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Original Studies |
Division and Research Unit of Endocrinology (I.C., M.T., V.T., A.S.), Division of Internal Medicine (V.C.), Departments of Clinical Pathology (S.F., M.P., A.D.G.), Radiology (G.G., M.C.), and Nuclear Medicine (S.Mo.), Scientific Institute Casa Sollievo della Sofferenza, San Giovanni Rotondo; Division of Endocrinology, Istituto Auxologico Italiano (A.L.), Scientific Institute San Giuseppe, Piancavallo; and Istituto di Clinica Medica II, Policlinico Umberto I (S.Mi.), Rome, Italy
Address all correspondence and requests for reprints to: Dr. Iacopo Chiodini, Division and Research Unit of Endocrinology, IRCCS Ospedale Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo (FG), Italy.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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In CS patients, serum bone Gla protein, a marker of osteoblastic function, was reduced (3.28 ± 2.3 vs. 6.47 ± 2.5; P < 0.01), and bone resorption was increased, as indicated by increased urinary hydroxyproline (36.6 ± 12 vs. 29.0 ± 9.1, P < 0.05) and urinary deoxypyridinoline (22.1 ± 8.0 vs. 16.4 ± 6.3; P < 0.05). BMD was significantly (P < 0.05 or P < 0.01) reduced at all sites, except cortical forearm, in CS patients compared to controls. By comparing z-scores of reduced BMD in CS patients, spinal trabecular BMD was found to be the most severely affected. Furthermore, disease activity, as measured by urinary free cortisol, was significantly correlated with bone Gla protein (r = -0.57; P < 0.02), urinary hydroxyproline (r = 0.57; P < 0.02), urinary deoxypyridinoline (r = 0.48, P < 0.05), and BMD measured at spine and femur.
Our results show that compared to matched control subjects, female eumenorrheic CS patients have reduced osteoblastic function, increased bone resorption, and reduced BMD, and that the severity of these abnormalities is statistically related to the severity of disease activity, as indicated by urinary free cortisol. Moreover, our data suggest a site and tissue specificity of the effect of glucocorticoid excess on bone mass.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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These questions are unresolved mainly due to the following reasons. Firstly, some of the previous studies conducted on the effect of glucocorticoid on the skeleton have been carried out in patients with glucocorticoid excess secondary to exogenous glucocorticoid treatment of disorders (i.e. rheumatoid arthritis and systemic lupus erythematosus), which also affect bone turnover and mass by themselves (2, 8, 21, 22, 23, 24, 25, 26, 27). In these studies, therefore, it has been difficult to identify the specific role of glucocorticoid excess on bone mass and turnover. Secondly, all previous studies in patients with Cushing’s syndrome (CS) have been carried out in both eugonadal and hypogonadal subjects from both sexes (15, 28). The results of these studies, therefore, have been potentially biased by the different impacts of different gonadal status and/or sex on the effect of glucocorticoid on bone turnover and mass. Thirdly, bone resorption in patients with glucocorticoid excess has been studied using poorly specific markers, such as urinary hydroxyproline (Hp) and serum type I cross-linked C telopeptide (5, 7, 10, 13, 14, 15, 16).
To study the effect of "pure" glucocorticoid excess on the skeleton, without the possible influence of different gonadal status and sex, we measured bone mineral density (BMD) at several skeletal sites and assessed specific biomarkers of bone turnover in 18 eugonadal female patients with CS.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Eighteen eugonadal (
10 menstrual cycles/yr) female patients
with active CS were studied. The clinical features of the studied
patients are shown in Table 1
. CS was
suspected from the clinical picture, and diagnosis was based on the
following biochemical data: 24-h urinary free cortisol (UFC) levels
higher than 100 µg/24 h and morning cortisol serum levels exceeding 5
µg/dL after overnight 1-mg dexamethasone suppression test. CS was
sustained by adrenal adenomas in 6 patients and by ACTH-secreting
pituitary adenomas in 12 patients, with the differential diagnosis
based on morning serum ACTH levels and percent decrease in morning
serum cortisol levels after overnight 8 mg dexamethasone administration
(>50% or <50%). In all cases, both imaging techniques and
pathological reports after surgery confirmed the previous etiological
diagnosis. At the time of the study no patient underwent pituitary or
adrenal surgery. All patients menstruated regularly and had no signs of
other hormonal abnormalities.
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10 menstrual cycles/yr) women, matched
for age and body mass index (Table 1
), served as controls. The BMD
values of control subjects and their z-scores lie within the normal
distribution of the reference population of our center. None of the
subjects studied took substances affecting bone metabolism or had
evidence of vertebral fractures on lateral x-ray of the spine. In all
subjects serum estradiol levels, measured in the early follicular
phase, were in the normal range and were not different between patients
and controls (Table 2).
