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CLINICAL STUDY
Restoration of the coupling process and normalization of bone
mass following successful treatment of endogenous Cushing’s
syndrome: A prospective, long-term study
Cybe
`le Kristo
1,2
, Rune Jemtland
1
, Thor Ueland
1,2
, Kristin Godang
1
and Jens Bollerslev
1
1
Section of Endocrinology and
2
Research Institute of Internal Medicine, Department of Medicine, Rikshospitalet University Hospital, N-0027 Oslo, Norway
(Correspondence should be addressed to C Kristo; Email: cybele.kristo@rikshospitalet.no)
Abstract
Objective: Endogenous Cushing’s syndrome (CS) is associated with bone loss and an increased risk of
fractures. However, the long-term outcome of treatment on bone health has not been adequately
clarified.
Design: We followed 33 patients with active CS prospectively before and twice after treatment (mean
follow-up 33 (n¼25) and 71 months (n¼18), respectively). The patients were compared to age-,
sex- and body mass index (BMI)-matched controls, also followed longitudinally.
Methods: Bone mineral indices (bone mineral density (BMD), bone mineral content (BMC) and bone
area) were evaluated in the lumbar spine (LS), femoral neck (FN), and total body (TB) by dual-energy
X-ray absorptiometry (DXA). Biochemical markers of bone turnover were assessed by serum levels of
osteocalcin and C-terminal telopeptides of Type-1 collagen (CTX-1).
Results: Mann–Whitney rank sum tests showed that BMD of the LS, FN and TB was reduced by 14.8%
(P,0.001), 15.7% (P,0.001), and 9.2% (P,0.001) in CS vs. controls at baseline, with markedly
reduced serum osteocalcin (P¼0.014) and increased CTX-1 (P¼0.012) levels, but no correlation
between markers. At first follow-up, BMD was increased in LS (7.9%, P,0.001) and FN (3.5%,
P¼0.003) compared to baseline. The time-dependent rise in BMD (LS (r¼0.59; P¼0.002) and
FN (r¼0.52; P¼0.007); Spearman’s rank correlation), in CS was paralleled by increased osteocalcin
(275%, P,0.001) and correlation between biochemical markers (r¼0.92, P,0.001; Pearson’s cor-
relation). TB BMD did not increase significantly before the second follow-up, when BMD Z-scores were
normalized in all three compartments.
Conclusion: Our observations demonstrate restoration of coupled bone remodeling and normalization
of bone mineral density in all measured skeletal compartments of treated CS patients after prolonged
recovery, first significant in predominantly trabecular bone (i.e. lumbar spine).
European Journal of Endocrinology 154 109–118
Introduction
Glucocorticoid (GC)-induced bone loss is a frequent and
serious complication in patients with endogenous
Cushing’s syndrome (CS) (1, 2 – 11) as well as in
patients on GC therapy (12), leading to an increased
risk of low-energy fractures (8, 13–15). While patients
receiving GC therapy usually suffer from a primary dis-
ease that may have negative influence on bone mass by
itself, CS (i.e. hypercortisolism, caused by a tumor in the
pituitary gland or in the adrenal cortex) is not associ-
ated with an underlying primary condition known to
affect bone metabolism directly. Thus, CS is considered
to be a useful clinical model to investigate the ‘pure’
effects of excess GC on bone metabolism, minimizing
other confounding factors. Previous reports have pro-
vided evidence that bone loss due to hypercortisolism
(exogenous or endogenous) is most pronounced in
areas with trabecular bone such as the lumbar spine
(predominantly trabecular bone) and femoral neck
(FN) (less trabecular bone, more cortical bone)
(1– 11, 16). Accordingly, fracture risk appears to be
highest in the vertebrae and ribs of these patients
(8, 13, 14). In contrast, the effect of GC on skeletal sites
with predominantly cortical bone is less well-documen-
ted (5, 9, 16, 17). Few reports have assessed restoration
of bone mass after treatment of patients with active CS
and there is a scarcity of long-term prospective studies
(1, 3, 11, 18), reflecting the low incidence of CS (1–10
per million per year) (19). Most previous studies have
described partial restoration of bone mass after treat-
ment (1, 3, 11). However, little is known about the
long-term outcome of bone health in CS, because
most studies have re-evaluated the patients after
a short (3 – 6 months) or intermediate (12 – 24
months) time of follow-up (3, 6, 11). Moreover, it has
European Journal of Endocrinology (2006) 154 109–118 ISSN 0804-4643
q2006 Society of the European Journal of Endocrinology DOI: 10.1530/eje.1.02067
Online version via www.eje-online.org
not been established whether bone mass at skeletal sites
with predominantly trabecular or cortical bone is
affected differently by correction of hypercortisolism
after successful surgical treatment.
The exact mechanism by which GC influences bone
metabolism is still not completely understood (12).
While there is evidence that bone loss due to excess
GC, at least in part, may be secondary to hypogonad-
ism, decreased intestinal calcium absorption and
renal calcium reabsorption, in addition to loss of
muscle mass and strength, it is obvious that GCs have
direct potent effects on bone cells. The skeletal effects
of GC excess dominate over those of orchidectomy in
mice, indicating that bone loss in this model occurs
independently of changes in gonadal status (20). A
characteristic feature of GC-induced bone loss is a
depression of bone turnover, which is likely to be related
to reduced bone formation (3, 4, 9, 16, 21 – 26),
although bone resorption has also been shown to be
increased in some studies (27). Accordingly, animal
studies and in vitro experiments have shown that
chronic GC administration decreases the birth rate
and function of osteoblasts, increases apoptosis of
mature osteoblasts and osteocytes (28, 29 – 32) and
stimulates and prolongs osteoclast formation, activity
and survival (27, 33, 34).
