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Special Hematology Laboratory, Veterans Affairs Medical Center, Louisville, KY 40206.
a Address correspondence to this author at: Department of Veterans Affairs Medical Center, 800 Zorn Ave., Louisville, KY 40206. Fax 502-894-6191;
pat.streipts{at}med.va.gov
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Abstract |
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Methods: 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).
Results: 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.
Conclusions: 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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here we further document the clinical value of an improved immunoassay method for selectively measuring TRAcP 5b activity and introduce the concept that TRAcP 5a may have its own clinical significance. We did this by separating TRAcP isoforms 5a and 5b from pooled sera of healthy subjects with column chromatography and studying their properties independently. We compared the electrophoretic properties of serum isoforms to that of normal osteoclastic TRAcP and evaluated the quantitative changes and the clinical significance of TRAcP 5a and 5b in serum from patients with rheumatoid arthritis (RA) and end-stage renal disease (ESRD).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Individual serum specimens were collected from 27 healthy donors, 35 patients with RA, and 30 patients with ESRD obtained before hemodialysis. Before the blood samples were taken, informed consent was obtained from each healthy donor and from each ESRD patient. RA sera were waste specimens obtained after they had served their clinical purposes. All sera were stored at -70 °C within 8 h of collection. These studies were approved by the Human Studies Subcommittee of the VA Medical Center, Louisville.
TRAcP ASSAYS
Total TRAcP activity was determined using 4-nitrophenyl
phosphate (4-NPP) as substrate according to the microplate assay
method of Lau et al. (20) with modifications. Sample (50
µL) was incubated for 1 h at 37 °C with 150 µL of substrate
consisting of 7.6 mmol/L 4-NPP in 100 mmol/L sodium acetate buffer
containing 50 mmol/L sodium tartrate and adjusted to pH 4.9, 5.5, or
6.1. The reaction was stopped by the addition of 50 µL of 3
mol/L NaOH. A calibration curve was constructed from solutions
of 4-nitrophenol containing 10–0.16 µmol/min per liter of sample
(U). Absorbance was measured at 405 nm in a Bio-Rad Model 550
Microplate Reader.
Immunoassays for type-5 TRAcP activity and protein were performed simultaneously according to previously published methods (16). A common anti-TRAcP monoclonal antibody (14G6) was used to coat duplicate microwells. Samples were incubated in coated wells overnight at 4 °C. After washing, TRAcP activity was measured by adding the 4-NPP substrate at pH 5.5 or 6.1. TRAcP protein was determined with a second, peroxidase-conjugated anti-TRAcP antibody, J1B (provided by Dr. J. Halleen, University of Turku, Finland).
chromatographic separation and identification of
TRAcP 5a AND 5b
TRAcP isoforms 5a and 5b were separated from each serum
pool by published methods of cation-exchange chromatography
(17) with modifications. Serum was acidified to pH 5.0 by
dropwise addition of glacial acetic acid. Precipitated proteins were
removed by centrifugation at 10 000g for 15 min. The
supernatant was applied to a column (1.6 x 10 cm) of Sepharose S
fast flow (Amersham Pharmacia) equilibrated with 10 mmol/L
sodium acetate, pH 5.0. The column was washed with 200 mL of
equilibration buffer followed by 200 mL of equilibration buffer
containing 0.3 mol/L NaCl to remove weakly bound proteins. TRAcP
isoforms were then eluted by a linear NaCl gradient from 0.3 to 0.75
mol/L NaCl; 100 equal fractions were collected in a total volume
approximately equal to the serum sample to facilitate direct
comparisons of TRAcP isoform yield. The identities of eluted TRAcP
enzyme peaks, as well as purified bone TRAcP (a generous gift from Dr.
J. Halleen, University of Turku, Finland), were confirmed by
nondenaturing polyacrylamide gel electrophoresis (21). A
5-µL aliquot from the fraction at the apex of each enzyme peak was
mixed with 45 µL of 100 g/L bovine albumin and 50 µL of
sample gel. Electrophoresis was carried out at pH 4.0 for 90 min to
enhance the separation of TRAcP isoforms. Gels were immediately stained
for TRAcP activity in 100 mmol/L sodium acetate-50 mmol/L sodium
tartrate (pH 5.5) containing 2 g/L naphthol ASBI-P and 0.02 g/L Fast
Garnet GBC.
