Clinical Chemistry 46: 469-473, 2000;
(Clinical Chemistry. 2000;46:469-473.)
© 2000 American Association for Clinical Chemistry, Inc.
Development of a Kinetic Assay for Band 5b Tartrate-resistant Acid Phosphatase Activity in Serum
Mamoru Nakanishi1,a,
Kousei Yoh2,
Toshihide Miura3,
Tatsuya Ohasi3,
Shiba Kumar Rai4 and
Kagehiro Uchida1
1
FALCO Biosystems Co., 17-1 Nishiarami Tai, Kumiyama-cho, Kuze-gun, Kyoto 613-0036, Japan.
2
Department of Orthopedic Surgery, Hyogo College of
Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 633-8501, Japan.
3
Nitto Boseki Co. Ltd, 1 Shiojima, Fukuhara, Koriyama,
Fukusima 963-8061, Japan.
4
Department of Medical Zoology, Kobe University School of
Medicine, 7-5-1 Kusuniki-cho, Chuo-ku 650-0017, Japan.
a Author for correspondence. Fax 0774-46-1101; e-mail falconpd{at}alles.or.jp
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Abstract
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Background: Band 5 tartrate-resistant acid phosphatase (TrACP; EC
3.1.3.2) consists of two isoenzymes, bands 5a and 5b, of which band 5b
TrACP is considered to be derived from bone. However, no kinetic method
for the specific measurement of band 5b TrACP in serum is available.
Our aim was to develop a kinetic assay method for the specific
measurement of band 5b TrACP in serum.
Methods: Band 5b TrACP was measured kinetically in serum as
tartrate-resistant fluoride-sensitive heparin-resistant ACP with
2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.6.
Results: Heparin inhibited band 5a TrACP but had no effect on
band 5b TrACP in serum or in bone extract. The presence of EDTA or
ascorbic acid had no effect, but dithiothreitol inhibited enzyme
activity. The within-run (n = 20) and between-run (n = 20)
CVs of band 5b TrACP activity were 3.3–5.8% and 5.0–7.3%,
respectively. The mean ± SD values of band 5b TrACP activity in
males (n = 25) and females (n = 57) 20–29 years of age by
this method were 8.0 ± 2.2 U/L and 6.4 ± 1.8 U/L,
respectively. The band 5b TrACP value was significantly higher in
females >50 years of age compared with the younger subjects (20–29
years). The highest band 5b TrACP values were among children younger
than 15 years.
Conclusions: This kinetic assay is a simple and specific method
for the measurement of band 5b TrACP in serum samples and is useful in
the evaluation of bone turnover activity.
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Introduction
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Serum band 5 tartrate-resistant acid phosphatase
(TrACP;1
EC 3.1.3.2) usually is expressed in certain differentiated
cells of the mononuclear phagocyte system, notably osteoclasts and
alveolar macrophages (1), and pathologically in
Gaucher cells (2) and in the hairy cells of leukemia
reticuloendotheliosis (3). Serum band 5 TrACP is one of the
most abundant enzymes in osteoclasts (4) and is regarded as
a marker (5)(6). Thus, the serum concentrations
of band 5 TrACP constitutes a potential index for the bone resorption
rate (7)(8).
On the basis of the catalytic and ionic properties and carbohydrate
content, two isoenzymes of band 5 TrACP, bands 5a and 5b, have
been identified (9). Band 5a TrACP is sialylated, whereas
band 5b lacks sialic acid. It has been shown that the removal of sialic
acid from band 5a by sialidase changes band 5a to 5b (10).
Increased band 5b TrACP is liberated from osteoclasts during normal
physiologic bone growth among healthy children and during
metastasis of bone (11). The presence of band 5b TrACP in
Gaucher serum in spite of the presence of band 5a TrACP in the
spleen has been reported (10). Thus, the measurement
of band 5b TrACP appears to be a specific marker for osteoclastic
activity.
However, no kinetic method for the specific measurement of band 5b
TrACP in serum has been available. Here we report a simple kinetic
method for the measurement of this osteoclastic activity marker (band
5b TrACP) by modifying the method we described previously for the
measurement of tartrate-resistant fluoride-sensitive acid
phosphatase (TrFsACP) (12).
