Blood Cells, Molecules and Diseases 52 (2014) 91–94
Contents lists available at ScienceDirect
Blood Cells, Molecules and Diseases
journal homepage: www.elsevier.com/locate/bcmd
Thalassemic osteopathy: A new marker of bone deposition
M. Baldini a,b,⁎, S. Forti c, A. Orsatti a,b, A. Marcon a,b, F.M. Ulivieri d, L. Airaghi b,e, L. Zanaboni a,b, M.D. Cappellini a,b
a
U.O. Medicina Interna 1A, Fondazione IRCCS “Ca' Granda” Ospedale Maggiore Policlinico, Milano, Italy
Dipartimento di Medicina Interna, Università degli Studi di Milano, Milano, Italy
c
U.O. Audiologia, Fondazione IRCCS “Ca' Granda” Ospedale Maggiore Policlinico, Milano, Italy
d
U.O. Medicina Nucleare, Servizio di Mineralometria Ossea Computerizzata-Ambulatorio Malattie Metabolismo Minerale e Osseo, Fondazione IRCCS “Ca' Granda” Ospedale Maggiore Policlinico,
Milano, Italy
e
U.O. Medicina Interna 1B, Fondazione IRCCS “Ca' Granda” Ospedale Maggiore Policlinico, Milano, Italy
b
a r t i c l e
i n f o
Article history:
Submitted 21 May 2013
Revised 22 July 2013
Available online 3 October 2013
(Communicated by G. Stamatoyannopoulos,
M.D., Dr. Sci., 02 August 2013)
Keywords:
Thalassemia
Osteoporosis
Bone turnover
P1NP
a b s t r a c t
Osteopathy represents a prominent cause of morbidity in patients with beta-thalassemia major (TM) and manifests as osteopenia/osteoporosis. Biochemical turnover markers (BTMs) are considered a useful, non-invasive
tool for the clinical follow-up of osteoporotic patients; they can provide a dynamic view of the remodeling process and give information on the metabolic activity of bone tissue as well as on the pathogenesis of bone loss. The
amino-terminal pro-peptide of type I procollagen (P1NP) is a recently introduced marker that is considered the
most sensitive index of bone formation. Although demonstrated in several categories of patients with bone disease, there is little information on the clinical usefulness of this bone formation index in thalassemic patients. We
evaluated the P1NP levels of 53 adult patients with b-thalassemia major (21 males and 32 females, mean age
34.5 ± 5.7, range 22–46 years) and associated osteopathy. We investigated the correlation between P1NP and
bone condition as examined by dual X-ray photon absorptiometry and with BTMs expressing bone resorption
and bone mineralization (carboxyterminal collagen cross-linked (CTX) terminal regions of type I collagen and
osteocalcin, respectively). P1NP serum levels were correlated with CTX levels (r = 0.545, p b 0.001); the results
were unchanged when males and females, as well as osteoporotic and osteopenic subgroups, were considered
separately. No correlation was demonstrated neither between OC and CTX (r = 0.17, p = ns), nor between
P1NP and OC levels (r = 0.11, p = ns). No correlation was demonstrated among the P1NP/CTX ratio and age,
OC or densitometric values and no difference was found in the same ratio between osteopenic (0.19 ± 0.16)
and osteoporotic (0.15 ± 0.14) patients. Similar results were obtained for the OC/CTX ratio, as it was not correlated with age, P1NP or densitometric values.
This is the first report of circulating P1NP in patients with TM-associated osteoporosis. P1NP and CTX assays show
good precision and low analytical CV, and, compared to other markers, they can acceptably reflect bone metabolic
processes and promptly respond to antiosteoporotic treatments. We trust that this sensitive marker can be useful
in the assessment of treatment efficacy and can overcome the pitfalls due to wide variability in the normal values
of most BTMs that create difficulty in pinpointing the individual patient's response.
© 2013 Elsevier Inc. All rights reserved.
Introduction
Osteopathy represents a promi'nent cause of morbidity in patients
with beta-thalassemia major (TM), even among well-treated ones, and
Abbreviations: TM, thalassemia major; BTMs, biochemical turnover markers; OC,
osteocalcin; P1NP, amino-terminal pro-peptide of type I procollagen; CTX, carboxyterminal
collagen cross-linked; SDS, standard deviation score; BMD, bone mineral density; BMC,
bone mineral content.