biochemical characterization of TRAcP ISOFORMS5a AND 5b
Column fractions of each enzyme peak were pooled, and the
volumes were recorded. The pH optimum of each TRAcP isoform was
determined at pH 4.0–6.5, using the total activity assay. The yield of
enzymatic activity in each peak was calculated from peak volume and
results of total activity assay at its pH optimum. The yield of enzyme
protein in each peak was calculated from the peak volume and results of
a two-site immunoassay. Yields were normalized to a 1-L sample. The
specific activity of each TRAcP isoform at its pH optimum was
calculated as the ratio of activity/protein and expressed as
U/µg. The Km for each
partially pure enzyme was determined from Lineweaver-Burke plots after
assay of a single sample at its optimum pH at substrate concentrations
of 0.2–10 mmol/L 4-NPP.
other bone markers
Serum bone alkaline phosphatase (BAP) activity was
determined in individual sera as a measure of bone formation
(22) by a method (Metra Biosystems) based on the
enzyme-capture immunoassay principle. N-telopeptides of type-I collagen
(NTx) were measured in individual sera as a measure of bone resorption
(23) by a method (Ostex International) based on a
competitive antigen-binding principle.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 1
, showed that serum TRAcP consisted of two distinct activity
peaks. The first peak contained 87% of the total TRAcP protein but
only 55% of the TRAcP activity because of its relatively low specific
activity. Polyacrylamide gel electrophoresis showed that the first peak
was the slower migrating isoform 5a and that the second peak was the
faster migrating isoform 5b, which had an electromobility identical to
the purified bone TRAcP (Fig. 2
). Isolated TRAcP 5a had a pH optimum of 5.2 and a
Km of 1.9 mmol/L 4-NPP. Isolated TRAcP
5b had a pH optimum of 5.8 and a Km of
7.0 mmol/L 4-NPP. When TRAcP activity was measured at pH 4.9, which
favored 5a activity, or at pH 6.1, which favored 5b activity, the
apparent proportions of the two isoforms differed dramatically (Fig. 3
). At pH 4.9, TRAcP 5a and TRAcP 5b contributed almost equally
to total activity. At pH 6.1, TRAcP 5a activity was reduced twofold,
whereas TRAcP 5b activity was increased twofold. Therefore, by
increasing the pH of TRAcP activity assays to 6.1, preferential
detection of osteoclastic 5b was achieved.
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evaluation of TRAcP ISOFORMS IN PATHOLOGIC CONDITIONS
TRAcP 5a and 5b were separated chromatographically twice using
pooled rheumatic disease serum and once using selected RA serum with
similar results. Fig. 4
illustrates the results from RA serum, in which TRAcP 5a
accounted for 90% of the TRAcP protein and 63% of TRAcP activity.
Both TRAcP isoforms were increased in absolute amounts over the
concentrations seen in the control serum (Table 1
). The specific
activities of both isoforms 5a and 5b in serum from patients with
rheumatic diseases and RA were approximately one-half the activities in
the serum from healthy subjects. The electrophoretic mobilities
of isoforms 5a and 5b in rheumatic diseases and RA were the same as for
the isoforms from healthy subjects. The
Km of isoform 5a was increased to 3.2
mmol/L 4-NPP, whereas that of isoform 5b was unchanged.
Therefore, the significant increase in TRAcP protein we observed
previously in rheumatic diseases (16) and RA (24)
was attributable to an increase in the amount of both isoforms 5a and
5b with a concomitant decrease in the specific activities of both.
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Compared with pooled control serum, chromatographic separation of TRAcP
5a and 5b from pooled ESRD serum showed marked relative and absolute
increases in 5b protein and activity (Table 1
and Fig. 5
). In this condition, TRAcP 5a accounted for 60% of total TRAcP
protein and 27% of activity. In absolute terms, isoform 5a protein was
unchanged, whereas isoform 5b protein was increased almost fivefold
compared with control serum. In contrast to RA serum, the specific
activity of isoform 5a in ESRD serum was similar to that of the control
serum (0.47 vs 0.58 U/µg of TRAcP, respectively), whereas the
specific activity of isoform 5b was reduced by 42% (1.90 vs 3.30
U/µg of TRAcP, respectively). The electrophoretic properties
of the TRAcP isoforms in ESRD were similar to the control serum. The
Km of isoform 5a was slightly
increased to 3.7 mmol/L 4-NPP, whereas that of isoform 5b decreased
slightly to 4.4 mmol/L 4-NPP. Therefore, the increased TRAcP
activity we had observed previously in serum from ESRD patients
(16)(25) was attributable to an absolute
increase of circulating 5b isoform. Its reduced specific activity
suggests that serum from ESRD patients may contain substantial amounts
of inactive isoform 5b.