The aim of the present study was to evaluate long-
term effects of successful surgical treatment on bone
densitometric parameters and bone turnover in
patients with CS. Patients were evaluated longitudin-
ally over an interval of mean 71 months, and were
also compared to a closely matched control group,
also followed longitudinally. Moreover, bone mass and
projected bone area was evaluated in different compart-
ments in order to assess whether GCs exert differential
effects at skeletal sites with predominantly cortical or
trabecular bone. Finally, we estimated alterations in
biochemical markers of bone turnover, to explore poss-
ible mechanisms involved in the pathogenesis of GC-
induced bone loss in CS patients with active disease
and during the recovery phase after cure.
Material and methods
Subjects
CS patients with pituitary adenoma (Cushing’s disease)
or adrenal cortex adenoma diagnosed in our center
between 1994 and 1999 were consecutively recruited
to this study, after giving signed informed consent (5,
16). The study was approved by the local ethical com-
mittee and conducted according to the Declaration of
Helsinki II.
All patients were diagnosed with CS based on a
thorough clinical evaluation and biochemical work-up,
as previously reported (5). Prior to surgical treatment,
a baseline population of 33 CS patients (24 women, 9
men) was systematically evaluated and matched with
33 healthy controls. Of these, 25 patients (17 women,
8 men) were followed longitudinally for a mean of 33
months (range 5 – 69 months), and among these, 18
patients (13 women, 5 men) were further re-evaluated
at a second follow-up, 71 months (range 43 – 108
months) after treatment. The controls, whom were
matched for age, sex, and body mass index (BMI), were
evaluated at baseline and then after a mean of 50
months (range 26 –56 months). Prior to operative treat-
ment, none of our patients received medication in order
to block steroid synthesis. Patients with Cushing’s dis-
ease were treated by transsphenoidal microsurgery; of
these, four patients with relapse were subsequently sub-
jected to bilateral adrenalectomy and were substituted
with cor tisone acetate 37.5 mg/day and fludrocortisone
0.1 mg/day. GC substitution was stopped for 24 hours
before blood sampling in the patients on this medication.
At baseline, none of the male patients had hypogonadism
defined as a testosterone/SHBG (sex hormone binding
globulin) ratio of less than 25% (mean ratio in the popu-
lation 55%, normal range ¼25– 200%). Six women
diagnosed with CS had regular menstruation, three
had entered menopause (two were hysterectomized)
and fifteen had amenorrhea for a mean of 27 months
(range 6 – 60 months). None of the patients were substi-
tuted with sex hormones, thyroxine or growth hormone
(GH). At the second follow-up, one of two men who had
developed partial pituitary deficiency was substituted
with testosterone and the other with GH. Five women
had still regular menstruation, whereas two of eight
women who had entered the menopause were substi-
tuted with hormone replacement therapy and two with
dehydroepiandrosterone (DHEA).
Methods
All patients had clinical and biochemical findings of CS
at baseline. The diagnosis was confirmed by an increase
in daily urinary cortisol excretion, increase in basal
serum cortisol concentrations with lack of physiological
diurnal rhythm, lack of urinary and serum cortisol sup-
pression after 2-day dexamethasone suppression test
(DST, 0.5 mg £4 per day, sampling 2 h after the last
tablet), and a low-dose DST (1.0 or 1.5 mg depending
on weight at follow-up) according to international
standards (35). For patients with Cushing’s disease
the diagnosis was confirmed by inappropriately high
plasma adrenocorticotropin (ACTH) concentrations
and magnetic resonance imaging (MRI) of pituitary
gland (eventually followed by sinus petrosus sampling),
and for patients with adrenal cortex adenoma, sup-
pressed plasma ACTH and computed tomography (CT)
scanning of the adrenal glands.
Following treatment (first and second follow-up), all
patients presented with normalized diurnal variation
of serum cortisol, in addition to levels of 24-h urine
free cortisol within the normal range. DST was con-
sidered normal when serum cortisol was suppressed
110 C Kristo and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
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to less than 40 nmol/l. For ten patients at first follow-up
and six patients at second follow-up, serum cortisol was
.40 nmol/l at DST. However, none of these patients
showed differences in biochemical parameters or bone
mass indices (bone mineral density (BMD), bone min-
eral content (BMC) and area), as compared to patients
with DST ,40 nmol/l, nor did they present with any
clinical signs of relapse. All patients had normal liver
and kidney function, as judged by serum levels of
ASAT (aspartat aminotransferase), ALAT (alanin ami-
notransferase), albumin and creatinine.
Osteodensitometric measurements
BMD (g/cm
2
), BMC (g) and projected bone area (cm
2
)
were measured in the LS (L2-L4, anterior-posterior pro-
jection), the left FN and total body (TB), using DXA
(Dual-Energy X-ray Absorptiometry; Lunar DPX-L, soft-
ware version 4.6c, Lunar Corporation, Madison, WI)
(36). BMD Z-scores are given with reference to norma-
tive data provided by the manufacturer. The precision
error of LS was about 1%, FN 1.5% and TB 0.5%, inde-
pendent of the operator (16).
Blood sampling
Blood samples were drawn between 0800 h and
1000 h, after overnight fasting. For collection of
serum, blood was drawn into pyrogen-free tubes
(Becton Dickinson, San Jose, CA) without additives.
The tubes were immediately immersed in ice water,
allowed to clot for 2 hours, and centrifuged at 1000 g
at 4 C for 10 minutes; serum was stored at 2808C
until analyzed. All samples were thawed three times,
and samples from a given individual were run in the
same assay to minimize run-to-run variability.