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Materials and Methods
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chemicals
2,6-Dichloro-4-acetylphenyl phosphate (DCAPP) was obtained from
Nitto Boseki Co. Ltd. Sodium L-(+)-tartrate and
heparin were obtained from Sigma, and polybrene was obtained from
Aldrich. Carboxymethyl-agarose (CM-Sepharose) was obtained from
Pharmacia Fine Chemicals. All other analytical grade chemicals were
purchased from Wako Pure Chemical Co.
reaction solution
The reaction solutions we reported previously (12) were
used with some modification. Briefly, buffer solution I consisted of
150 mmol/L 2-(-hydroxy-3-morpholino) propanesulfonic acid, 60 mmol/L
sodium L-(+)-tartrate, 23 kIU/L heparin,
and 5 g/L bovine albumin (pH 6.6). Buffer solution II consisted of
buffer solution I plus sodium fluoride (45 mmol/L). The substrate
solution consisted of 45 mmol/L DCAPP and 50 mmol/L Tris (pH 4.0).
serum samples
Blood samples collected from 516 apparently healthy Japanese
subjects, ages 5–79 years (210 males and 306 females), by clean
venipuncture were allowed to clot at room temperature for 2–4 h and
centrifuged at 1000g for 10 min at room temperature. Serum
thus separated was transferred into 1.5-mL tubes and stored at
-80 °C until use (within 6 months).
bone extract
Bone extract samples were prepared as described previously
(12). In brief, bovine tibia after the removal of soft
tissue was cut into small cubes. The marrow and blood were removed, and
the bone was ground to powder and homogenized in a solution containing
Triton X-100, potassium chloride, phenylmethylsulfonyl fluoride,
benzamidine, and aminocaproic acid. The extract was collected by
centrifugation at 10 000g for 20 min at 4 °C and stored
at -80 °C until use.
assay procedure
TrACP activity was measured using a centrifugal automatic
analyzer (Cobas Fara; Hoffmann-La Roche). Briefly, 150 µL of
buffer solution I was added to 15 µL of sample, and the mixture was
incubated at 37 °C for 5 min. The enzyme-substrate reaction was
initiated by the addition of 60 µL of substrate solution. The change
in absorbance at 340 nm was monitored at 20-s intervals for 5 min. The
millimolar absorptivity of the hydrolysis product
(2,6-dichloro-4-acetylphenol) is 21.49
L · mmol-1 · cm-1
at 340 nm. One IUB unit (1 U) of TrACP activity is defined as 1 µmol
of DCAPP hydrolyzed per minute at 37 °C in the presence of 40 mmol/L
sodium L-(+)-tartrate and 23 kIU/L heparin at pH
6.6. Tartrate-resistant fluoride-resistant acid phosphatase
activity was assayed using buffer solution II instead of buffer
solution I. Band 5b TrACP activity was estimated by subtracting
tartrate-resistant fluoride-resistant acid phosphatase activity from
TrACP activity.
fractionation on cm-sepharose
For biochemical characterization, serum collected from children
was pooled and chromatographed on CM-Sepharose. The CM-Sepharose was
packed into glass tubing [60 x 0.9 cm (i.d.)] and washed with
10 mmol/L acetate buffer, pH 5.0. The acidity of the serum sample was
adjusted to pH 5.0 by the addition of acetic acid, and the coagulated
proteins were removed by centrifugation at 20 000g for 15
min. The supernatant fraction was passed through the column at 0.5
mL/min. The enzyme, which was retained on the column, was then eluted
with a linear concentration gradient of sodium chloride: 0–1 mol/L in
299 mL of elution buffer (10 mmol/L acetate buffer, pH 5.0).
Fractionated serum was subjected to TrFsACP measurement with added
heparin or polybrene in reaction buffers I and II.
electrophoresis
Acid phosphatase isoenzymes were analyzed by electrophoresis on
acidic polyacrylamide gel. After the electrophoresis was completed, the
gel columns were washed with 0.1 mol/L acetate buffer (pH 5.2) and
stained with 
-naphthyl phosphate-Fast Garnet GBC as
described previously (9).
pH OPTIMUM
The optimum pH values for band 5a and 5b TrACP were determined
using 150 mmol/L 2-(-morpholino)ethanesulfonic acid, pH 5.0–6.4, and
150 mmol/L 2-(-hydroxy-3-morpholino)propanesulfonic acid, pH 6.6–7.2,
buffers in buffer solution I with added polybrene or heparin.
statistics
All results were expressed as means ± SD. Statistical
significance was evaluated by the Student t-test for
unpaired data.