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Serum and urinary samples were collected and stored at -70 C until assayed. In all subjects serum total calcium (Ca), phosphorus, creatinine (Cr), and total alkaline phosphatase activity were determined by a multichannel autoanalyzer.
In all patients, serum cortisol concentrations and UFC levels (after dichloromethanol extraction) were evaluated using a commercially available immunofluorometric assay (TDX-FLX Abbott, Diagnostika, Wiesbaden-Delkenheim, Germany); serum ACTH levels were measured using an immunoradiometric assay (Brahms Diagnostica, Berlin, Germany); dehydroepiandrosterone sulfate (DHEAS) was evaluated by RIA (DHEA-S-7, Diagnostic System Laboratories, Webster, TX). In all subjects serum estradiol levels were measured by RIA (Coat-A-Count Estradiol, Diagnostic Products Corp., Los Angeles, CA), and serum intact PTH levels were measured by a two-site chemoluminometric immunoassay (Chiron Diagnostics, East Walpole, MA).
In all subjects serum bone GLA protein (BGP) was assayed by immunoradiometric assay for the intact molecule (ELSA-OST-NAT, Cis Biointernational, Gif-sur-Yvette, France); intra- and interassay coefficients of variation were 3.8% and 4.7%, respectively. Ca, Hp, and deoxypyridinoline (D-Pyr) were also evaluated in fasting spot urine samples and corrected for creatinine excretions (Ca/Cr, Hp/Cr, and D-Pyr/Cr, respectively). Hp was assessed by spectrophotometric detection, and D-Pyr was determined by fluorometric detection after reverse phase high pressure liquid chromatography using commercial kits (Bio-Rad Laboratories, Segrate, Milan, Italy). The intra- and interassay coefficients of variation were 3% and 5.1% for the Hp assay, and 6.6% and 12.3% for the D-Pyr assay, respectively.
Bone densitometry
In all subjects BMD was evaluated by means of different devices at axial and appendicular skeletal sites, characterized by different compositions of cortical and trabecular tissue. Spinal BMD was measured by both single energy quantitative computed tomography of L1–L4 (QCT; Toshiba CT Xpeed, Toshiba Medical Systems Division, Tokyo, Japan), which is able to detect selectively trabecular true density (in vivo precision, 1.8%), and dual x-ray absorptiometry of L2–L4 (DXA; Norland XR-26, Norland Instruments, Fort Atkinson, WI), which assesses BMD of total vertebral bodies (in vivo precision, 1.0%). BMD was also measured by peripheral QCT (pQCT; Stratec XCT 960, Stratec Medizintechnik, Pforzheim, Germany) at an ultradistal radial site of the nondominant arm in 12 patients and 23 controls; such an instrument (in vivo precision, 1.2%) allows simultaneously the measurement of integrated value, trabecular, and cortical BMD. BMD was also evaluated by DXA at three femoral sites: neck (FN), Ward’s triangle (WT), and great trochanter (TR) (in vivo precision, respectively, 2.1%, 3.5%, and 2.4%). Our DXA machine does not perform a total hip measurement.
Statistical analysis
The results are reported as the mean ± SD. P < 0.05 was considered significant. For each variable, the normality of distribution was tested with the W statistic of Shapiro-Wilk. Normally distributed data were compared, after testing homogeneity of variance, by Student’s t test with pooled or Dixon-Massey modified variance as appropriate. The Mann-Whitney test was used when the normality test failed.
The associations between variables were tested by Pearson’s product-moment correlation or Spearman’s rank order correlation, as appropriate. Multiple regression analysis was used to evaluate the relative influence of disease activity, PTH, DHEAS, and estimated disease duration on BMD and turnover markers values after logarithmic transformation of not normally distributed variables.
To compare the effects of glucocorticoid excess on bone mass at different skeletal sites with different devices, individual BMD levels of CS patients were normalized to those of the reference population of our center (29) and expressed as SD units (z-score values). The mean z-score values were then compared by one-way ANOVA. Bonferroni’s test for multiple comparisons was performed when significant differences were found.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Comparisons between patients and control subjects are shown in
Table 2. Serum BGP and PTH levels were significantly different (with
BGP being lower and PTH being higher) in patients than in controls. In
addition, urinary Ca/Cr, Hp/Cr, and D-Pyr/Cr levels were significantly
higher in patients than in controls. In contrast, serum Ca, phosphorus,
Cr, and alkaline phosphatase did not differ between patients and
controls.
Bone mass
As shown in Table 3, spinal BMD
measured by both QCT and DXA, forearm trabecular BMD, and femoral BMD
at each site, were significantly lower in patients than in controls. In
contrast, forearm cortical and integrated BMD did not significantly
differ between patients and controls.