Biochemical markers of bone turn-over
Osteocalcin was measured by immunoradiometric assay
(IRMA) with a commercial kit (Incstar Corporation,
Stillwater, MI), that measures intact osteocalcin 1 – 49
. Degradation products of the C-terminal telopeptides of
Type-I collagen (CTX-1) were measured with an
enzyme immunoassay (EIA) (Crosslaps; Osteometer Bio-
Tech A/S, Herlev, Denmark). Serum and free cortisol in
24-h urine was measured by radioimmunoassay (RIA),
using commercial kits from Orion Diagnostica, (Espoo,
Finland). Cortisol and dehydroepiandrosterone levels
were analysed using standard laboratory methods.
The intra- and inter-assay coefficients of variation were
,7% for all assays.
Statistical analysis
Statistical analyses were performed by SPSS forWindows
(version 12.0; SPSS Inc., Chicago, IL). Wilcoxon signed
rank sum test was used to analyze changes in bone
mass at different observation times. Mann – Whitney
Rank-Sum test was used to compare variables of patients
and controls. Relationships between two variables in
BMD, BMC and bone area were tested using Spearman’s
rank correlation test and Pearson’s correlation test for
bone turnover markers. P,0.05 was considered stat-
istically significant.
Results
The clinical characteristics and biochemical findings of
the study populations at baseline and at regular intervals
throughout the observation period are given in Table 1.
Time-dependent changes in bone mass indices
at multiple skeletal sites before and after
successful treatment of CS
BMD, BMC and bone area were determined in
CS patients by DXA at baseline and at two consecutive
follow-ups (means 33 and 71 months respectively)
Table 1 Clinical characteristics and laboratory data before and after surgical treatment of CS patients, and during longitudinal follow-up
in controls.
CS Controls
Baseline
(n¼33)
First follow-up
(n¼25)
Second follow-up
(n¼18)
Baseline
(n¼33)
Follow up
(n¼25)
Gender (Female/male) 33 (24/9) 25 (17/8) 18 (13/5) 33 (24/9) 25 (17/8)
Age (years) 43^247^250^343^247^2
Diagnosis (pituitary/adrenal) 26/7 20/5 14/4
BMI kg/m
2
29.0^1 27.3^1 26.9^1 27.6^1 27.5^1
Body weight (kg) 82.6^2.5 78^3 75.6^4 81.2^2.4 83.8^2.9
Serum cortisol (nmol/l) 0800 h 627^42** 259^28† 422^47 409^33 393^361
Urinary cortisol (nmol/24 h) 1446^345 193^21 179^15
DHEAS (mg/dl) 298^58*60^11 142^13
Osteocalcin (mg/L) 3.1^0.3*13.5^1.6†† 11.0^1.1 4.26^0.4 6.97^0.4
CTX-1 (Crosslaps) (ng/ml) 0.53^0.04*0.75^0.13
ns
0.73^0.08 0.39^0.03 0.58^0.06
Data are presented as mean^S.E.M. DHEAS, dehydroepiandrosterone. *P,0.05, **P,0.001, CS patients vs. controls at baseline.
†
P,0.05,
††
P,0.001, CS patients vs. controls at first follow-up. ns, not significant.
Bone mass and turnover in Cushing’s syndrome: a prospective study 111EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
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after surgical treatment, as shown in Fig.1 and Table 2.
At baseline, bone mass (BMD and BMC) was reduced in
all compartments of CS patients relative to controls, as
was the TB projected area (13%, P¼0.01) (Table 2).
Already at the first follow-up (Fig. 1), highly significant
improvements were demonstrated at the LS (BMD, 7.9%
(P,0.001); BMC, 17.5% (P¼0.001); area, 3.1%
(P¼0.022)) and FN (BMD, 3.5% (P¼0.003); BMC,
5.0% (P¼0.025); area, 24.5% (ns)) relative to pre-
treatment values. Further gain in bone mass indices
were observed at the second follow-up (relative to the
first follow-up) at the LS (BMD, 10.7% (P¼0.039);
BMC, 7.8% (P¼0.001); area, 6.9% (P¼0.003)) and
FN (BMD, 4.6% (P¼0.004); BMC, 3.6% (P¼0.001);
area, 20.2% (ns)). Whereas a trend towards increased
BMC (12.6%, (ns)) and bone area (11.8%, (ns)) was
observed for TB at first follow-up (relative to baseline),
BMD was unaltered (0.9%, (P¼0.16)). By the second
follow-up, a highly significant increase was observed
in TB BMD (5.1%, (P,0.001)), with unchanged or
only modest alterations in BMC (0.8%, (ns)) and bone
area (21.8%, (ns)). Thus, increases in BMD were
observed at all measured skeletal sites in patients after
treatment, however the changes (P,0.05) appeared
earlier at the LS and FN compared to TB, where the
improvements reached statistical significance at the
second follow-up.
A highly significant positive correlation between the
changes in BMD and time after surgical treatment
(mean 33 months) was demonstrated in the LS
(r¼0.59; P¼0.002) and FN (r¼0.52; P¼0.007),
but not in TB (r¼0.27; P¼0.30) (Fig. 2).
At baseline there was no correlation between serum
cortisol 0800 h and osteodensitometric measurements.
At the first follow-up we found a negative correlation
between serum cortisol 0800 h and BMC at TB
(r¼20.48; P¼0.017), TB projected bone area
(r¼20.47; P¼0.021) and serum osteocalcin
(r¼20.41; P¼0.04). Moreover, there was a negative
correlation between serum cortisol and LS BMD at the
second follow up (r¼20.50; P¼0.03) (data not
shown).