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Results
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separation of TrACP BANDS 5a AND5b BY CATION-EXCHANGE CHROMATOGRAPHY
Chromatography of sera from children showed at least four TrFsACP
activity peaks (I, II, III, and IV) when polybrene was added to the
reaction buffer (Fig. 1
). However, only the fourth peak appeared when heparin was added
to the reaction buffer. The activity of the fourth peak was the same
with either polybrene or heparin added. Electrophoresis of peaks III
and IV on acidic polyacrylamide gels (9) yielded band 5a and
5b TrACP, respectively (Fig. 2
). These fractions were used to study the effect of heparin and
pH on band 5a and 5b TrACP.
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Figure 1. CM-Sepharose chromatographic pattern of serum TrFsACP.
Serum (5 mL) was adsorbed on the column and eluted by a linear NaCl
concentration gradient as described in the text. The elute was assayed
for TrFsACP activity, with added heparin ( ) or polybrene (•) in
the reaction mixture.
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Figure 2. Electrophoresis of peaks III and IV of TrFsACP on
polyacrylamide gel.
Lane 1, peak III; lane 2, peak IV;
lane 3, serum pool from children.
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effect of heparin on band 5a AND 5b TrACPAND BONE EXTRACT
The effect of heparin on band 5a and 5b TrACP, bone extract, and
serum is shown in Fig. 3
. These fractions were collected after cation-exchange
chromatography as mentioned above. Band 5a TrACP was
significantly inhibited by heparin (P <0.001),
whereas band 5b TrACP was not. However, a slight inhibition in bone
extract and serum by heparin was observed.
effect of pH ON BAND 5a AND 5b
TrACP
The optimal reaction pH for band 5a TrACP (peak III) was 
5.8,
whereas band 5b TrACP (peak IV) showed maximum activity at pH 6.6. The
addition of heparin in the reaction mixture selectively reduced band 5a
TrACP activity to 0 at pH 6.6 (Fig. 4
).
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Figure 4. Effect of pH on band 5a and 5b TrACP activity.
The enzymes separated on a CM-Sepharose column were assayed at
different pHs. Band 5a TrACP (peak III; fractions 54–56;
circles) and band 5b TrACP (peak IV; fractions 68–72;
triangles) were measured with added heparin (open
symbols) or polybrene (closed symbols) in the
reaction mixture.
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effect of storage
In view of the labile nature of band 5b TrACP activity, we
examined the effect of storage temperature (room temperature vs
-80 °C) on the stability of band 5b TrACP activity in serum samples
collected from five healthy individuals. Band 5b TrACP activity in
these samples did not change within 12 h at room temperature or
after 12 months at -80 °C.
effects of various effectors on band 5b TrACP
The effects of potential activators and inhibitors of band 5b
TrACP are summarized in Table 1
. The presence of EDTA or ascorbic acid had no effect, but
dithiothreitol inhibited enzyme activity.
assay imprecision
The within- and between-run imprecision was evaluated with two
different serum samples assayed 20 times each. Aliquots of the serum
samples were stored at -80 °C until use. The within-run CVs of band
5b TrACP activity were 5.8% (at 11.3 U/L) and 3.3% (at 29.9 U/L),
respectively. The between-run CVs were 7.3% (at 11.3 U/L) and 5.0%
(at 29.9 U/L), respectively. The upper limit of linearity of the
present band 5b TrACP assay, determined by measuring serum, was 110.0
U/L. The Km value of band 5b TrACP
activity was 4.7 mmol/L when DCAPP was used as the substrate.
method comparisons
We compared the present method for band 5b TrACP (with heparin, pH
6.6) with the previously reported method for TrFsACP (with polybrene,
pH 6.2), using randomly selected apparently healthy children (n =
22) and adults (n = 28). The correlation coefficient
(r) between band 5b TrACP (x) and TrFsACP
(y; with polybrene) was 0.97 [Deming regression
equation: y = (0.92 ± 0.03)x +
(10.0 ± 0.61); n = 50; Sy|x =
2.26]. For children, the correlation was r =
0.92 [y = (0.92 ± 0.08)x + (10.0
± 2.26); n = 22; Sy|x = 2.65]. For adults,
the correlation was r = 0.78 [y =
(1.16 ± 0.18)x + (8.03 ± 1.54); n = 28;
Sy|x = 1.86; Fig. 5
].
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Figure 5. Correlation between band 5b TrACP (with heparin) and
TrFsACP (with polybrene) in children () and adults (•).