⁎ Corresponding author at: U.O. Medicina Interna 1A, Fondazione IRCCS “Ca' Granda”
Ospedale Maggiore Policlinico, via F. Sforza 35, 20122 Milano, Italy.
E-mail addresses: marina.baldini@policlinico.mi.it (M. Baldini),
stella.forti@policlinico.mi.it (S. Forti), alessandra.orsatti@unimi.it (A. Orsatti),
alessia.marcon81@gmail.com (A. Marcon), ulivieri@policlinico.mi.it (F.M. Ulivieri),
lorena.airaghi@policlinico.mi.it (L. Airaghi), laura.zanaboni@policlinico.mi.it (L. Zanaboni),
maria.cappellini@unimi.it (M.D. Cappellini).
1079-9796/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bcmd.2013.09.008
manifests as osteopenia/osteoporosis [1]. The underlying mechanism
is unbalanced bone turnover, with increased resorption vs deposition
rates. Chronic anemia with bone marrow expansion, iron toxicity and endocrine complications are recognized as major contributing factors [1,2].
Biochemical turnover markers (BTMs) are considered a useful, noninvasive tool for the clinical follow-up of osteoporotic patients that can
provide a dynamic view of the remodeling process, especially during
treatment with bisphosphonates, and give information on the metabolic
activity of bone tissue as well as on the pathogenesis of bone loss. An increase in bone resorption markers and a reduction in bone deposition
markers have been attributed to osteoblast “poisoning” by iron overload
and have been described as the prominent causes of bone loss in TM
[3,4].
Classically, bone formation has been studied with bone alkaline phosphatase, a marker of osteoblastic activity, or with osteocalcin (OC), which
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M. Baldini et al. / Blood Cells, Molecules and Diseases 52 (2014) 91–94
is a marker of bone mineralization. Although these are undoubtedly valuable tools, the laboratory test results for both are influenced by technical
aspects that impair sensitivity and increase variability, leading to reduction of clinical usefulness [5].
The amino-terminal pro-peptide of type I procollagen (P1NP) is a recently introduced marker that is considered the most sensitive index of
bone formation in patients with bone disease of varying origins [6–12].
As information about circulating P1NP in patients with TM-associated
osteoporosis is lacking, we evaluated the P1NP levels of a group of thalassemic patients with osteopenia or osteoporosis and investigated their
correlation with other BTMs and bone conditions.
Patients and methods
Patients
From a large cohort of 150 adult patients with b-thalassemia major
followed up at the Hereditary Anemia Center of Fondazione IRCCS Ca'
Granda Policlinico of Milan, we considered the data of 53 patients
(21 males and 32 females, mean age 34.5 ± 5.7, range 22–46 years)
with osteopathy who underwent a complete bone evaluation for clinical
purposes. All of the patients were regularly transfused and sufficiently
chelated; furthermore, all were in a satisfactory control from the endocrinological point of view with treatment for hypogonadism, hypoparathyroidism, hypothyroidism and vitamin D deficiency.
Methods
Fasting blood samples for BTMs were obtained in the morning for all
subjects via venipuncture and centrifuged within 1 h of blood sampling;
the serum was frozen at −80 °C. Bone resorption was evaluated by
measuring the carboxyterminal collagen cross-linked (CTX) terminal
regions of type I collagen (CTX ELISA, IDS Nordin, Boldon Colliery,
UK), which has normal values in post-menopausal women of 0.142–
1.351 ng/mL, in pre-menopausal women of 0.112–0.738 ng/mL, and
in males of 0.115–0.748 ng/mL. Bone formation was assessed by the
serum levels of the N-terminal propeptide of type I procollagen or
P1NP (UniQ P1NP RIA, ORION Diagnostica, Espoo, Finland), which has
normal values of 19–102 μg/L in females and 21–78 μg/L in males.
Finally, bone mineralization was evaluated by assaying the levels of
osteocalcin (N-MID Osteocalcin ELISA, IDS Nordin, Boldon Colliery,
UK), which has normal values in post-menopausal women of 8.4–
33.9 ng/mL, in pre-menopausal women of 12.8–55.0 ng/mL, and in
males of 9.6–40.8 ng/mL. The assays were performed according to the
manufacturer's instructions, and a standard curve was included in each
assay plate.