Normalization of bone mineral density at
multiple skeletal sites in patients with CS
after prolonged recovery
Data are presented as Z-scores (25, 75 percentiles) BMD
Z-scores for the various bone compartments in patients
and healthy controls are given in Fig. 3. Patients with
active CS had reduced BMD Z-scores at all measured
sites (LS, 21.50 (22.49, 20.76); FN, 21.23
(21.95, 20.52); and TB, 21.37 (22.00, 20.71)) at
baseline. By comparison, BMD Z-scores for the controls
Figure 1 BMD (A-C), BMC (D-F) and
projected bone area (G-I) before treat-
ment and during longitudinal follow-up
(mean 33 and 71 months, respectively)
after surgical treatment of patients with
CS. BL, baseline; 1.FU, first follow-up;
2.FU, second follow-up. Data are given
as median and 25 and 75 percentiles.
*P,0.05, **P,0.01, ***P,0.001;
first follow-up vs. baseline.
#
P,0.05,
##
P,0.01,
###
P,0.001; second vs.
first follow-up.
112 C Kristo and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
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Table 2 Osteodensitometric parameters at various skeletal sites (see methods) before and after surgical treatment in CS patients and age-, sex-, and BMI-matched controls.
Cushing’s Controls
Osteodensitometry Baseline (n¼33) First follow-up (n¼25) Second follow-up (n¼18) Baseline (n¼33) Follow-up (n¼25) CS vs. controls
LS BMD (g/cm
2
) 1.05 (0.94, 1.13) 1.15 (1.03, 1.26) 1.22 (1.11, 1.39) 1.23 (1.09, 1.33) 1.20 (1.06, 1.35) P,0.001 (BL)
P¼0.149 (FU)
LS Z-score (S.D.)21.50 (22.49, 20.76) 20.50 (21.45, 0.25) 0.30 (20.80, 1.33) 20.03 (20.934, 0.87) 20.10 (20.95, 1.15) P,0.001 (BL)
P¼0.159 (FU)
LS BMC (g) 45.73 (37.81, 53.72) 53.09 (41.88, 62.81) 56.87 (46.44, 66.10) 59.46 (48.61, 66.52) 60.65 (46.61, 68.190 P¼0.001 (BL)
P¼0.051 (FU)
LS (area (cm
2
) 42.42 (39.44, 50.25) 43.83 (41.66, 52.47) 46.58 (41.21, 55. 52) 46.15 (41.19, 52.39) 46.36 (41.33, 53.13) P ¼0.17 (BL)
P¼0.135 (FU)
FN BMD (g/cm
2
) 0.86 (0.74. 0.91) 0.88 (0.76, 0.96) 0.92 (0.80, 1.00) 0.95 (0.88, 1.11) 1.00 (0.90, 1.09) P,0.001 (BL)
P¼0.001 (FU)
FN Z-score (S.D.)21.23 (21.95, 20.52) 20.70 (21.40, 0.05) 0.10 (20.85, 0.63) 0.28 (20.76, 0.67) 0.10 (20.95, 0.70) P,0.001 (BL)
P¼0.008 (FU)
FN BMC (g) 4.27 (3.83, 4.65) 4.20 (3.84, 4.89) 4.32 (3.94, 5.69) 5.26 (4.46, 5.7) 5.10 (4.66, 5.50) P,0.001 (BL)
P¼0.001 (FU)
FN area (cm
2
) 5.12 (4.76, 5.49) 4.97 (4.58, 5.44) 4.86 (4.63, 5.56) 5.9 (4.88, 5.99) 5.07 (4.37, 5.62) P,0.462 (BL)
P¼0.613 (FU)
TB BMD (g/cm
2
) 1.10 (1.01, 1.13) 1.09 (1.04, 1.15) 1.17 (1.12, 1.22) 1.19 (1.13, 1.28) 1.19 (1.14, 1.26) P,0.001 (BL)
n¼23 n¼16 n¼11 n¼23 n¼16 P,0.001 (FU)
TB Z-score (S.D.)21.37 (22.00, 20.71) 20.70 (21.55, 0.05) 0.45 (20.60, 1.13) 0.22 (20.35, 0.89) 0.40 (20.35, 1.20) P,0.001 (BL)
n¼23 n¼16 n¼11 n¼23 n¼16 P¼0.002 (FU)
TB BMC (g) 2348 (2035, 2635) 2511 (2197, 2798) 2544 (2354, 3293) 2828 (2557, 3366) 2783 (2491, 3181) P¼0.001 (BL)
n¼23 n¼16 n¼11 n¼23 n¼16 P¼0.006 (FU)
TB area (cm
2
) 2091 (1892, 2422) 2260 (2094, 2499) 2218 (2072, 2734) 2396 (2223, 2693) 2387 (2160, 2601) P¼0.010 (BL)
n¼23 n¼16 n¼11 n¼23 n¼16 P¼0.062 (FU)
Patients were evaluated at baseline and at two successive follow-ups, mean 33 and 71 months after surgical treatment respectively. Controls were also followed longitudinally and evaluated at baseline and
follow-up, mean 50 months after inclusion. Data are given as median and 25 and 75 percentiles. Cross-sectional data: differences between CS patients and controls at baseline (BL) and first follow-up (FU),
respectively, are presented as Pvalues in the last column.
