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Review Series

Volume 3, April 06
Immunodiagnostic Systems (IDS).

Jussi Halleen

For Medical Educational Purposes Only.

Tartrate-resistant Acid Phosphatase 5b


(TRACP 5b) as a Marker of Bone Resorption
Jussi Halleen
INTRODUCTION
Tartrate-resistant acid phosphatase (TRACP) is an enzyme that is expressed in high amounts by bone-resorbing osteoclasts,
inflammatory macrophages and dendritic cells. Two forms of TRACP circulate in human blood, TRACP 5a derived from
macrophages and dendritic cells, and TRACP 5b derived from osteoclasts. Recent data have demonstrated the utility of
TRACP 5b as a marker of bone resorption and osteoclast number, and serum TRACP 5a is a marker of inflammatory
conditions. This review summarizes the scientific knowledge on the role of TRACP in osteoclastic bone resorption, the
mechanism of TRACP 5b generation in osteoclasts and its secretion into the blood circulation, the methodology of measuring
TRACP 5b, clinical evidence for the use of TRACP 5b as a resorption marker, and advantages of TRACP 5b compared to
other commonly used bone turnover markers.

STRUCTURE AND FUNCTION OF TRACP


Tartrate-resistant acid phosphatase (TRACP), also
known as purple acid phosphatase, is an iron-containing
enzyme with unknown biological function. Despite its
discovery in the early 1970s and the development of
knock-out and overexpressing mouse models,1,2 the
biological function of TRACP is not known. High
amounts of TRACP are expressed in bone-resorbing
osteoclasts, alveolar macrophages of the lung and
dendritic cells.3-5 TRACP is synthesized as one
polypeptide chain with a molecular weight of
approximately 35 kD.6 The structure of TRACP contains
two possible sites for N-linked oligosaccharides, a
disulfide bond, an exposed protease-sensitive loop
peptide, and a binuclear iron center.7-1 2 The binuclear
iron center contains two ferric (3+) ions, one of which is
stabilized into the ferric form.8 The other ferric iron is
redox-active and capable of reducing to ferrous (2+)
form. Redox-state of the environment is an important
regulator of the activity of TRACP, as it has been shown
that reduction of both the redox-active iron and the
disulfide bond increases its enzymatic activity.7 Another
important regulatory mechanism of TRACP is excision
of the protease-sensitive loop peptide, which has also
been shown to increase the enzymatic activity.1 0-12 In
addition, TRACP has been shown to contain high
mannose-type oligosaccharides that may also have a role
in regulating the enzymatic activity.13,14

TRACP has two distinct enzymatic activities. It can function


as a phosphatase at acidic pH, and as a generator of reactive
oxygen species (ROS) at neutral pH. 15 The phosphatase
activity prefers substrates where phosphate is linked to an
aromatic ring, such as the widely used synthetic compound
4-nitrophenyl phosphate (4-NPP).7,16-18 The most important
naturally occurring compound with such structure is
phosphotyrosine, and it would be convenient to suggest that
TRACP would function as a tyrosine phosphatase in vivo.
However, no biological substrates have been identified for
this activity. Instead, it has been shown that the two
phosphoserine-containing bone matrix proteins osteopontin
and bone sialoprotein may be biologically relevant substrates
for TRACP.19 Also, it has been demonstrated that TRACP
becomes an efficient ATPase upon cleavage of the loop
peptide.20 The redox-active iron of TRACP can facilitate
generation of ROS through Fentons reaction in the presence
of hydrogen peroxide.21-23 A biological role has been
suggested for the ROS-generating activity in both osteoclasts
and macrophages. ROS generated by TRACP have been
suggested to participate in finalizing degradation of bone
matrix components in transcytotic vesicles of osteoclasts,23,24
and in degradation of foreign compounds in antigen
presentation route of macrophages.25-27 Other suggested
biological functions for TRACP include transporting iron
from the mother to the developing fetus in pigs,28 and a
coupling factor of bone formation and bone resorption
through activation of osteoblasts. 29,30

_________________________________________________________________________________________________________________________
*Dr. Jussi Halleen is a consultant to Immunodiagnostic Systems (IDS). All correspondence to
IDS Ltd, 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD. UK

Review Series
Volume 3, April 06
Immunodiagnostic Systems (IDS).

Jussi Halleen
For Medical Educational Purposes Only.