The bone densitometry scan was performed by dual X-ray photon absorptiometry (Hologic Bone Densitometer QDR Discovery A, Version
12.7.3.1 WALTHAM, MA, USA) at the lumbar spine and femur in all of
the patients; bone mineral density (BMD) was calculated as a ratio
between bone mineral content (BMC) and area in square centimeters.
The bone mineral density values were expressed as T- and Z-scores; the
T-score was calculated as a standard deviation score (SDS) from a normal
reference population database, while the Z-score was calculated as an SDS
from an age- and sex-matched population. Data were classified according
to the WHO report (WHO Technical Report, ISCD Official Position Paper
2007) as follows: T-score N −1 = normal, −1 N T-score N −2.5 = low
bone density (osteopenia), T-score b −2.5 = osteoporosis.
was performed to evaluate the same correlations controlling for gender
and bone mineralization status (osteopenia/osteoporosis). Finally, in
consideration of the small size of the subgroups the nonparametric
Mann–Whitney test was used to analyze the differences between genders and between hypogonadal/eugonadal patients. p values b 0.05
were considered significant. All statistics were calculated using the Statistical Package for the Social Sciences 20.0 for Windows software package (SPSS Inc., Chicago, IL).
Results
Among 53 TM patients, 41 (77%) were affected by osteoporosis and
12 (23%) by osteopenia. Eight patients (14.5%), 4 males and 4 females,
were taking alendronate, which was prescribed in the presence of
DXA values in the range for osteoporosis associated to high risk for fractures or previous fragility fractures.
The average values of BTM and bone mineral density are shown in
Tables 1 and 2, respectively. There were 41 hypogonadal patients
(77%), 17 males and 24 females, all on sex steroid replacement. There
were no differences in the BTM levels neither between males and females nor between hypogonadal and eugonadal patients.
As shown in Fig. 1, P1NP serum levels were correlated with CTX
levels (r = 0.54, p b 0.001); the results were unchanged when males
and females, as well as osteoporotic and osteopenic subgroups, were
considered separately. No correlation was demonstrated neither between OC and CTX (r = 0.17, p = ns), nor between P1NP and OC levels
(r = 0.11, p = ns).
No correlation was demonstrated among the P1NP/CTX ratio and age,
OC or densitometric values and no difference was found in the same ratio
between osteopenic (0.19 ± 0.16) and osteoporotic (0.15 ± 0.14) patients. Similar results were obtained for the OC/CTX ratio, as it was not
correlated with age, P1NP or densitometric values.
Discussion
There is great interest in the etiology and management of thalassemic osteopathy due to the high prevalence of this complication even
in well-treated thalassemic patients. The determination of biochemical
markers of bone turnover provides important information about bone
metabolism disorders and treatment responses; thus, their use should
be implemented. Among the greatest advantages of these markers are
their non-invasiveness, repeatability and ability to change quickly in
response to treatment. Although recent laboratory improvements have
increased the ability of BTM assays to characterize bone disease, both biological variability and laboratory variability compromise their usefulness
in reflecting bone metabolism [3,5,8,13,14]. The expert opinion is that no
perfect marker exists [5].
Initially, the total alkaline phosphatase in serum and urinary hydroxyproline were adopted as bone turnover indices, but they were
later replaced by bone-specific alkaline phosphatase and OC. The former
Table 1
Biochemical bone turnover markers in 53 patients with thalassemic osteopathy (means ±
SD, median and range).
Males (n = 21)
Females (n = 32)
Statistical analysis
Descriptive data (frequency, mean, standard deviation, median,
range) were calculated for all variables; moreover, for P1NP variable
percentiles also calculated. Pearson's index was used to evaluate the
correlations among CTX, OC, P1NP, bone mineralization, as expressed
by T and Z-scores, and BTMs. Furthermore, partial correlation analysis
Osteoporosis (n = 41)
Osteopenia (n = 12)
Osteocalcin (ng/mL)
P1NP (μg/L)
CTX (pg/mL)
18.9 ± 19.3
13.1
(3.6–97.5)
22.3 ± 18.6
18.0
(3.8–80.1)
19.4 ± 15.5
14.8
(3.6–80.1)
26.2 ± 27.5
15.6
(7.3–97.5)
57.2 ± 53.1
49.9
(13.6–263.8)
60.2 ± 41.5
46.4
(18.5–207.6)
56.6 ± 39.6
43.7
(13.6–207.6)
67.2 ± 64.8
52.8
(18.5–263.8)
487.6 ± 339.1
456.3
(41.4–1556.0)
490.5 ± 309.4
420.6
(50.0–1400.0)
499.6 ± 297.2
473.6
(41.4–1400.0)
454.4 ± 394.9
335.6
(135.9–1556.0)
M. Baldini et al. / Blood Cells, Molecules and Diseases 52 (2014) 91–94
93
Table 2
Densitometric characteristics (mean ± SD, median and range) of thalassemia major patients.
