Decreased Bone Density, Elevated Serum Osteoprotegerin,
and ?-Cross-Laps in Wilson Disease
D. HEGEDUS, V. FERENCZ, P.L. LAKATOS, S. MESZAROS, P. LAKATOS,
C. HORVATH, and F. SZALAY
Osteopathia has been reported in Wilson disease (WD), but bone density has not been measured; therefore,
we performed bone mineral density (BMD), bone mineral content (BMC), and quantitative bone ultrasound
(QUS) assessments, as well as measured the serum levels of osteocalcin (OCN), ?-cross-laps (?-CTx’s), and the
recently discovered osteoprotegerin (OPG) and its ligand RANKL to investigate the underlying mechanism of
osseous disorders. Serum OCN, ?-CTx, OPG, and RANKL levels were measured by ELISA in 21 WD patients
and in 20 age- and gender-matched healthy subjects. BMD, BMC, and QUS parameters were also determined.
Osteoporosis was present in 9/21 (43%) WD patients. Abnormal QUS parameters were found in 7 (33%) of
the patients. Although serum OCN levels were similar in patients and controls (29.93 ? 24.65 mg/ml vs.
29.84 ? 6.89 mg/ml), ?-CTx and OPG levels were significantly increased in WD compared with the healthy
controls (625.4 ? 312.3 pg/ml vs. 423.6 ? 144.3 pg/ml and p ? 0.022 and 7.2 ? 3.4 pM vs. 3.5 ? 1.0 pM and
p < 0.001, respectively). No difference was observed in the RANKL level. There was a positive correlation
between OCN and ?-CTx (r ? 0.55; p ? 0.01). We proved high occurrence of osteoporosis in WD. Negative
bone remodeling balance is a consequence of increased bone resorption, which is indicated by elevated ?-CTx.
The novel finding of elevated serum OPG may reflect a compensatory reaction to enhanced osteoclast activity,
despite the normal OCN level. (J Bone Miner Res 2002;17:1961–1967)
Key words:Wilson disease, osteoprotegerin, RANKL, osteocalcin, ?-cross-laps
transporting ATP7B protein gene located on the 13th chro-
mosome. The first studies appeared in the 1950s.(1,2)Min-
subarticular cysts, and marginal fragmentation in 33 out of
38 patients.(3)Aksoy reported that the bone changes ranged
from mild to severe and appeared to be age related.(4)The
changes they observed included osteochondritis dissecans
and subchondral changes, marginal bone fragmentation,
KELETAL CHANGES have been reported in Wilson disease
(WD), which is an inborn error of the copper-
cystic changes, renal rickets, and Milkman pseudofractures.
Other changes such as osteomalacia, osteoporosis, osteo-
chondritis of spine, osteoarthritis, spontaneous fractures and
para-articular calcifications, angulation of carpal bones, and
squaring of metacarpal heads have also been described.(1,2,5)
However, to describe the degree of the osteopathy, pre-
vious studies have not investigated the bone mineral density
(BMD) and bone mineral content (BMC) in WD, which
recently have become important parameters in the assess-
ment of the bone quantity. Biochemical markers of bone
turnover are also important in the assessment of osteoblastic
and osteoclastic functions. Osteocalcin (OCN) is a noncol-
lagenous protein secreted by osteoblasts and is widely ac-
cepted as a marker for osteoblastic activity(6)and hence,
The authors have no conflict of interest.
First Department of Medicine, Semmelweis University, Budapest, Hungary.
JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 11, 2002
© 2002 American Society for Bone and Mineral Research
bone formation,(7)whereas serum ?-cross-laps (?-CTx’s),
which is a collagen-degradation product, is a marker of bone
The recent discovery of osteoprotegerin (OPG; OPG/
osteoclastogenesis inhibitory factor/tumor necrosis factor
receptor [OPG/OCIF/TNFR]) and its ligand (OPG ligand/
RANKL]), which play a crucial role in the balance of bone
remodeling, provides new insights into the regulation of
osteoclastogenesis,(9)bone formation, and normal bone de-
velopment. RANKL is an endogen osteoclast-activating
factor, secreted mostly by osteoblasts and T and B cells as
well as monocytes. OPG acts as a decoy receptor for
RANKL and prevents its function.(10)Increased serum OPG
levels are reported in renal osteodystrophy,(11)rheumatoid
arthritis,(12)Cushing’s disease, human immunodeficiency
virus (HIV) patients, and common variable immunodefi-
ciency (CVI)(13)in patients with advanced prostate cancer
with bone metastasis(14)and in primary biliary cirrhosis.(15)
Reduced OPG levels were found in patients with multiple
myeloma with lytic bone disease.(16)
WD is inherited in an autosomal recessive fashion, and
?200 mutations have been described. The prevalence of the
disease is ?1/30 000. The defect in the copper transporter
protein results in an excessive accumulation of copper in
many organs: liver, central nervous system, cornea, kidney,
and skeletal tissues. The disease most often manifests in
hepatic disorders ranging from acute hepatitis, chronic ac-
tive hepatitis, fulminant hepatic failure, cirrhosis; in neuro-
logical symptoms such as tremor, choreoathetoid movement
disorders, dystonia, dysarthria, dysphagia; and in psychiat-
Our goal was to investigate the underlying mechanism of
bone abnormalities in WD. Because bone disorders are
common in WD, we determined the BMD and BMC pa-
rameters, as well as some quantitative ultrasound (QUS)
parameters. QUS can provide further information on bone
density, as well as on bone structure.(17–19)We also mea-
sured serum OCN, ?-CTx, OPG, and RANKL levels in
patients with WD.