TRACP ISOFORMS

ASSAYS FOR THE MEASUREMENT OF TRACP

TRACP was discovered in the early 1970s as the most


cathodic of five separate acid phosphatase bands in nondenaturing polyacrylamide gel electrophoresis at acidic
pH, and it was hence named type 5 acid phosphatase.31
Later, this band could be separated to two distinct
bands, referred to as 5a and 5b.32 The difference in
migration was due to the presence of sialic acid residues
in band 5a that were not present in band 5b.33 Treatment
of band 5a with sialidase changed the migration of the
band similar to the migration of band 5b, suggesting that
there would be no other structural differences between
the two forms. However, the pH optimum of the two
forms was different, being 5.2 for band 5a and 5.8 for
band 5b.32,34 Later, it was discovered that band 5a
consisted of a single polypeptide chain with a molecular
weight of 35 kD, while band 5b consisted of two
subunits with molecular weights of 16 kD and 23 kD.34,35
This was due to proteolytic cleavage of the proteasesensitive loop-peptide by cysteine proteases such as
trypsin or various cathepsins. As a result of the cleavage,
the specific activity of band 5b was substantially higher
than that of band 5a. Although osteoclasts, macrophages
and dendritic cells contain both forms, osteoclasts
appeared to secrete only 5b and macrophages and
dendritic cells appeared to secrete only 5a. 35 Thus,
circulating TRACP 5a originates from macrophages and
dendritic cells, and circulating TRACP 5b originates
from osteoclasts. This means that TRACP 5a is a marker
of inflammatory conditions and TRACP 5b is a marker
of bone resorption.

In the early days, serum TRACP activity was measured by


kinetic assays that detected both TRACP 5a and 5b, and also
other, non-type 5 tartrate-resistant acid phosphatase
enzymes, such as those derived from platelets and
erythrocytes. 36,37 Specificity of the kinetic assays was further
improved by using selective inhibitors such as fluoride and
heparin,3 8 and selective substrates such as -naphthyl
phosphate. 39 Various immunoassays specifically detecting the
type-5 TRACP have also been developed. Such assays
include assays where TRACP activity bound to an antibody
is measured using pNPP as a substrate at pH 5.5 (so-called
TRAP5 assays), 40-4 3 competitive assays using polyclonal
antibodies, 44-4 6 and sandwich-type assays using two
monoclonal antibodies.42,43,47,48 None of these assays were,
however, specific for the osteoclast-derived TRACP 5b
form, and their clinical use for determining bone resorption
was poor.

Table 1: Differences between TRACP 5a and


TRACP 5b
Property
Presence of
sialic acid
pH optimum
Loop
peptide
Specific
activity
Secreted by
Marker of

TRACP 5a
YES

TRACP 5b
NO

5.2
Intact

5.8
Cleaved

Low

High

Macrophages,
dendritic cells
Inflammatory
conditions

Osteoclasts
Bone resorption

TRACP 5b is secreted from osteoclasts as an enzymatically


active form. This active TRACP 5b looses its iron content
and is thereby inactivated and degraded to fragments that are
eventually cleared from the circulation through the liver. 49
Therefore, secreted TRACP 5b activity describes accurately
the amount of intact TRACP 5b enzyme molecules that have
been freshly released from osteoclasts. It has been estimated
that only approximately 10% of TRACP circulates as intact
enzymatically active molecules in human blood, while the
remaining approximately 90% circulates as inactive
fragments.46,48 On the other hand, approximately 87% of
TRACP molecules in human blood circulate as the nonosteoclastic form TRACP 5a, while only approximately 13%
circulate as the osteoclast-derived TRACP 5b.50 Therefore, a
diagnostic TRACP-method describing bone resorption
should measure only enzymatically active TRACP 5b,
without measuring enzymatically active TRACP 5a or
interfering inactive fragments.
Two immunoassays with high specificity for TRACP 5b have
been developed, one using the selective substrate naphthyl
ASBI phosphate,51 and the other using pH-selection of the
enzymatic reaction to distinguish TRACP 5b from TRACP
5a.52 The pH-selective immunoassay has been
commercialized (BoneTRAP assay, SBA-Sciences, Oulu,
Finland), and the technique involved has been patented
worldwide. The BoneTRAP assay uses a monoclonal antiTRACP antibody O1A that binds only enzymatically active
TRACP molecules, without binding any interfering inactive
fragments.5 2 The BoneTRAP assay has been widely studied