Bone mass and turnover in Cushing’s syndrome: a prospective study 113EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
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were not significantly different from the normative data
of the reference population throughout the study. Fol-
lowing treatment, BMD Z-scores for the patients
increased at the LS (20.50 (21.45, 0.25),
P,0.001) and FN (20.70 (21.40, 0.05),
P,0.001) during the first follow-up (relative to base-
line), with a trend towards an improvement also in TB
20.70 (21.55, 0.05), P¼0.083). Thus, at the first
follow-up there were no significant (P¼0.16) differ-
ences between LS BMD Z-score values in CS patients
and controls, indicating normalization of bone mass
at this site, whereas Z-scores were still significantly
lower at FN (P¼0.010) and TB (P¼0.001). At the
second follow-up, we observed a further rise in BMD
Z-score at the LS (0.30 (20.80, 1.33), P,0.001),
FN (0.10 (20.85, 0.63), P,0.001), and TB (0.45
(20.60, 1.13), P,0.001), indicating normalization
of BMD in all measured skeletal compartments with
time.
Biochemical markers of bone turnover:
restoration of the bone remodeling process
after successful treatment of CS
To determine the rate of bone turnover, we also
measured serum levels of osteocalcin (OC), a marker
for bone formation, and cross-laps (CTX-1), a marker
for bone resorption, both during longitudinal follow-
up in CS patients (Fig. 4), and in relation to matched
controls (Table 1). Compared to controls at baseline,
serum OC levels were reduced by 26.9% (P¼0.014)
in the patients, whereas their serum CTX-1 levels
were increased by 35.9% (P¼0.012). At follow-up,
levels of OC were markedly elevated (approx. 2-fold)
in CS patients compared to controls (P,0.001),
whereas CTX-1 levels were only moderately enhanced
(29.1%, P¼0.68). The longitudinal data (Fig. 4)
show that serum osteocalcin levels increased markedly
in the patients (275%, P,0.001) at the first follow-up
compared to baseline, and were sustained at high levels
throughout the observation period. However, serum
levels of CTX-1 tended to increase only modestly
during the same time period. Notably, while no corre-
lation was found between levels of bone formation
and resorption markers before treatment in the patients
(r¼0.32, P¼0.19), serum levels of osteocalcin
and CTX-1 were significantly correlated both at
first (r¼0.92, P,0.001) and second follow-up
(r¼0.90, P,0.001) after treatment, indicating that
coupling between formation and resorption had been
restored.
Discussion
The present study, which contains both longitudinal
and cross-sectional data is, to our knowledge, the first
to demonstrate normalization of bone mineral density
in multiple skeletal compartments of patients with
endogenous Cushing’s syndrome after prolonged recov-
ery time (mean 71 months). Whereas biochemical mar-
kers of bone turnover indicated uncoupled remodeling
in the state of active disease, coupling seemed to be
restored following treatment, leading to a marked
increase in bone mass first appreciated in
compartments rich in the metabolically active trabecu-
lar bone as in the LS, and most recently in the TB skel-
eton constituting of mainly (80%) cortical bone.
Our baseline data demonstrate that patients with
active CS displayed marked deficits in bone mass not
only at the spine and FN, as generally appreciated
(3– 7, 9, 11, 16, 23), but also in the TB skeleton. The
most pronounced bone loss was observed at the LS,
consistent with the view that trabecular bone is a prin-
cipal target for the detrimental effects of excess GC in
the skeleton (12). Our longitudinal data demonstrating
improvement in bone mass following treatment of
CS extend previous findings by showing that the gain
in LS BMD and FN BMD was positively related to
follow-up time (r¼0.59 and r¼0.52, respectively).
Figure 2 Correlations (r) between changes in BMD in (A) LS, (B) FN and (C) TB, and recovery time (mean 33 months) after surgical
treatment in patients with CS. Data are given as individual observations.
114 C Kristo and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
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This suggests that bone loss in CS is reversible after suc-
cessful treatment, and is supported by normalization of
BMD Z-scores in both sexes at all measured sites in CS
patients with prolonged recovery (mean 71 months).
This occurred despite the expected physiological age-
dependent bone loss, which was relatively modest in
the study population (Fig.3) consisting mainly of pre-
menopausal women. However, the time for significant
measurable improvement in bone mass differed
between the various skeletal compartments, and
became significant first in the LS (mainly trabecular
bone), then in the FN (less trabecular bone, more corti-
cal bone), and lastly in TB (mainly cortical bone), poss-
ibly reflecting that a higher remodeling rate at the
spine may have caused an earlier bone recovery than
at cortical sites in the appendicular skeleton.
Interestingly, TB projected area was reduced com-
pared to controls at baseline. In the follow-up period
bone area increased significantly in the LS (and a simi-
lar trend in TB) throughout the observation period
most likely reflecting periosteal apposition of new
bone, in accordance with the persistent increase in
serum levels of the bone formation marker osteocalcin.
Decreased bone area has previously been demonstrated
in active CS (5, 16), and altered bone area has also
been found in other endocrine disorders (37 – 39).
While we are not aware of any earlier reports showing
that successful cure of CS affects bone area, treatment
with parathyroid hormone was recently shown to
increase vertebral cross-sectional area in postmenopau-
sal women with GC-induced osteoporosis (40). The
anabolic effect of parathyroid hormone in their study
was explained by increased bone turnover and in par-
ticular bone formation, similar to the changes in bone
turnover markers observed in our study after correction
of excess cortisol levels. However, it is important to con-
sider that investigations by DXA are limited by its two-
dimensional acquisition plane (of a 3 dimensional
structure) for determining structure and geometric par-
ameters, and that a threshold effect on edge determi-
nation due to changes in body composition can not
be ruled out. Moreover, in relation to BMD measure-
ments and Z-scores patients with CS are especially
prone to vertebral fractures, most often manifested
clinically as back pain or loss of height due to com-
pression of the vertebrae, DXA measurements may
record inappropriately high BMD values in the spine.