MATERIALS AND METHODS
Twenty-one patients with WD (9 women and 12 men;
mean age, 30.8 years; range, 14–46 years) from the Hepa-
tological Outpatient Unit of Semmelweis University,
Budapest were involved in the study. Mean duration of
d-penicillamine treatment was 110.7 ? 163.6 months. The
diagnosis of WD was based on characteristic clinical labo-
ratory findings, presence of Kayser-Fleischer ring, and ge-
netic testing. To confirm the diagnosis, the presence of
excessive copper accumulation in the liver biopsy specimen
and increased 24-h urinary copper excretion (?100 ?g/day)
were required in some cases. Serum ceruloplasmin level
was low in each patient. Abnormal liver tests and signs of
liver disease without neurological disorder were present
in 8 patients; neurological symptoms such as tremor,
choreoathetoid movements, coordination disturbances, and
Parkinson-like symptoms were present in 13 patients; and
psychiatric and behavioral disturbances were present in 5
patients. In most patients, characteristically, more than one
abnormality was present. Kayser-Fleischer ring by slit-lamp
examination, investigated by an experienced ophthalmolo-
gist, was present in 13 patients. The H1069Q gene mutation
of ATP7B gene, which is the most common mutation in
Hungary(20)among the ?200 described mutations in WD,
was assessed by a semi-nested polymerase chain reaction
(PCR)–based restriction fragment length polymorphism
(RFLP) assay. Eight patients were homozygous, 7 patients
were heterozygous, and 6 patients were negative for
Among the 21 patients with WD, a 45-year-old female
patient had compression fracture of the thoracic vertebrae at
the age of 44 years, but none of the other patients had
pathological bone fractures. However, 2 more patients com-
plained about articular pain (back pain and large joint pain).
A 40-year-old woman had prominent spondyloarthrosis and
a 14-year-old boy had severe knee pain. This boy was
investigated and treated by rheumatologists throughout 2
years when WD was diagnosed. The X-ray showed de-
creased articular gap of the right knee and calcification of
both menisci. A radiographic study was not performed on
any WD patients, except for the aforementioned three cases
in which patients had actual skeletal complaints.
The control group was recruited from 20 age- and gender-
matched (10 men and 10 women) individuals with a mean
age of 28.3 ? 8 years (range, 19–51 years). We also
involved a second control group, which comprised 74 post-
menopausal osteopenic (?2.5 ? T score ? ?1), otherwise
healthy, women (mean age, 55.7 years; range, 47–71 years).
The reason for the latter control group was to assess whether
the elevated OPG level is associated merely with decreased
bone density or if it is a consequence of WD. The study
protocol conforms to the ethical guidelines of the 1975
Declaration of Helsinki and was approved by the local
Regional Committee of Science and Research Ethics.
Bone densitometry and QUS
BMD (g/cm2), assessed by Norland XR26 (Norland, Fort
Atkinson, WI, USA) DXA, was performed at the lumbar
L2–L4 vertebrae and the left femoral neck (FN); BMD, Z
score, and T score were determined for each patient and
control. The distal radius was investigated by measurements
of BMC (g/cm) by single photon absorptiometry (SPA)
method (Gamma NK-364; Gamma, Budapest, Hungary).
Heel broadband ultrasound attenuation (BUA) and speed of
sound (SOS) were assessed by DTU-1 (Osteometer Medi-
Tech, Hawthorne, CA, USA) device.
According to the World Health Organization (WHO)
guidelines,(21)osteoporosis is defined as the BMD or BMC
values being ?2 SD below the average values of the age-
matched healthy population (Z score ? ?2).(22)The same
criterion (Z score ? ?2) was used for the QUS parameters.