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significance of differential expression of TRAcPISOFORMS IN PATHOLOGIC CONDITIONS
In both RA and ESRD, an adjustment to pH 6.1 for
determination of total enzymatic activity selectively increased the
proportion of isoform 5b detected (Fig. 6
). Peak activity of TRAcP 5b increased twofold, and peak
activity of TRAcP 5a decreased twofold. Measuring TRAcP activity in the
enzyme capture immunoassay at pH 6.1, therefore, provided greater
specificity for isoform 5b, as shown previously in postmenopausal women
(19).
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To determine whether this adjustment produced a stronger association
between TRAcP activity and bone turnover in other diseases, we
performed immunoassays for total type-5 TRAcP activity at pH 5.5,
isoform 5b activity at pH 6.1, and TRAcP protein in a group of 27
control subjects, 35 RA patients, and 30 ESRD patients on hemodialysis.
Serum BAP and NTx were also determined as independent markers of bone
formation and resorption (Table 2
). Mean total TRAcP activity (pH 5.5) was not increased in
either RA or ESRD; however, a subpopulation of ESRD patients who had
increased total TRAcP caused the wide distribution and larger standard
deviation. On the other hand, mean TRAcP 5b activity (pH 6.1) and TRAcP
protein were significantly increased in both cohorts. BAP was increased
in both RA and ESRD, whereas NTx was increased only in ESRD. If TRAcP
activity measured at pH 6.1 is selective for osteoclastic isoform 5b
and related to bone turnover, it should correlate with serum BAP or
NTx. The calculated correlation coefficients for the tested markers are
summarized in Table 3
. Whereas no significant correlations were found between TRAcP
and bone markers in RA, both total TRAcP and TRAcP 5b activities
correlated significantly with bone markers in ESRD. No significant
correlations were obtained between TRAcP protein and bone markers in
any disease.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our present results confirm earlier findings (18) showing
that TRAcP 5a and 5b are antigenically related; both react with the
antibodies used for immunoassays. We demonstrated that they have
different specific activities and that their absolute and proportional
amounts differed in disease-specific ways. We had previously reported
that the specific activity of TRAcP in serum samples from healthy
subjects and from patients was 
10-fold lower than estimated here. We
have since found that the affinity-purified TRAcP 5b used to calibrate
the two-site immunoassay in our previous work contained substantial
amounts of denatured TRAcP protein. Therefore, the amount of
immunoreactive TRAcP protein in the calibrator was lower than the total
protein quantified by ultraviolet absorbance, which led to
overestimation of serum TRAcP protein by the two-site immunoassay. For
this work, affinity-purified TRAcP 5b was freshly prepared from hairy
cell leukemia spleen for use as the calibrator. Despite these
differences between studies in the absolute amounts of measured TRAcP
protein, the relationships between TRAcP specific activity and disease
and the biological significance of differential isoform expressions are
unchanged.
TRAcP is believed to be a biomarker of osteoclastic activity. Its activity is increased in the serum of growing children (17)(26) and in patients with various metabolic bone diseases (6)(7)(8)(27). Lam et al. (18) showed that the TRAcP of giant cell tumor was isoform 5b and that it had immunologic identity to serum TRAcP 5b. Here we confirm and extend those findings by showing that serum TRAcP 5b is the same as normal bone TRAcP, as originally defined by electrophoresis. Serum TRAcP activity often is increased in patients with ESRD undergoing hemodialysis (7)(16)(27)(28). This is presumed to result from increased bone resorption. Results from our chromatographic and electrophoretic studies are strong evidence that increased TRAcP activity in ESRD serum is in fact attributable to increased TRAcP 5b. The reduced specific activity of TRAcP 5b in ESRD serum suggests some partial inactivation of isoform 5b in this disease.