_________________________________________________________________________________________________________________________
*Dr. Jussi Halleen is a consultant to Immunodiagnostic Systems (IDS). All correspondence to
IDS Ltd, 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD. UK

Review Series
Volume 3, April 06
Immunodiagnostic Systems (IDS).
and shown to be an excellent method for determining
the bone resorption rate.
STABILITY OF TRACP 5B IN SERUM SAMPLES
Some time ago there was a common belief in the
scientific community that serum TRACP activity was
extremely labile, which causes problems in using it as a
marker. This belief was mostly due to a
misunderstanding that started in the early days when
kinetic assays were used. Later it was discovered that the
lability was actually due to an interfering non-type 5
tartrate-resistant acid phosphatase isoenzyme derived
from erythrocytes that the assays also measured.53 In
fact, with proper pre-incubation of serum samples, the
interfering enzyme was inactivated, which improved
clinical perform ance of the kinetic assay as a method for
determining bone resorption.53,54
Stability of serum TRACP 5b has been studied in detail
using the BoneTRAP assay, showing that serum
TRACP 5b is stable for routine laboratory
measurements. 52,55 TRACP 5b is stable for at least 2 days
at room temperature (+25C) and 3 days in the
refrigerator (+4C). However, care should be taken with
long-term storage of serum samples. To avoid problems
in long-term storage, freshly collected, separated serum
samples should be stored in aliquots at -70C
temperatures (or lower), where TRACP 5b is stable for
years. Serum samples can be stored at -20C for up to
one month, but after that the enzyme starts to inactivate.
Also, re-freezing of serum samples after thawing is not
recommended, as TRACP 5b looses activity rapidly
during the re-freezing period even at -70C.55 Table 2
summarizes stability of serum TRACP 5b in different
conditions.
Table 2: Stability of TRACP 5b in human serum
samples
Storage condition
Room temperature (+25C)
Refrigerator (+4C)
Frozen at -20C
Frozen at -70C
Re-freezing after thawing

Stability
2 days
3 days
1 month
Several years
Not recommended

Jussi Halleen
For Medical Educational Purposes Only.
TRACP 5B AS A MARKER OF BONE RESORPTION
Bone resorption starts when acid and proteolytic enzymes,
most importantly cathepsin K, are secreted from the
osteoclast into the space between the cell membrane and
bone surface, known as the resorption lacuna.56 Acid
dissolves the mineral phase of the bone matrix and provides
the optimal acidic environment for cathepsin K to initiate
degradation of the organic bone matrix. Initial organic matrix
degradation products are then endocytosed together with
cathepsin K into the osteoclast and transported through the
cell in transcytotic vesicles. 57,58 Vesicles containing TRACP
are fused into these vesicles, whose acidic pH allows
cathepsin K to cleave the loop-peptide, generating TRACP
5b with high specific activity. 24,59 When the vesicles move
away from the resorption lacuna their pH is changed to
neutral, providing an optimal environment for the ROS
generating activity of TRACP.15,24 TRACP then generates
ROS that are targeted to finalize degradation of the organic
matrix components during their transcytosis.23 Finally, the
matrix degradation products are released from the osteoclast
into the blood circulation together with TRACP 5b through
a functional secretory domain (FSD) in the basolateral
membrane. 60
As described previously, TRACP 5b is generated in
transcytotic vesicles of osteoclasts by cleavage of the
protease-sensitive loop-peptide by cathepsin K, and secreted
from the osteoclast through FSD at the end of the
transcytotic route. Based on this, it could be assumed that
secreted TRACP 5b would be a marker of osteoclast activity.
Surprisingly, this appears not to be the case. Instead, secreted
TRACP 5b has been shown in numerous studies to be an
accurate marker of osteoclast number. This concept was
introduced already in 1983 by Stepan and co-workers, who
observed that plasma TRACP activity was continuously
decreased after removal of parathyroid adenomas from
patients with primary hyperparathyroidism.36 Later, a strong
correlation of secreted TRACP 5b with osteoclast number
was observed in mouse osteoclast cultures where nonresorbing osteoclasts were cultured on plastic surface in the
absence of bone. 61 It was also noticed that serum TRACP 5b
had a strong correlation with histologically determined
number of osteoclasts in patients with renal bone disease, 62
and in orchidectomized rats.63 Secreted TRACP 5b has also
been shown to correlate with osteoclast number in patients
with autosomal dominant osteopetrosis type 2 (ADO2),64
various osteopetrotic rat strains and human osteoclast
cultures.65,66 These data together (summarized in table 3)
suggest that TRACP 5b could be secreted from osteoclasts at