While not all the patients fulfilled strict criteria for
cure according to the low dose DST, all patients in
the present study were without clinical symptoms of
active disease at follow-up, with normal diurnal vari-
ation in serum cortisol levels and normal urinary
excretion of free cortisol. Moreover, biochemical par-
ameters (Table 1) and bone mass indices (BMD, BMC,
area) showed no differences between patients with
DST ,40 nmol/l as compared to those with DST
.40 nmol/l. Interestingly however, indices of bone
mass and turn-over were not correlated to cortisol
levels at baseline, whereas negative correlations were
demonstrated after treatment for various indices
of bone mass and osteocalcin. The limited size of
the study population did not allow for stratification
of eugonadal and hypogonadal patients. However, we
did not find evidence for a high bone-turnover state
in CS patients as seen in patients with postmenopausal
osteoporosis caused by estrogen deficiency. On the con-
trary, we observed a low bone turnover state and indi-
cations of un-coupling of the remodeling process in
patients with active CS. Thus, the principal mediator
of bone loss in active CS is likely to be excess GC and
not hypogonadism. This is in accordance with obser-
vations in an experimental mouse model in which
Figure 3 Z-score BMD values for (A) LS, (B) FN and (C) TB
before (i.e. baseline) and after longitudinal follow-up in patients
with CS (B), and in age-, sex-, and BMI-matched controls (A) also
evaluated longitudinally. Data are presented as median ^S.E.M.
*
P,0.05, **P,0.01, ***P,0.001; first follow-up vs. baseline in
CS.
#
P,0.05,
#
P,0.01,
###
P,0.001; second vs. first follow-up
in patients with CS (see Table 2).
a
P,0.001,
b
P¼0.001,
c
P,0.05,
d
P¼ns; CS patients vs. controls.
Bone mass and turnover in Cushing’s syndrome: a prospective study 115EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
www.eje-online.org
excess GC seems to override the effect of sex-steroids on
the skeleton (20). Moreover, we did not find evidence
for anabolic skeletal effects of the androgen DHEAS in
our CS population, as was suggested by a recent
study in adrenal vs. pituitary CS (8), because DHEAS
levels did not correlate (data not shown) with measured
bone mass parameters in our study. However, these
results must be interpreted with caution as we were
not able to match controls and patients according to
gonadal status.
Our data further suggest that the observed bone loss
in active CS is due to uncoupled bone remodeling and
suppressed bone formation. This is in agreement with
previous reports that excess GCs cause reduction in
bone formation and, to a lesser degree, increased
bone resorption, implying a state of low bone turnover
(2–4, 9, 16, 18, 22, 23, 25). While the importance of
suppressed bone formation secondary to reduced osteo-
blast number and/or activity is considered to be a cen-
tral component in GC-induced bone loss, reports on
effects on bone resorption have been less consistent,
as there is evidence for increased (3, 9), decreased (4,
23), or unchanged (11) bone resorption. The apparent
controversy as to the importance of increased bone
resorption as an important component of GC-induced
bone loss may in part be related to the study popu-
lation; in contrast to hospitalized patients starting up
GC therapy, patients with CS are likely to have
manifested disease long (several years) before diagnosis
and treatment. Hence, the initial resorptive phase may
easily have been missed in the large majority of CS
patients when they are diagnosed and selected for clini-
cal studies. Whereas the biochemical markers of bone
turnover showed no relationship at baseline, a highly
significant correlation between the formative and
resorptive serum markers was observed during follow-
up, suggesting a normalization of the coupling process
with the balance in favor of increased bone formation
after treatment.
In conclusion, this study demonstrates generalized
pronounced bone loss in active CS, not only at sites con-
sisting mainly of trabecular bone, but also in predomi-
nantly cortical bone compartments. Biochemical
markers of bone turnover indicate uncoupling of the
remodeling process in active CS and association of
reduced bone mass with suppressed bone formation
and increased resorption. Our longitudinal data add
to previous findings by showing a positive correlation
between gain in BMD and follow-up time after cure at
the LS and FN respectively. The accompanying changes
in bone turnover markers, showing restoration of the
coupling process and a marked increase in serum osteo-
calcin levels, indicate that enhanced bone formation is
mainly responsible for improvement of bone mass after
correction of excess cortisol levels. Finally, our data
demonstrating successive normalization of BMD first
Figure 4 Longitudinal changes in serum levels of (A) osteocalcin and (B) CTX-1 at baseline (BL) and first follow-up (1.FU) after surgical
treatment in CS patients (n ¼25). Correlations (r) between serum levels of osteocalcin and CTX-1 (C) before and (D) after surgical
treatment, respectively. Data are given as individual observations.
116 C Kristo and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
www.eje-online.org
at the LS, then at the FN, and finally in TB, suggest that
bone loss in active CS is reversible in all measured bone
compartments after prolonged recovery.
References
1 Di Somma C, Colao A, Pivonello R, Klain M, Faggiano A,
Tripodi FS, Merola B, Salvatore M & Lombardi G. Effectiveness
of Chronic Treatment With Alendronate in the Osteoporosis of
Cushing’s Disease. Clinical Endocrinology 1998 48 655– 662.
2 Di Somma C, Pivonello R, Loche S, Faggiano A, Marzullo P,
Di Sarno A, Klain M, Salvatore M, Lombardi G & Colao A.
Severe Impairment of Bone Mass and Turnover in Cushing’s Dis-
ease. Clinical Endocrinology 2002 56 153– 158.
3 Di Somma C, Pivonello R, Loche S, Faggiano A, Klain M,
Salvatore M, Lombardi G & Colao A. Effect of 2 Years of Cortisol
Normalization on the Impaired Bone Mass and Turnover in Ado-
lescent and Adult Patients With Cushing’s Disease: a Prospective
Study. Clinical Endocrinology 2003 58 302 – 308.