T-score
Males (n = 21)
Females (n = 32)
Osteoporosis (n = 41)
Osteopenia (n = 12)
Eugonadic (n = 12)
Hypogonadic (n = 41)
Z-score
Vertebral
Femoral total
Femoral neck
Vertebral
Femoral total
Femoral neck
−2.7 ± 0.9
−2.4
(−4.3–(−1.3))
−2.3 ± 1.1
−2.3
(−4.3–0.1)
−2.8 ± 0.8
−2.7
(−4.3–(−0.6))
−1.2 ± 0.6
−1.3
(−1.9–0.1)
−2.4 ± 1.0
−2.3
(−4.3–0.1)
−2.5 ± 1.1
−2.5
(−4.3–(−0.6))
−2.0 ± 0.9
−1.8
(−3.9–(−0.2))
−1.8 ± 1.1
−2.0
(−3.9–1.7)
−2.1 ± 1.0
−2.3
(−3.9–(1.7))
−1.2 ± 0.6
−1.2
(−1.9–(−0.2))
−1.9 ± 1.0
−1.9
(−3.9–1.7)
−1.7 ± 0.9
−2.0
(−3.2–(−0.2))
−1.9 ± 1.0
−1.7
(−3.5–(−0.2))
−1.8 ± 1.1
−1.7
(−4.8–1.1)
−2.0 ± 1.1
−2.2
(−4.8–1.1)
−1.2 ± 0.7
−1.4
(−2.1–(−0.1))
−1.8 ± 1.1
−1.7
(−4.8–1.1)
−1.7 ± 0.8
−1.6
(−3.0–(−0.3))
−2.7 ± 0.9
−2.4
(−4.3–(−1.3))
−2.2 ± 1.1
−2.2
(−4.0–0.2)
−2.7 ± 0.8
−2.6
(−4.3–(−0.3))
−1.2 ± 0.6
−1.2
(−1.8–0.2)
−2.4 ± 1.0
−2.2
(−4.0–0.2)
−2.5 ± 1.1
−2.5
(−4.3–(−0.4))
−1.9 ± 0.9
−1.8
(−3.8–(−0.2))
−1.8 ± 0.9
−1.9
(−3.8–(−0.2))
−2.1 ± 0.8
−2.2
(−3.8–(−0.2))
−1.1 ± 0.5
−1.1
(−1.8–(−0.2))
−1.9 ± 0.9
−1.8
(−3.8–(−0.3))
−1.7 ± 0.9
−1.9
(−3.1–(−0.2))
−1.6 ± 0.9
−1.4
(−3.30–(−0.1))
−1.7 ± 0.9
−1.5
(−4.70–0.1)
−1.8 ± 0.9
−1.9
(−4.7–(−0.1))
−1.0 ± 0.6
−1.1
(−1.8–0.1)
−1.6 ± 0.9
−1.4
(−4.7–0.1)
−1.6 ± 0.8
−1.5
(−2.8–(−0.3))
reflects the cellular activity of osteoblasts, while the latter is considered
a formation marker whose exact function remains unclear [5]. Unfortunately, the high within-method interlaboratory variability (CV 16–48%
and 16–42%, respectively) [15] and major drawbacks of these assays
impose considerable limitations on their clinical application. In particular, the reliability of bone alkaline phosphatase is impaired by crossreactivity between bone and liver isoforms. However, the rapid degradation of OC in serum and incorporation into the bone matrix lead to the
degradation of the native peptide in heterogeneous fragments, which
can be detected by commercial kits even with poorly defined specificity
and cross-reactivity [5]. Furthermore, OC is greatly influenced by genetics
and, though related to fracture risk, is not considered a responsive indicator of bone metabolism changes [16,17].