_________________________________________________________________________________________________________________________
*Dr. Jussi Halleen is a consultant to Immunodiagnostic Systems (IDS). All correspondence to
IDS Ltd, 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD. UK

Review Series
Volume 3, April 06
Immunodiagnostic Systems (IDS).
a constant rate despite the resorbing activity of the cells.
A hypothesis of generation and secretion of TRACP 5b
in non-resorbing and resorbing osteoclasts is presented
in Figure 1.

Jussi Halleen

For Medical Educational Purposes Only.


Table 3: Summary of the evidence showing that TRACP 5b
is a marker of osteoclast number
Study setup
Hyperparathyroidism
Mouse osteoclast cultures

Reference
Stepan et al. 1983 (Ref. 36)
Alatalo et al. 2000 (Ref. 61)

Histology from human Chu et al. 2003 (Ref. 62)


subjects
Rat orchidectomy model
Alatalo et al. 2003a (Ref. 63)
ADO2 patients
Osteopetrotic rats
Human osteoclast cultures

Figure 1: Hypothesis for generation and secretion of


TRACP 5b A) in non-resorbing osteoclasts and B) in
resorbing osteoclasts. A) Vesicles containing cathepsin K
fuse into TRACP containing vesicles, and cathepsin K
cleaves TRACP to produce TRACP 5b that is secreted
through the basolateral membrane; B) Cathepsin K
containing vesicles are first transported into the ruffled
border membrane and cathepsin K is secreted into the
resorption lacuna where it starts degradation of the

Alatalo et al. 2004 (Ref. 64)


Alatalo et al. 2003b (Ref. 65)
Ylipahkala et al. 2005 (Ref.
66)

organic bone matrix. Cathepsin K is then endocytosed into


the osteoclast together with the initial matrix degradation
products, and the endocytosed vesicles fuse to the TRACP
containing vesicles. Cathepsin K then cleaves TRACP to
produce TRACP 5b, and the activated TRACP 5b generates
ROS that finalize the matrix degradation inside the vesicles.
Cleaved TRACP 5b is finally secreted through a functional
secretory domain in the basolateral membrane (Figure:
Hannele Ylipahkala).

_________________________________________________________________________________________________________________________
*Dr. Jussi Halleen is a consultant to Immunodiagnostic Systems (IDS). All correspondence to
IDS Ltd, 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD. UK

Review Series
Volume 3, April 06
Immunodiagnostic Systems (IDS).
Changes in bone resorption are usually associated with
changes in osteoclast number, suggesting that secreted
TRACP 5b may be a useful marker of bone resorption.
In fact, numerous clinical studies (summarized in table 4)
have demonstrated that TRACP 5b is elevated in bone
diseases such as postmenopausal osteoporosis,67- 69 breast
and prostate cancer bone metastases,67,70- 78 renal bone
disease,79-83 multiple myeloma,84-87 and Pagets disease of
bone.67 Additionally, it has been shown that TRACP 5b
values can predict future fracture risk. 88 Antiresorptive
treatment with estrogen and various bisphosphonates
resulted in decreased TRACP 5b levels (summarized in
table 5).52,68,70,71,73,77,84,86,87,89 - 94 Studies using human in vitro
osteoclast cultures have demonstrated that secreted
TRACP 5b activity correlates significantly with both
osteoclast number and bone resorption in the presence
of various antiresorptive agents, further demonstrating
that secreted TRACP 5b is a useful marker for
monitoring the efficacy of antiresorptive treatment.6 6 In
patients with ADO2 who have highly elevated number
of non-functional osteoclasts, serum TRACP 5b values
are exceptionally elevated, being much higher than in
any condition with increased bone resorption such as
osteoporosis and bone metastases. 64 In fact, the
exceptionally high TRACP 5b values can be used to help
diagnose ADO2. In this very rare disease, serum
TRACP 5b is a marker of osteoclast number, but
contrary to conditions of increased bone resorption such
as osteoporosis and bone metastases, it does not
demonstrate bone resorption.
Table 4: Summary of clinical studies showing that
TRACP 5b is elevated in bone diseases
Condition
Postmenopausal
osteoporosis
Breast cancer bone
metastases
Prostate cancer bone
metastases
Renal bone disease

Multiple myeloma
Pagets disease of bone
Fracture risk prediction

References

Halleen et al. 2001 (Ref. 67), Halleen et al.