4 Francucci CM, Pantanetti P, Garrapa GG, Massi F, Arnaldi G &
Mantero F. Bone Metabolism and Mass in Women With Cushing’s
Syndrome and Adrenal Incidentaloma. Clinical Endocrinology
2002 57 587– 593.
5 Kristo C, Godang K, Ueland T, Lien E, Aukrust P, Froland SS &
Bollerslev J. Raised Serum Levels of Interleukin-8 and
Interleukin-18 in Relation to Bone Metabolism in Endogenous
Cushing’s Syndrome. European Journal of Endocrinology 2002
146 389– 395.
6 Luisetto G, Zangari M, Camozzi V, Boscaro M, Sonino N & Fallo F.
Recovery of Bone Mineral Density After Surgical Cure, but Not by
Ketoconazole Treatment, in Cushing’s Syndrome. Osteoporosis
International 2001 12 956– 960.
7 Manning PJ, Evans MC & Reid IR. Normal Bone Mineral Density
Following Cure of Cushing’s Syndrome. Clinical Endocrinology
1992 36 229– 234.
8 Ohmori N, Nomura K, Ohmori K, Kato Y, Itoh T & Takano K.
Osteoporosis Is More Prevalent in Adrenal Than in Pituitary
Cushing’s Syndrome. Endocrine Journal 2003 50 1–7.
9 Chiodini I, Carnevale V, Torlontano M, Fusilli S, Guglielmi G,
Pileri M, Modoni S, Di Giorgio A, Liuzzi A, Minisola S,
Cammisa M, Trischitta V & Scillitani A. Alterations of Bone Turn-
over and Bone Mass at Different Skeletal Sites Due to Pure Gluco-
corticoid Excess: Study in Eumenorrheic Patients With Cushing’s
Syndrome. Journal of Clinical Endocrinology and Metabolism 1998
83 1863–1867.
10 Minetto M, Reimondo G, Osella G, Ventura M, Angeli A &
Terzolo M. Bone Loss Is More Severe in Primary Adrenal Than
in Pituitary-Dependent Cushing’s Syndrome. Osteoporosis Inter-
national 2004 15 855–861.
11 Hermus AR, Smals AG, Swinkels LM, Huysmans DA, Pieters GF,
Sweep CF, Corstens FH & Kloppenborg PW. Bone Mineral Density
and Bone Turnover Before and After Surgical Cure of Cushing’s
Syndrome. Journal of Clinical Endocrinology and Metabolism 1995
80 2859– 2865.
12 Canalis E & Giustina A. Glucocorticoid-Induced Osteoporosis:
Summary of a Workshop. Journal of Clinical Endocrinology and
Metabolism 2001 86 5681– 5685.
13 Vestergaard P, Lindholm J, Jorgensen JO, Hagen C, Hoeck HC,
Laurberg P, Rejnmark L, Brixen K, Kristensen LO, Feldt-
Rasmussen U & Mosekilde L. Increased Risk of Osteoporotic Frac-
tures in Patients With Cushing’s Syndrome. European Journal of
Endocrinology 2002 146 51– 56.
14 van Staa TP, Leufkens HG, Abenhaim L, Zhang B & Cooper C. Oral
Corticosteroids and Fracture Risk: Relationship to Daily and
Cumulative Doses. Rheumatology 2000 39 1383– 1389.
15 Vestergaard P, Olsen ML, Paaske Johnsen S, Rejnmark L,
Sorensen HT & Mosekilde L. Corticosteroid Use and Risk of Hip
Fracture: a Population-Based Case-Control Study in Denmark.
Journal of Internal Medicine 2003 254 486– 493.
16 Godang K, Ueland T & Bollerslev J. Decreased Bone Area, Bone
Mineral Content, Formative Markers, and Increased Bone Resorp-
tive Markers in Endogenous Cushing’s Syndrome. European Jour-
nal of Endocrinology 1999 141 126–131.
17 Karavitaki N, Ioannidis G, Giannakopoulos F, Mavrokefalos P &
Thalassinos N. Evaluation of Bone Mineral Density of the Periph-
eral Skeleton in Pre- and Postmenopausal Women With Newly
Diagnosed Endogenous Cushing’s Syndrome. Clinical Endocrin-
ology 2004 60 264– 270.
18 Sartorio A, Conti A, Ferrario S, Passini E, Re T & Ambrosi B.
Serum Bone Gla Protein and Carboxyterminal Cross-Linked Telo-
peptide of Type I Collagen in Patients With Cushing’s Syndrome.
Postgraduate Medical Journal 1996 72 419–422.
19 Boscaro M, Barzon L, Fallo F & Sonino N. Cushing’s Syndrome.
Lancet 2001 357 783–791.
20 Weinstein RS, Jia D, Powers CC, Stewart SA, Jilka RL, Parfitt AM
& Manolagas SC. The Skeletal Effects of Glucocorticoid Excess
Override Those of Orchidectomy in Mice. Endocrinology 2004
145 1980– 1987.
21 Lukert BP, Higgins JC & Stoskopf MM. Serum Osteocalcin Is
Increased in Patients With Hyperthyroidism and Decreased in
Patients Receiving Glucocorticoids. Journal of Clinical Endocrin-
ology and Metabolism 1986 62 1056– 1058.
22 Sartorio A, Ambrosi B, Colombo P, Morabito F & Faglia G. Osteo-
calcin Levels in Cushing’s Disease Before and After Treatment.
Hormone and Metabolic Research 1988 20 70.
23 Cortet B, Cortet C, Blanckaert F, d’Herbomez M, Marchandise X,
Wemeau J-L, Decoulx M & Dewailly D. Quantitative Ultrasound
of Bone and Markers of Bone Turnover in Cushing’s Syndrome.