However, there are no QUS guidelines for diagnosing
secondary osteoporosis in young patients, although there
are several studies involving postmenopausal osteoporo-
1962HEGEDUS ET AL.
Laboratory testing was performed by an Olympus AU600
autoanalyzer (Olympus, Ltd., Shizouka, Japan). Serum bil-
irubin and the liver function tests (aspartate aminotransfer-
ase [AST], alanine aminotransferase [ALT], gamma glu-
tamil transferase [GGT], and alkaline phosphatase [ALP])
as well as the serum calcium and phosphate levels were
investigated in each patient. We also measured the OCN
level for each patient and control with a commercially
available N-MID OCN Electrochemiluminescence Immu-
noassay (ECLIA) kit (Roche Diagnostics GmbH, Mann-
heim, Germany), as well as determined the serum levels of
type 1 collagen cross-linked C-telopeptide (?-CTx) by the
Elecsys ?-Cross-laps CalSet commercially available immu-
noassay kit (Roche Diagnostics GmbH). Both measure-
ments were performed for patients and controls by Elecsys
2010 immunoassay system (Hitachi, Tokyo, Japan).
OPG level measurement
For the measurement of serum OPG, blood was drawn
from fasting subjects into 5-ml vacuum containers with
added potassium-EDTA as anticoagulant. Blood was cen-
trifuged at 3000 rpm for 10 minutes at ?4°C (Rotina 35 R;
Hettich, Tuttingen, Germany) and the plasma was collected
in 2-ml plastic tubes. After the separation of the plasma, we
stored the samples at ?20°C until the measurement. Serum
OPG level was measured with a commercially available
ELISA kit according to the protocol of the manufacturer.
(Osteoprotegerin Kit; Biomedica GmbH, Vienna, Austria).
The assay was performed blind to the subject group. The
absorption was determined with an ELISA reader (Lab-
systems Multiscan MS; Helsinki, Finland) at 450 nm
against 690/620 nm.
RANKL level measurements
The same serum samples were used for RANKL mea-
surements as those used for OPG measurements. Serum
RANKL level was also measured with a commercially
available ELISA kit according to the protocol of the man-
ufacturer. (sRANK Kit; Biomedica GmbH). The assay was
performed blind to the subject group. The absorption was
determined with an ELISA reader (Labsystems Multiscan
MS) at 450 nm against 690/620 nm.
OPG levels were compared with Kruskal-Wallis one way
ANOVA on ranks and Dunn’s method was used for the
pairwise multiple comparison procedures. OCN, ?-CTx,
and RANKL levels in patients with WD were compared
with the healthy controls with Mann-Whitney rank sum test.
Associations between age, bilirubin, liver function tests,
OCN, ?-CTx, OPG, RANKL level, and BMD were tested
with Spearman rank order correlation. Data are presented as
mean ? SD if otherwise not stated.
Densitometry and QUS
According to the densitometric measurements, osteopo-
rosis was found in 9 (43%) out of 21 patients with WD.
Z-score ? ?2 was measured in 4/14 (29%) patients who
were ?40 years old, and in 4/7 (71%) patients who were
?40 years old. According to the QUS values, 7/21 (33%)
patients had BUA Z scores of ? ?2. Heel SOS Z scores
were in the normal range for each patient. The mean ? SD
for BMD and BMC score is summarized in Table 1, and the
QUS parameters are summarized in Table 2. Bone density
parameters did not correlate with age or liver function tests.
Laboratory liver tests
Most patients had normal or mildly elevated serum liver
enzyme levels; the serum bilirubin was in the normal range.
The calcium and phosphate levels were in the reference
range for each patient.
TABLE 1. BMD (G/CM2) AND BMC (G/CM) PARAMETERS IN PATIENTS WITH WD (n ? 21) SORTED ACCORDING TO AGE
Densitometric parametersMean ? SD
No. of patients with Z score
? ?2 (age ? 40; n ? 7)
No. of patients with Z score
? ?2 (age ? 40; n ? 14)
1.08 ? 0.22
?0.59 ? 1.89
?0.51 ? 1.8743
0.99 ? 0.10
?0.53 ? 1.72
?0.31 ? 1.5841
0.96 ? 0.29
?0.92 ? 1.72
?0.87 ? 1.3721
1963OPG, RANKL, OCN, ?-CTx, AND BONE DENSITY IN WD
OCN and ?-CTx’s
Serum levels of OCN did not differ statistically in pa-
tients and controls (29.93 ? 24.65 ng/ml and 29.84 ? 6.89
ng/ml, respectively). However, ?-CTx levels were signifi-
cantly higher in patients with WD (625.4 ? 312.3 pg/ml;
p ? 0.022) compared with the controls (423.6 ? 144.3
pg/ml). Moreover, OCN levels correlated with ?-CTx levels
in patients with WD (r ? 0.55; p ? 0.01). The correlation
is illustrated in Fig. 1.