2002 (Ref. 68), Rosenbrock et al. 2002 (Ref.
69)
Martinetti et al. 2002 (Ref. 70), Capeller et
al. 2003 (Ref. 71), Koizumi et al. 2003a
(Ref. 72), Koizumi et al. 2003b (Ref. 73),
Mose et al. 2003 (Ref. 74), Chao et al. 2004
(Ref. 75), Lyubimova et al. 2004 (Ref. 77)
Jung et al. 2004 (Ref. 76), Lyubimova et al.
2004 (Ref. 77), Salminen et al. 2005 (Ref.
78)
Takahashi et al. 2000 (Ref. 79), Albertini et
al. 2002 (Ref. 80), Chu et al. 2003 (Ref. 81),
Reichel et al. 2003 (Ref. 82), AvbersekLuznik et al. 2005 (Ref. 83)
Terpos et al. 2003a (Ref. 84), Terpos et al.
2003b (Ref. 85), Terpos et al. 2003c (Ref.
86), Terpos et al. 2004 (Ref. 87),
Halleen et al. 2001 (Ref. 67)
Gerdhem et al. 2004 (Ref. 88)

Jussi Halleen

For Medical Educational Purposes Only.


Table 5: Summary of clinical studies showing that TRACP
5b is decreased during antiresorptive treatment
Treatment
Estrogen
Raloxifene
Alendronate
Risedronate
Pamidronate
Clodronate
Ibandronate
Bisphosphonate
(Not identified)

References
Halleen et al. 2000 (Ref. 52), Halleen et al.
2002 (Ref. 68)
Hansdottir et al. 2004 (Ref. 91)
Hannon et al. 2004 (Ref. 90), Nenonen et al.
2005 (Ref. 92), Vlimki and Thtel 2005
(Ref. 94)
Vlimki and Thtel 2005 (Ref. 94)
Martinetti et al. 2002 (Ref. 70), Terpos et al.
2003a (Ref. 84), Terpos et al. 2003c (Ref.
86), Voskaridou et al. 2003 (Ref. 89)
Thtel et al. 2005 (Ref. 93)
Terpos et al. 2003c (Ref. 86),
Capeller et al. 2003 (Ref. 71), Koizumi et al.
2003b (Ref. 73), Lyubimova et al. 2004 (Ref.
77), Terpos et al. 2004 (Ref. 87)

MONITORING ANTIROSORPTIVE TREATMENT


The most important and accepted use of bone resorption
markers is their ability to monitor the efficacy of
antiresorptive treatment. For such monitoring, a baseline
value should be obtained from each individual prior to the
initiation of treatment. During treatment, marker
measurements should be repeated, for example at 3-month
intervals, and the values obtained should be compared with
the baseline value. If the treatment is effective, the marker
value should decrease to a new steady-state level within the
first 3 months of treatment, and stay in this reduced level
thereafter (see Figure 2 for TRACP 5b as an example). If the
marker value increases towards the baseline level at some
point during treatment, it means that for some reason, the
treatment is no longer effective. One important reason to
use bone markers in treatment monitoring is because bone
marker values show the patient that the treatment is working,
which increases their compliance to the treatment.
A recent study compared TRACP 5b with other commonly
used markers of bone turnover, including the bone
resorption markers serum C-terminal cross-linked
telopeptides of type I collagen (S-CTX) and total urinary
deoxypyridinoline (U-DPD), and the bone formation
markers serum procollagen I N-terminal propeptide (SPINP), serum total osteocalcin (S-OC) and serum bonespecific alkaline phosphatase (S-BAP) for monitoring
treatment with alendronate.9 2 Serum TRACP 5b values
decreased significantly during treatment (Figure 2). Table 6
demonstrates that although serum TRACP 5b values did not
decrease as much as the values for some other markers
during treatment, the sensitivity, area under ROC curve
(AUC) and signal-to-noise ratio were all higher for TRACP

_________________________________________________________________________________________________________________________
*Dr. Jussi Halleen is a consultant to Immunodiagnostic Systems (IDS). All correspondence to
IDS Ltd, 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD. UK

Review Series
Volume 3, April 06
Immunodiagnostic Systems (IDS).