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assays of samples from apparently healthy subjects
Band 5b TrACP activity in apparently healthy Japanese males and
females, ages 20–39 years, ranged from 3.6 to 12.4 U/L and 2.8 to 10.0
U/L, respectively. Both sexes showed an increase in band 5b TrACP
activity with age (Table 2
). Band 5b TrACP activity was significantly higher in males >60
years and females >50 years compared with the younger subjects (20–29
years). Band 5b TrACP activity in females was slightly lower than in
males at ages 20–39 years. However, the activity was highest in
children <15 years of age.
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Discussion
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We previously reported an improved method for the measurement of
TrFsACP, based on its fluoride-sensitive property, using DCAPP as
substrate. The improved method, however, measured TrFsACP originating
from osteoclasts and other macrophages and excluded TrACP originating
from erythrocytes and platelets (12). To detect band 5b
TrACP specifically, we introduced heparin into the reaction mixture
instead of polybrene. In our previous report, we showed a marked
reduction in TrFsACP activity when heparin was added at the
concentration of 100 kIU/L, but the
activity did not decrease further with increases in the heparin
concentration. The unaffected TrFsACP appeared to be band 5b
TrACP, and the affected TrFsACP appeared to be band 5a and other
unknown TrFsACP isoenzymes, as shown by CM-Sepharose
chromatography performed separately with added heparin or polybrene.
Our present finding showed a small inhibitory effect of heparin on bone
extract as well as indicating that bone extract also contains a small
amount of non-band 5b TrACP.
Modder (13) observed a maximal inhibitory effect of heparin
at the concentration of 50 kIU/L, but this was
accompanied by maximal turbidity in the reaction mixture. This
turbidity presumably occurred because of the globulin-precipitating
effect of heparin. However, no such turbidity was observed in our
reaction mixture. This appears to be related to the low pH (4.9) of the
reaction mixture used by Modder compared with ours (pH 6.6). At pH 6.6,
we found a good correlation between band 5b TrACP (with heparin) and
TrFsACP (with polybrene). The intercept of 10.0 observed in the added
polybrene method appeared to include band 5a and other unknown TrACP
isoenzymes.
Fukushima et al. (14), however, have suggested that
intracellular TrACP is highly diffusible at a pH below 6.0 in an
inactive form. When secreted to the extracellular acidic compartment
(resorption lacunae), TrACP is transformed into an active form and
infiltrates cartilage and bone matrices. Under light and electron
microscopes, the TrACP at pH 6.5, using a lead-salt method, was found
to be localized in cartilage and bone matrices, osteoclasts, and
chondroclasts. The most favorable pH was 6.5. At pH 6.6, a peak of band
5b TrACP was observed in both the heparin- and polybrene-added reaction
mixtures, whereas the band 5a TrACP activity was near 0 when heparin
was added. The optimum pH of band 5a TrACP was between 5.6 and 5.8. Lam
et al. (15) also reported a relatively lower optimal pH
(5.0) for band 5a TrACP than for band 5b (5.5–6.0) in acidic
polyacrylamide gel electrophoresis. These findings show that the
optimal pH for band 5b is relatively higher than that for band 5a.
Previously, Allen et al. (16) and Cheung et al.
(17) showed no inhibitory effect of EDTA on TrACP extracted
from human bone and cord blood. On the other hand, dithiothreitol at
low concentrations has been found to enhance enzyme activity but to
inhibit activity at higher concentrations and prolonged exposure
(17). In our study, we also observed no effect of EDTA on
band 5b TrACP but a massive inhibitory effect of dithiothreitol
even at low concentrations. The explanation for this discrepancy is not
available.
Using this improved method, we also measured serum band 5b TrACP
activity in apparently healthy Japanese. Band 5b TrACP activity in
females increased and reached a plateau at >40 years of age; activity
in males increased more slowly than in females and reached the same
values at >70 years of age. The higher band 5b TrACP values observed
children younger than 15 years was in agreement with TrFsACP values we
reported previously (12) but at a relatively low
activity. The low values obtained by this method appear to be
attributable to the inhibition of band 5a and other unknown TrACP
isoenzymes by heparin.
In conclusion, the improved method we reported previously
(12) can be used for the specific measurement of band 5b
TrACP with some modification (added heparin instead of polybrene at
slightly higher pH). This method is simple and may be useful in the
evaluation of bone turnover.
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Footnotes
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1 Nonstandard abbreviations: TrACP, tartrate-resistant acid phosphatase; TrFsACP, tartrate-resistant fluoride-sensitive acid phosphatase; and DCAPP, 2,6-dichloro-4-acetylphenyl phosphate. 
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Top
Abstract
Introduction
, band 5b TrACP
(peak IV; fractions 68–72); •, bone extract; 
, serum.