We found that serum OPG levels were significantly ele-
vated in patients with WD (7.2 ? 3.4 pM; p ? 0.001)
compared with both healthy controls (3.5 ? 1.0 pM) and
osteopenic, otherwise healthy controls (4.0 ? 1.0 pM; p ?
0.001). Results are illustrated in Fig. 2. OPG did not show
a correlation with either bone density scores or OCN and
On the other hand, there was no statistical difference in
serum RANKL level in patients with WD (0.6 ? 1.2 pM)
compared with healthy subjects (0.2 ? 0.4 pM). In 15
individuals from the 21 patients with WD and in 14 con-
trols, RANKL levels were undetectable, what we consid-
ered 0 pM RANKL concentration. There was no correlation
between RANKL levels and OPG levels, bone density pa-
rameters, OCN, and ?-CTx levels. No RANKL measure-
ments were performed in the osteopenic, otherwise healthy,
OCN, ?-CTx, OPG, and RANKL values are summarized
in Table 3.
Major findings of this study are the high-serum OPG and
?-CTx levels without change in RANKL and OCN levels
and the significantly decreased BMD by osteodensitometric
and QUS measurements. This is the first report on BMD and
bone ultrasound in WD. Furthermore, there have been no
OPG, RANKL, OCN, and ?-CTx measurements reported in
As in previous reports, our study confirms the high rate of
skeletal abnormalities in WD. Forty-three percent of pa-
tients with WD (9/21) had osteoporosis according to the
densitometric parameters. The BUA values showed abnor-
malities in 7 patients (33%). The large number of abnormal
BUA findings supports the osteoporosis diagnosis found by
DXA. As a result of abnormal densitometric and ultrasound
values, there is an increased frequency of osteoporosis as
well as an increased fracture risk(24)in patients with WD.
Although Aksoy suggested that the duration of the disease
may be an important factor in the occurrence of bone lesions
with the symptoms being milder in younger patients,(4)we
did not find a correlation between the BMDs of the patients
and their ages, which is in concordance with that of other
The reason for the decreased BMD may be the toxic
effect of copper damaging the tissues. Skeletal copper con-
rum OCN and serum ?-CTx’s in
patients with WD (Spearman cor-
relation, r ? 0.55 and p ? 0.01).
Correlation between se-
TABLE 2. QUS PARAMETERS IN PATIENTS
WITH WD (n ? 21)
QUS parameters Mean ? SD
No. of patients
with Z score
44.1 ? 7.1
?1.84 ? 1.07
?0.92 ? 1.72
1562 ? 9
?0.10 ? 0.69
0.29 ? 0.76
1964 HEGEDUS ET AL.
tent is reported to be increased about four times.(5)Menerey
described that the excessive toxic copper in the skeletal
system may mediate oxygen free radical release and thus
damage tissue.(25)Some studies propose that changes in the
calcium and phosphate plasma levels may contribute to the
high incidence of osteoporosis.(5,26,27)However, all of our
patients had serum calcium and phosphate levels in the
To investigate the underlying mechanism of decreased
BMD and BMC in patients with WD, we measured the
serum levels of the newly discovered OPG and RANKL, as
well as the serum level of OCN and ?-CTx’s, which are
markers of bone turnover. The mature OPG is a hydropho-
bic polypeptide consisting of 380 amino acids.(28)Although
OPG is secreted mainly by osteoblasts and immune com-
petent cells (T and B cells and monocytic cells), high
mRNA levels were detected in fibroblasts, endothelial cells,
and smooth muscle cells as well.(29)The biological effect of
OPG is inhibition of both: the terminal stages of osteoclas-
togenesis from osteoclast precursors and activity of mature
osteoclasts.(30–33)High levels of OPG mRNA have been
detected in the lung, heart, kidney, liver, stomach, intestine,
skin, brain and spinal cord, thyroid gland, and bone.(28,30)
RANKL, which was discovered later,(31,34)is a 317-amino
acid–containing ligand in the form of a type II transmem-
brane protein, as well as a soluble molecule derived form
the cell-associated form by post-translational processing.
RANKL gene expression by marrow stromal cells and os-
teoblasts is most abundant in the skeleton and lymphoid
tissues(31,34–36)and plays a role in the differentiation and
activation of osteoclasts by binding to its high affinity
receptor (osteoclast differentiation and activation receptor
[ODAR]/RANK) located on the surface of osteoclasts.(37,38)
The effect is counterbalanced by OPG, which acts as a
decoy receptor competing with RANK for RANKL.