Osteoporosis International 2001 12 117– 123.
24 Osella G, Terzolo M, Reimondo G, Piovesan A, Pia A, Termine A,
Paccotti P & Angeli A. Serum Markers of Bone and Collagen
Turnover in Patients With Cushing’s Syndrome and in Subjects
With Adrenal Incidentalomas. Journal of Clinical Endocrinology
and Metabolism 1997 82 3303– 3307.
25 Piovesan A, Terzolo M, Reimondo G, Pia A, Codegone A, Osella G,
Boccuzzi A, Paccotti P & Angeli A. Biochemical Markers of Bone
and Collagen Turnover in Acromegaly or Cushing’s Syndrome.
Hormone and Metabolic Research 1994 26 234– 237.
26 Sartorio A, Conti A, Ferrero S, Giambona S, Re T, Passini E &
Ambrosi B. Evaluation of Markers of Bone and Collagen Turnover
in Patients With Active and Preclinical Cushing’s Syndrome and
in Patients With Adrenal Incidentaloma. European Journal of Endo-
crinology 1998 138 146– 152.
27 Takuma A, Kaneda T, Sato T, Ninomiya S, Kumegawa M &
Hakeda Y. Dexamethasone Enhances Osteoclast Formation Syner-
gistically With Transforming Growth Factor-Beta by Stimulating
the Priming of Osteoclast Progenitors for Differentiation into
Osteoclasts. Journal of Biological Chemistry 2003 278 44667—
44674.
28 O’Brien CA, Jia D, Plotkin LI, Bellido T, Powers CC, Stewart SA,
Manolagas SC & Weinstein RS. Glucocorticoids Act Directly on
Osteoblasts and Osteocytes to Induce Their Apoptosis and
Reduce Bone Formation and Strength. Endocrinology 2004 145
1835– 1841.
29 Ohnaka K, Taniguchi H, Kawate H, Nawata H & Takayanagi R.
Glucocorticoid Enhances the Expression of Dickkopf-1 in
Human Osteoblasts: Novel Mechanism of Glucocorticoid-Induced
Osteoporosis. Biochemical and Biophysical Research Communications
2004 318 259– 264.
30 Weinstein RS, Jilka RL, Parfitt AM & Manolagas SC. Inhibition of
Osteoblastogenesis and Promotion of Apoptosis of Osteoblasts and
Osteocytes by Glucocorticoids. Potential Mechanisms of Their
Deleterious Effects on Bone. The Journal of Clinical Investigation
1998 102 274– 282.
31 Pereira RM, Delany AM, Durant D & Canalis E. Cortisol Regulates
the Expression of Notch in Osteoblasts. Journal of Cellular Biochem-
istry 2002 85 252– 258.
Bone mass and turnover in Cushing’s syndrome: a prospective study 117EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
www.eje-online.org
32 Eberhardt AW, Yeager-Jones A & Blair HC. Regional Trabecular
Bone Matrix Degeneration and Osteocyte Death in Femora of
Glucocorticoid- Treated Rabbits. Endocrinology 2001 142
1333– 1340.
33 Hirayama T, Sabokbar A & Athanasou NA. Effect of Corticoster-
oids on Human Osteoclast Formation and Activity. Journal of
Endocrinology 2002 175 155– 163.
34 Weinstein RS, Chen JR, Powers CC, Stewart SA, Landes RD,
Bellido T, Jilka RL, Parfitt AM & Manolagas SC. Promotion of
Osteoclast Survival and Antagonism of Bisphosphonate-Induced
Osteoclast Apoptosis by Glucocorticoids. The Journal of Clinical
Investigation 2002 109 1041– 1048.
35 Arnaldi G, Angeli A, Atkinson AB, Bertagna X, Cavagnini F,
Chrousos GP, Fava GA, Findling JW, Gaillard RC, Grossman AB,
Kola B, Lacroix A, Mancini T, Mantero F, Newell-Price J,
Nieman LK, Sonino N, Vance ML, Giustina A & Boscaro M. Diag-
nosis and Complications of Cushing’s Syndrome: a Consensus
Statement. Journal of Clinical Endocrinology and Metabolism 2003
88 5593– 5602.
36 Lunar corporation. Technical manual. Documentation version
3/95. DPX version 4.6c.
37 Akcay MN, Akcay G & Bilen H. The Effects of Calcitonin on Bone
Resorption in Hyperthyroidism: a Placebo-Controlled Clinical
Study. Journal of Bone and Mineral Metabolism 2004 22 90 – 93.
38 Chen Q, Kaji H, Iu MF, Nomura R, Sowa H, Yamauchi M,
Tsukamoto T, Sugimoto T & Chihara K. Effects of an Excess and
a Deficiency of Endogenous Parathyroid Hormone on Volumetric
Bone Mineral Density and Bone Geometry Determined by Periph-
eral Quantitative Computed Tomography in Female Subjects. Jour-
nal of Clinical Endocrinology and Metabolism 2003 88 4655–4658.
39 Wuster C, Harle U, Rehn U, Muller C, Knauf K, Koppler D,
Schwabe C & Ziegler R. Benefits of Growth Hormone Treatment
on Bone Metabolism. Bone Density and Bone Strength in Growth
Hormone Deficiency and Osteoporosis. Growth Hormone & IGF
Research 1998 8(Suppl A) 87– 94.
40 Rehman Q & Lane NE. Effect of Glucocorticoids on Bone Density.
Medical and Pediatric Oncology 2003 41 212– 216.
Received 20 June 2005
Accepted 12 October 2005
118 C Kristo and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2006) 154
www.eje-online.org