For Medical Educational Purposes Only.

5b than for the other markers studied. This was because


the biological and analytical variability of TRACP 5b was
lower than those of the other markers, making TRACP
5b more reliable and allowing a smaller decrease to be
significant. Determination of TRACP 5b levels in blood
samples would allow frequent, inexpensive and
quantitative evaluation to evaluate disease progression as
well as treatment effectiveness.
Placebo
Alendronate

4,0
3,5

S-TRACP5b (U/L)

3,0
2,5

***

***

***

2,0
1,5
1,0
0,5
0,0
0

12

Time (months)

Figure 2: Serum TRACP 5b values decrease to a new steadystate level at 3 months after start of alendronate treatment,
and stay at this new level thereafter (from Nenonen et al.
2005).92
Table 6: Comparison of the clinical use of serum TRACP 5b
(S-TRACP5b) and other commonly used bone turnover
markers S-CTX, U-DPD, S-PINP, S-OC and S-BAP for
m onitoring alendronate treatment.
Marker

Change

CVa

CVi

LSC

Jussi Halleen

SPEC

SENS

AUC

Table 7: Comparison of the clinical use of various TRACP


methods for monitoring alendronate treatment.
S/N

S-TRACP5b
S-CTX
U-DPD

- 40
- 64
- 34

2.1
2.6
4.3

12.5
22.4
13.8

29.5
52.5
33.7

89.0
98.6
95.9

82.7
78.7
56.0

0.88
0.87
0.78

3.17
2.82
2.31

S-PINP

- 54

3.6

17.9

42.5

93.2

73.3

0.86

2.95

S-OC
S-BAP

- 28
- 27

5.2
2.1

14.4
9.4

35.7
22.4

94.5
90.4

40.0
61.3

0.76
0.78

1.84
2.78

Change, change of marker level at 3 months after start of treatment (%); CVa,
Analytical variability (%); CVi, Biological variability (%); LSC, Least significant
change (%); SPEC, specificity (%); SENS, sensitivity (%); AUC, Area under
ROC curve; S/N, signal-to-noise ratio (from Nenonen et al. 2005).92

Table 7 compares the clinical value of four different


TRACP immunoassays for monitoring alendronate
treatment.9 5 The assays include an immunoassay using a
selective pH to measure specifically TRACP 5b activity
(BoneTRAP, SBA-Sciences, Oulu, Finland), 52 an
immunoassay measuring both TRACP 5a and TRACP
5b activity (TRAP5, Biovendor, Brno, Czech

Republic),40-43 an immunoassay using naphthol ASBI


phosphate as a selective substrate for TRACP 5b activity
(ASBI),51 and a sandwich-type immunoassay measuring
both TRACP 5a and 5b amount (Sandwich).48 The results
demonstrate the clinical performance superiority of the
BoneTRAP assay among the tested four TRACP
immunoassays, probably because BoneTRAP has the
highest specificity for osteoclast-derived TRACP 5b. Because
the TRAP5 assay detects also TRACP 5a derived from
inflammatory macrophages and dendritic cells, it causes two
problems. Firstly, because approximately 55% of total
TRACP activity is of the non-osteoclastic 5a form in normal
conditions, 5 0 the clinical performance of the assay is
substantially disturbed even in subjects who have no changes
in inflammatory conditions, as demonstrated in table 7.
Secondly, values measured with the TRAP5 assay are
probably elevated in inflammatory conditions, disturbing the
bone-specificity of the assay. The subjects included in this
study were healthy, suggesting that inflammatory conditions
did not interfere with the results. Thus, clinical performance
of the TRAP5 assay is probably considerably lower in
cases where inflammatory conditions are involved. Use of
the 5a-specific substrate naphthol ASBI substrate does not
seem to increase the clinical performance compared with the
TRAP5 assay that uses the non-specific compound 4-NPP
as substrate. The clinical performance of the sandwich assay
is clearly lowest because approximately 87% of serum
TRACP enzyme amount is of the low -activity form 5a.50