The insignificant statistical difference in the OCN levels
between the patients and controls reflects that there is nor-
mal osteoblastic function in WD. However, ?-CTx levels
were significantly increased in patients with WD, which
reflects increased bone resorption in the patients. The pos-
itive correlation between OCN and ?-CTx may suggest that
by increasing their activity, osteoblasts attempt to compen-
sate for decreased bone mass. However, because of the
small number of patients and the large SD values, it is not
appropriate to draw conclusions from this correlation.
The high OPG levels in patients with WD found in our
study may suggest that other tissues such as immune-
competent cells or fibroblasts in the cirrhotic liver may
contribute to the production of the molecule as a conse-
quence of the inflammatory process.(10)Although most pa-
tients had normal liver function tests and other laboratory
parameters as a result of proper treatment and good control
over the disease, we observed that patients with more severe
liver damage—based on initial presentation and/or liver
biopsy findings—had higher serum OPG concentrations.
The correlation between OPG and liver function tests was
not significant, but as mentioned before, all patients were
treated and the disease was well controlled in each patient;
therefore, liver function tests were normal.
Another explanation for high OPG level in patients with
WD is that osteoblasts alone produce increased amounts of
OPG as a compensatory response to bone loss, which is
neither effective nor adequate, as it is reflected in the
decreased bone densitometric values. Ueland(13)also hy-
pothesized that increased serum OPG levels found in Cush-
ing’s syndrome may reflect a compensatory response to
enhanced osteoclastic activity or negative bone-remodeling
balance or even an enhanced activity in the OPG system
possibly correlated to increased activity of other members
of the TNF family. We propose that the increased OPG
levels with decreased bone mass in patients with WD might
have a similar pathomechanism.
To further assess the underlying mechanism, we exam-
ined whether OPG is produced as a result of a compensatory
reaction to high RANKL levels and activity in patients with
WD by measuring serum RANKL. However, the insignifi-
cant difference of RANKL level between patients with WD
and controls contradicts the hypothesis that osteoporosis
results from increased activation of RANKL, which may be
insufficiently counterbalanced by OPG. Therefore, it seems
that RANKL production in WD is not affected, whereas
OPG serum levels are elevated. Based on the normal serum
RANKL level, we may conclude that increased bone re-
sorption is not a consequence of increased RANKL produc-
tion by osteoblasts. However, it is interesting to note that
both the WD gene and the RANKL gene are localized to
chromosome 13q14. Although RANKL level was not ele-
vated, disturbances in this area of the 13th chromosome may
cause altered RANKL transcription or regulation as well.
teopenic otherwise healthy controls (4.0 ? 1.0 pM), and patients with
WD (7.2 ? 3.4 pM).
Serum OPG in the healthy controls (3.5 ? 1.0 pM), os-
TABLE 3. OCN, ?-CTX, OPG, AND RANKL SERUM
CONCENTRATIONS IN PATIENTS WITH WD
AND HEALTHY CONTROLS
(n ? 21)
(n ? 20)
29.93 ? 24.65 29.84 ? 6.89
625.4 ? 312.3 423.6 ? 144.3
7.2 ? 3.4
0.6 ? 1.2
3.5 ? 1.0
0.2 ? 0.4
1965OPG, RANKL, OCN, ?-CTx, AND BONE DENSITY IN WD
Another interesting observation is that the cleavage of the
membrane-bound RANKL and the release in the soluble
form is carried out by a specific metalloprotease. Altered
function of metalloproteases as a result of copper-zinc im-
balance may also play an important role in WD.(39)
In summary, we found a high rate of osseous disorders in
patients with WD. Based on the high serum ?-CTx and
OPG levels with normal OCN and RANKL levels found in
our study, we conclude that decreased bone density in
patients with WD is the consequence of increased bone
resorption unrelated to RANKL production; and the under-
lying mechanism is still unknown. We also hypothesize that
either normally functioning osteoblasts and/or immune-
competent cells produce an increased amount of OPG as a
compensatory reaction to increased bone loss in WD. Ad-
ditional studies are warranted to disclose the underlying
mechanism of bone disorders in patients with WD.
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Address reprint requests to:
Ferenc Szalay, M.D., Ph.D.
First Department of Medicine
Kora ´nyi S 2/A
H-1083 Budapest, Hungary
Received in original form May 15, 2002; in revised form June 12,
2002; accepted June 18, 2002.
1967 OPG, RANKL, OCN, ?-CTx, AND BONE DENSITY IN WD