-matched females. Serum TRACP 5a activity and protein were determined in 29 cases of rheumatoid arthritis. RESULTS: Serum matrix interference in both TRACP 5a assays required dilution to 10% serum to approach linearity. Intra-assay and inter-assay CV% were <10%. Mean serum TRACP 5a activity and protein were significantly higher in healthy men than women. There was a slight, but significant age related increase in both serum TRACP 5a and 5b among females, but not males, from age 20 to 70 years. TRACP 5a activity was positively correlated to TRACP 5a protein in healthy sera. Neither TRACP 5a activity nor protein was correlated strongly to TRACP-5b activity. TRACP 5a protein was significantly increased in 8/29 RA sera, whereas TRACP 5a and 5b activities were not. TRACP 5a activity and protein were not significantly correlated in RA sera. CONCLUSIONS: Although TRACP 5a and 5b are related biosynthetically, their circulating levels in healthy humans were independent, suggesting differential regulation of expression. In chronic diseases, increased TRACP 5a may represent pathological processes of inflammation unrelated to bone metabolism.

Tartrate-resistant acid phosphatase 5b is a useful serum marker for extensive bone metastasis in breast cancer patients.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSION: Serum TRACP5b activity is a useful diagnostic marker for extensive BM in patients with BC.

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

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

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.

Tartrate-resistant acid phosphatase 5b as a serum marker of bone metastasis in breast cancer patients.

Diagnosis and follow-up of bone metastases in breast cancer patients usually rely on symptoms and imaging studies. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is a specific marker of osteoclasts and is herein proposed as a marker of bone metastasis in breast cancer patients. An immunoassay using a monoclonal antibody, 14G6, was used to measure the activity of serum TRACP 5b at pH 6.1 in 30 early breast cancer patients without bone metastasis and in 30 aged-matched breast cancer patients with bone metastasis. Another 60 normal volunteers were recruited as controls. Bone alkaline phosphatase (BAP), a traditional marker of bone turnover, was also measured in selected cases. The overall mean TRACP 5b activity in normal women was 2.83 +/- 1.1 U/l, and it increased with age. The mean TRACP 5b activity in early breast cancer patients did not differ from that of the normal group (2.93 +/- 0.64 vs. 2.83 +/- 1.1 U/l; p = 0.66), whereas it was significantly higher in breast cancer patients with bone metastasis (5.42 +/- 2.5 vs. 2.83 +/- 1.1 U/l; p < 0.0001). BAP activity was significantly higher in breast cancer patients with bone metastasis than in early breast cancer patients (p = 0.004). Serum TRACP 5b activity correlated well with BAP activity in breast cancer patients with bone metastasis (p < 0.0001), but not in normal individuals or in patients without bone metastasis. TRACP 5b activity can be considered a surrogate indicator of bone metastasis in breast cancer patients.

Disease-specific expression of tartrate-resistant acid phosphatase isoforms.

The association between elevated serum type 5 TRACP activity and metabolic bone diseases has been recognized for many years. However, serum type 5 TRACP exists as two related isoforms: 5a and 5b. Only isoform 5b is osteoclast-derived; the origin and significance of isoform 5a has hardly been explored. We have used simultaneous immunoassays for type-5 TRACP activity and total type-5 TRACP protein in conjunction with non-denaturing gel electrophoresis and column chromatography to investigate the nature and significance of TRACP isoforms 5a and 5b in end-stage renal disease (ESRD) and rheumatoid arthritis (RA). Our studies have shown that TRACP activity and protein are elevated in approximately 50% of sera from ESRD patients, which is caused by osteoclastic isoform 5b. We have also shown that total TRACP protein, but not TRACP activity, is elevated in approximately 30% of sera from RA patients, which is caused by non-osteoclastic isoform 5a. When macrophages or dendritic cells (DC) were cultured in vitro, abundant TRACP 5a was secreted into the culture medium, whereas TRACP 5b was retained intracellularly by both cell types. This implicates macrophages and DC as potential sources of elevated TRACP 5a in RA. Because TRACP isoform expression may be disease-specific, it is important to be able to distinguish TRACP 5a from 5b. There are four criteria by which to do so: (1) TRACP 5a bears sialic acid residues while TRACP 5b does not; (2) the pH optimum for TRACP 5a is 5.2 while that for TRACP 5b is 5.8; (3) the specific activity of TRACP 5a is significantly lower than that of TRACP 5b; and (4) TRACP 5a is as an uncleaved polypeptide, whereas TRACP 5b is a proteolytically nicked disulfide-linked "heterodimer." The differences in biochemical properties and disease-specific expression of TRACP isoforms 5a and 5b suggest that they are regulated differently and perform separate functions in a tissue-specific manner.

Flow cytoenzymology of intracellular tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 x 10(5)/100 micro l test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia.

Correlation between histomorphometric parameters of bone resorption and serum type 5b tartrate-resistant acid phosphatase in uremic patients on maintenance hemodialysis.