Our previous work with simultaneous assays of TRAcP activity and protein in rheumatic diseases showed a strong association between RA and increased TRAcP protein with low specific activity (24). In this study, we showed that most of this low-activity TRAcP is isoform 5a. The amount of TRAcP 5a protein was increased threefold in RA, whereas 5a activity was increased only 43%. The amount of TRAcP 5b protein in RA sera was twice that of control sera, but its specific activity was also reduced. Increased TRAcP 5b protein in RA sera may reflect RA-associated osteoporosis or bone erosion by osteoclasts (29); however, its reduced activity remains to be explained. RA is an autoimmune disease with chronic inflammation. Activated macrophages are abundant in rheumatoid synovial tissues (30) and are known to contain TRAcP (31). The origin of serum TRAcP 5a is unknown. It coexists with, but is distinct from TRAcP 5b. The abnormal macrophages of Gaucher disease accumulate abundant TRAcP, which has been characterized as isoform 5a (32). It is conceivable that the increased amounts of both TRAcP 5a and 5b in the circulation of RA patients could be derived from activated macrophages rather than, or in addition to, osteoclasts.
The fact that TRAcP 5b often contributes much toward total activity but little toward total protein explains why TRAcP activity assays correlate better with other markers of bone turnover than do TRAcP protein assays. TRAcP 5b has a higher pH optimum than TRAcP 5a (17). Recently, this difference has been exploited to increase the specificity of a TRAcP immunoassay for isoform 5b (19). In that study, a significant decrease in TRAcP 5b activity (pH 6.1) was observed in response to hormone replacement therapy among postmenopausal women. Total TRAcP activity (pH 5.5) did not change significantly with hormone replacement therapy. It was concluded that measurement of TRAcP 5b activity at pH 6.1 more closely reflected bone resorption (19).
In the present study, we confirmed that immunoassay of TRAcP activity at pH 6.1 significantly increases the specificity for isoform 5b. We then examined the associations among total TRAcP activity at pH 5.5, TRAcP 5b activity at pH 6.1, and total TRAcP protein and other bone metabolic markers in RA and ESRD sera. Although TRAcP 5b activity was significantly increased in both RA and ESRD, it may have quite different clinical and biological significance in these diseases. In RA sera, TRAcP 5b activity did not correlate with BAP or NTx. This could be explained by the fact that most TRAcP in RA sera is isoform 5a and unrelated to bone resorption. Even when measured at pH 6.1, a large amount of isoform 5a would still contribute somewhat to the measurement of isoform 5b. Alternatively, TRAcP 5b in RA could be derived from inflammatory macrophages instead of osteoclasts. Some tissue macrophages contain abundant TRAcP activity (31)(33), and TRAcP has been purified from alveolar macrophages (31)(34) as well as from osteoclasts (14)(35). In this regard, it is noted that the mean NTx of RA sera (17.13 ± 3.86 nmol of bone collagen equivalents/L) was actually lower than that of our healthy group (19.33 ± 3.89 nmol of bone collagen equivalents/L), which suggests that our RA patients may not have ongoing increased bone resorption. In ESRD sera, TRAcP 5b activity did correlate significantly with both BAP and NTx, although the strength of the associations were weak. This could be attributable to the reduced specific activity of TRAcP 5b in ESRD. In addition, because NTx are usually cleared by the kidney, their serum concentrations in renal failure may be disproportionately high relative to actual bone resorption (36). The weak correlations notwithstanding, TRAcP 5b in ESRD by all accounts is osteoclast derived.
TRAcP is a biomarker of osteoclasts with emerging clinical importance in metabolic bone disease. However, TRAcP heterogeneity in serum has had a negative impact on the sensitivity and specificity of TRAcP as a marker of bone resorption. Although TRAcP isoform 5a retains some activity at pH 6.1, which may compromise the absolute specificity of the assay for TRAcP 5b, results from our studies provide further support for measuring TRAcP activity at pH 6.1 to increase the specificity of the TRAcP immunoassay for isoform 5b (19). Until isoform 5b-specific antibodies, substrates, or inhibitors are discovered to make TRAcP immunoassays absolutely bone specific, this strategy definitely improves the specificity of TRAcP immunoassays for diseases of increased bone resorption. It should be noted that TRAcP 5b is also produced by macrophages and could, in principle, become increased in serum of patients with inflammatory diseases such as RA. Until now, TRAcP 5a has been given little significance except as an interfering substance in TRAcP 5b measurements. Our results imply that TRAcP 5a may have practical, biological, and clinical significance of its own. Alterations in the absolute amounts and patterns of TRAcP isoform expression indicate that pathogenetic mechanisms leading to increased serum TRAcP 5a or 5b are different in metabolic bone disease and chronic inflammation.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: TRAcP, tartrate-resistant acid phosphatase; RA, rheumatoid arthritis; ESRD, end-stage renal disease; 4-NPP, 4-nitrophenyl phosphate; BAP, bone alkaline phosphatase; and NTx, N-telopeptides of type-I collagen. 
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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