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Disease activity, as indicated by UFC, correlated positively with
Ca/Cr (r = 0.49; P < 0.05), Hp/Cr (r = 0.57;
P < 0.02) and D-Pyr/Cr (r = 0.48;
P < 0.05) levels and negatively with BGP (r =
-0.57; P < 0.02) levels (Fig. 2). A significant negative correlation
was also found between UFC and BMD measured at the spine (QCT: r =
-0.5; P < 0.05; DXA: r = -0.61;
P < 0.002) and femur (WT: r = -0.56;
P < 0.02; FN: r = -0.59; P <
0.02; TR: r = -0.55; P < 0.05). Finally,
logUFC was significantly correlated with Ca/Cr, Hp/Cr,
D-Pyr/Cr, and BGP when data were analyzed by multiple regression
analysis, including PTH, DHEAS, and estimated duration of disease. In
contrast, PTH, DHEAS, and estimated duration of disease did not
correlate with any of the measured variables related to bone turnover
and/or mass by either single or multiple regression analysis.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our data are in agreement with previous reports showing reduced BGP, a sensitive marker of the effects of glucocorticoid on the osteoblast (6), in patients affected by CS (9, 10, 11, 12, 13, 14, 16). This is in line with in vitro (31) and in vivo histomorphometric (3, 4) studies showing decreased osteoblastic activity in response to supraphysiological glucocorticoid levels.
In contrast, as far as bone resorption is concerned, our data are conflicting, with most of the previous reports showing normal bone resorption in patients with glucocorticoid excess (10, 13, 14, 16). In fact, we demonstrate that urinary D-Pyr was clearly increased in CS patients, indicating, therefore, increased bone resorption, which was also suggested by the increase in a less specific marker of bone resorption, such as Hp/Cr. This discrepancy may be due to several factors, including the above-mentioned different selection of patients and the use of different markers of bone resorption (32). In fact, urinary D-Pyr is a highly specific marker of bone resorption that has recently become available (32).
When looking at individual data from CS patients, UFC correlated negatively with BGP and positively with both Hp/Cr and D-Pyr/Cr, suggesting a relationship between disease activity and altered bone turnover.
The patients we studied were affected by secondary hyperparathyroidism, as indicated by high PTH levels in the presence of normal plasma Ca levels. Similar data have been previously reported in patients affected by glucocorticoid excess (2, 4, 5, 6, 7, 8, 33, 34, 35, 36). Secondary hyperparathyroidism has been suggested to be the link between glucocorticoid excess and increased bone resorption (2, 4, 5, 6, 7, 8, 33, 34, 35, 36). However, this is unlikely in our series, because no correlation was observed between bone resorption markers and PTH levels. In contrast, as discussed previously, a positive correlation was observed between UFC and bone resorption markers, thus suggesting a direct role of glucocorticoid on bone resorption.
Given the concomitant presence of decreased osteoblastic function and increased bone resorption, the observation of reduced BMD at forearm, femur, and spine in CS patients was not surprising. Thanks to the device we used, which is able to measure independently the BMD of the two different bone tissues at the forearm, our data demonstrate that trabecular, but not cortical and integrated BMD, was significantly reduced in CS patients, suggesting different sensitivities of the two bone tissues to glucocorticoid excess at the forearm. These data indicate, therefore, that integrated BMD may not be the most appropriate measurement to quantify the negative effect of glucocorticoid on forearm bone, and this may partially account for the conflicting results previously reported on forearm bone mass in patients with glucocorticoid excess (19, 37). In addition, the finding of specific trabecular bone loss despite hyperparathyroidism, which is known to predominantly affect cortical bone (38), suggests that in CS patients, the excess of glucocorticoid, and not that of PTH, plays a predominant role in loss of bone mass. In contrast to that observed at the forearm, both trabecular (WT) and cortical (FN and TR) femoral bone were similarly reduced in CS patients, indicating, therefore, that the different sensitivities to glucocorticoid excess of the two different bone tissues are site specific (i.e. present at the forearm but not at the femur). In addition, by comparing the BMD z-score values for all affected sites in CS patients, spinal trabecular bone, as studied by QCT, was the most severely affected, as previously described (19, 39). It has been questioned that bone density at the spine, as measured by single energy QCT, might be underestimated due to increased bone marrow fat content in CS. However, this is unlikely, because no difference in bone marrow fat content has been reported between control subjects and CS patients (40).
Finally, an inverse correlation was found between BMD measured at the spine and femur and UFC. This finding, which to our knowledge has never been reported, suggests that the severity of BMD reduction in patients with CS is a function of glucocorticoid levels.
In conclusion, our data demonstrate that female eumenorrheic CS patients have reduced osteoblastic function, increased bone resorption, and reduced BMD that correlate with glucocorticoid levels. A site and tissue specificity of the negative effect on bone mass of glucocorticoid excess is also demonstrated.
Received December 2, 1997.
Revised February 23, 1998.
Accepted March 2, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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1 (I) procollagen mRNA by trascriptional
and post-trascriptional mechanism. J Cell Biochem. 57:488–494.[CrossRef][Medline]
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