BACKGROUND: Serum tartrate-resistant acid phosphatase 5b (TRACP) is a new marker of potential clinical use to monitor osteoclastic activity and bone resorption rate. The relationship between histomorphometric parameters of bone resorption and serum TRACP was evaluated in 14 chronically dialyzed patients and 6 healthy control subjects. METHODS: All patients underwent bone biopsies and serum biochemical testing for TRACP, intact parathyroid hormone (iPTH), pyridinoline cross-linked telopeptide domain of type I collagen (ICTP), total calcium, phosphorus, and albumin, which were measured at the time of biopsy. RESULTS: Bone histological examination showed predominant hyperparathyroid bone disease (HPT) in 6 patients, mixed uremic osteodystrophy in 3 patients, low-turnover osteomalacia in 1 patient, and adynamic bone disease in 4 patients. Mean TRACP activity was 3.25 +/- 0.59 U/L in control subjects. Median TRACP activity was significantly greater in patients with HPT (11.97 +/- 8.92 U/L) than those with other types of renal osteodystrophy (ROD; 2.17 +/- 0.61 U/L). Serum iPTH levels were greatest in all patients with HPT, but also were significantly elevated in 7 of 8 patients with other types of ROD. Serum ICTP levels also were significantly elevated in all patients with HPT and 6 of 8 patients with other types of ROD. Serum TRACP levels correlated more strongly with histological parameters of osteoclasts than those of erosion. Also, correlations between TRACP and histological parameters of osteoclasts were stronger than those of iPTH and ICTP levels. CONCLUSION: These early results suggest that serum TRACP levels correlate well with histological indices of osteoclasts and may serve as a specific marker for osteoclastic activity in patients with renal bone disease.

Stable expression of human tartrate-resistant acid phosphatase isoforms by CHO cells.

OBJECTIVE: In human serum, type-5 tartrate-resistant acid phosphatase (TRACP) exists as two closely related isoforms: 5a and 5b. Serum isoform 5b is an osteoclast product that reflects bone resorption rate and is frequently increased in diseases of increased bone turnover. Isoform 5a protein is often increased in rheumatoid arthritis (RA) sera and may be a product of inflammatory macrophages. Our objective was to compare the biochemical characteristics of TRACP 5a and 5b. METHODS: We transfected the human ACP 5 gene into CHO cells and cloned a stable cell line (CHO/TRACP 8F5) that expresses high levels of TRACP activity both intracellularly and as a secreted product. Both enzyme preparations were purified on an anti-TRACP antibody column. Their biochemical properties were compared to the natural serum isoforms using colorimetric assays for activity and total protein. Their structural properties were compared to natural serum isoforms using denaturing and nondenaturing polyacrylamide gel electrophoresis. RESULTS: Both enzyme preparations were heterogeneous. The combined secreted recombinant TRACPs (rTRACP(ex)) had all the characteristics of natural serum TRACP 5a. There were seven uncleaved glycoproteins with a pH optimum of 5.2, relatively low specific activity (278 U/mg) and differentially sialylated. The combined intracellular TRACPs (rTRACP(in)) had all the characteristics of natural serum TRACP 5b. They consisted of two proteins, one of which was a processed heterodimer, with a pH optimum of 5.8, a relatively high specific activity (887 U/mg) and lacked sialic acid. CONCLUSION: This cell line provides an avenue for the simultaneous study of the regulation, function and intracellular trafficking of separate TRACP isoforms and the identification of their physiologic substrates in a single uniform cell source.

Serum tartrate-resistant acid phosphatase isoforms in rheumatoid arthritis.

OBJECTIVES: Our objective was to evaluate the significance and source of serum tartrate-resistant acid phosphatase (TRACP) in patients with rheumatoid arthritis (RA). METHODS: Thirty-five RA, 32 osteoarthritis (OA) and 16 control subjects were studied. Serum TRACP-5b activity and total TRACP protein were determined by immunoassay. TRACP isoforms were analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Serum bone alkaline phosphatase (BAP), cross-linked N-terminal telopeptides (NTx), and C-terminal telopeptides (ICTP) of type I collagen were estimated as markers of bone turnover. C-reactive protein (CRP) was measured as a marker of chronic inflammation. Macrophages and dendritic cells (DC) were developed from peripheral blood monocytes. Cell lysates and culture supernatants were analyzed for TRACP isoforms by immunoassay and PAGE. RESULTS: In RA, mean TRACP-5b activity was normal, but median total TRACP protein was increased twofold (p<0.001). In OA, TRACP-5b activity and protein were normal. In RA, TRACP-5b activity correlated weakly with ICTP (r=0.56) while TRACP protein levels correlated weakly with NTx (r=0.43). Additionally, TRACP protein, but not TRACP-5b activity correlated significantly with CRP (r=0.42). Macrophage and DC lysates contained TRACP-5b, while tissue culture supernatants contained TRACP-5a. CONCLUSIONS: Increased total TRACP protein in RA sera was probably due to TRACP-5a and not derived from osteoclasts. Rather, it could be a secreted product of inflammatory macrophages and DC.