Recently, the serum concentrations of some byproducts of collagen
and proteins released during osteoid deposition were shown to reflect
the bone formation rate [18,19]. Among these byproducts, the aminoterminal propeptide (P1NP) of type 1 procollagen, which represents
95% of total bone collagen, is considered to possess the highest specificity
and sensitivity. Furthermore, as its serum concentrations are stoichiometrically equivalent to the new collagen molecules synthesized by osteoblasts [20], P1NP is considered to most accurately reflect changes in
new collagen synthesis [6]. In addition, although none of the collagen
Fig. 1. Correlation between amino-terminal pro-peptide of type I procollagen (P1NP) and
carboxyterminal cross-linking telopeptide of type I procollagen (CTX) serum levels in the
patients with thalassemia major.
formation byproducts are specific for bone, it is believed that most P1NP
is produced during bone deposition.
Although demonstrated in several categories of patients with bone
disease [7,9] there is little information on the clinical usefulness of this
bone formation index in thalassemic patients; though recognizing the
limits of the present study due to the low number of patients and the
wide range of results, we believe that our experience can be considered
an interesting first step to be continued with a larger study. A further
limit of the present study is the cross-sectional design; the absence of correlation between BMTs and bone mineral density can be explained as
bone metabolism is a dynamic multifactorial process, and a longitudinal
study could better demonstrate the relationships between the changes
in bone metabolism and the resulting mineral density.
We did not find significant differences between hypogonadic and
eugonadic thalassemic pts neither for bone turnover markers nor for
DXA parameters expressing bone mineralization. We observed, however, an interesting trend to higher levels in the two markers of bone deposition (P1NP and osteocalcin) in the hypogonadal group, where the
bone resorption marker CTX was even higher; it is likely that the increased bone turnover due to hypogonadism is not completely normalized despite an adequate replacement therapy. The lack of statistical
significance may be due to statistical reasons (low number of patients,
different numbers in the two groups); we believe that this finding warrants further exploration.
It is well known that bone resorption is physiologically coupled to reformation processes, with a net effect maintaining an unchanged bone
mass; this is crucial for the repair of microfractures and allows bone structure response to biomechanical impacts. According with bone physiology,
we found a correlation between P1NP and CTX in our series of thalassemic
patients. The absence of a correlation between OC and the other markers
evaluated (P1NP and CTX) can be explained by the lower reliability of
osteocalcin measurement; this could be related to the well-known
lower sensitivity of the assay and the pre-analytical problems due to molecule instability [7,8,10]. In contrast, P1NP remains stable for at least 48 h
in serum stored at room temperature [5].
Increased remodeling with subsequent bone loss is a feature of osteopathy in patients with TM [13]. This change is the rationale for the
therapeutic use of bisphosphonates in this disease, as well as the explanation for the increased effectiveness of these agents in TM over thalassemia intermedia (TI), where demineralization does not seem to be
caused by increased bone turnover [13]. In this preliminary study, we
did not find significant differences in the P1NP serum concentrations
between patients on alendronate and those not on bisphosphonate treatment, but the low number of patients treated and the cross-sectional
study protocol did not suit this aim. In the immediate future, we are
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M. Baldini et al. / Blood Cells, Molecules and Diseases 52 (2014) 91–94
planning a prospective study including a P1NP assay in TM before and
after antiresorptive treatment. We trust that this sensitive marker can reveal useful in the assessment of treatment efficacy and can overcome the
pitfalls due to wide variability in the normal values of most BTMs, that
create difficulty in pinpointing the individual patient's response.
Conclusion
P1NP and CTX are biochemical bone turnover markers widely used
for monitoring patients with primary and secondary osteoporosis, as
they can acceptably reflect bone metabolic processes and promptly respond to anti-osteoporotic treatments, and the assay methods are provided with good precision and low analytical CV [21,22]. To the best of
our knowledge, this is the first report of P1NP serum levels in patients
with TM-associated osteoporosis; the measurement of this sensitive
marker in a larger series of TM could be useful in the clinical followup of the changes in bone metabolism related to disease and drug response. Further studies on this topic are anticipated with great interest,
as they will help in the decision-making process.
Conflict of interest
None of the authors had actual nor potential conflict of interest.
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