Method

Change

CVa

CVi

LSC

SPEC

SENS

AUC

S/N

BoneTRAP

- 42

2.1

12.7

29.9

91.4

80.0

0.93

3.19

ASBI

- 33

3.3

12.1

29.1

97.0

60.0

0.84

2.56

TRAP5

- 27

3.2

9.7

23.7

95.5

60.0

0.86

2.63

Sandwich

-7

1.8

7.3

17.5

86.6

12.9

0.55

0.97

Change, change of marker level at 3 months after start of treatment (%); CVa,
Analytical variability (%); CVi, Biological variability (%); LSC, Least significant
change (%); SPEC, specificity (%); SENS, sensitivity (%); AUC, Area under ROC
curve; S/N, signal-to-noise ratio (from Fagerlund et al. 2005).95

ADVANTAGES OF TRACP 5B AS A RESORPTION


MARKER
Major problems of most serum markers of bone resorption
include their high diurnal variability and the effects of
feeding and renal function on the values obtained.96- 98
Because of the high diurnal variability, the serum samples

_________________________________________________________________________________________________________________________
*Dr. Jussi Halleen is a consultant to Immunodiagnostic Systems (IDS). All correspondence to
IDS Ltd, 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD. UK

Review Series
Volume 3, April 06
Immunodiagnostic Systems (IDS).
must be collected at exactly the same time of day. The
effect of feeding requires that the serum samples must
be collected after overnight fasting. Recent studies have
demonstrated that the diurnal variability and the effect
of feeding are caused by the gastrointestinal hormone
glucagon-like peptide-2 (GLP-2) that modulates
osteoclast activity.99 Since GLP-2 has no effect on
osteoclast number, it should not affect serum TRACP
5b levels. Thus, serum TRACP 5b levels should not be
affected by feeding, and the diurnal variability of serum
TRACP 5b should be low. These assumptions have been
verified by Hannon and co-workers, who showed that
contrary to serum CTX, a marker of osteoclast activity,
serum TRACP 5b measured using the BoneTRAP
assay has a low diurnal variability and is not affected by
feeding.90
Renal function affects the marker values because the
markers are cleared from the circulation through the
kidneys, and when the kidneys do not work properly, the
markers can not be cleared from blood circulation,
leading to increased marker levels. This can lead to
elevated marker levels in conditions where bone
turnover is normal.97 This is a particularly serious
problem in renal osteodystrophy, a bone disease
affecting the majority of patients with chronic renal
failure, causing confusion in interpretation of bone
marker results. TRACP 5b is cleared from the blood
circulation through the liver,49 and the function of the
kidneys has no effect on serum TRACP 5b values.67,90
TRACP 5b is secreted from osteoclasts as an active
enzyme that is inactivated and degraded to fragments
before clearance from the blood circulation by the liver.
Thus, in hepatic failure, the inactive fragments
accumulate in the circulating blood, while the
enzymatically active TRACP 5b molecules measured in
the BoneTRAP assay do not accumulate. 67
CONCL USION
TRACP 5b is probably the most reliable marker of bone
resorption available. TRACP 5b is derived exclusively
from bone-resorbing osteoclasts. Contrary to most other
commonly used bone markers, TRACP 5b does not
accumulate in blood circulation during renal or hepatic
failure, has a low diurnal variability and is not affected by
feeding. TRACP 5b describes the number of osteoclasts
in addition to their activity, and appears to be a highly
specific and sensitive marker of bone resorption. Serum
TRACP 5b is elevated in bone diseases such as

Jussi Halleen

For Medical Educational Purposes Only.


postmenopausal osteoporosis, breast and prostate cancer
bone metastases, multiple myeloma, renal bone disease and
Pagets disease of bone, and serum TRACP 5b values predict
future fracture risk. Antiresorptive treatment with estrogen,
raloxifene and bisphosphonates significantly decreases
TRACP 5b values. The clinical performance of TRACP 5b
for treatment monitoring is among the best of known bone
turnover markers. Because secreted TRACP 5b is an
accurate marker of osteoclast number, it can be conveniently
used to replace histomorphometric determination of
osteoclast number in animal models and microscopic
counting of osteoclasts in in vitro bone cell cultures.
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IDS Ltd, 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD. UK

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IDS Ltd, 10 Didcot Way, Boldon Business Park, Boldon, Tyne & Wear, NE35 9PD. UK