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Laboratory of Pediatric Endocrinology and Department of Pediatrics, San Raffaele Scientific Institute, Vita-Salute S. Raffaele University, 20132 Milan, Italy
Address all correspondence to: Stefano Mora, M.D., Laboratory of Pediatric Endocrinology, H. San Raffaele,Via Olgettina 60, 20132 Milano MI, Italy. E-mail: mora.stefano{at}hsr.it.
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Abstract |
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Objective: The objective of the study was to evaluate bone mineral density (BMD) and bone metabolism in CAH patients.
Design: This was a cross-sectional observational study.
Setting: The study was conducted at a referral center for pediatric endocrinology.
Patients and Other Participants: Thirty young patients with the classical form of CAH (aged 16.4–29.7 yr) treated with glucocorticoid from diagnosis (duration of treatment 16.4–29.5 yr) and 138 healthy controls (aged 16.0–30.0 yr) were enrolled.
Main Outcome Measures: BMD was measured in the lumbar spine and whole body by dual-energy x-ray absorptiometry. Bone formation and resorption rates were estimated by serum measurements of bone-specific alkaline phosphatase and C-terminal telopeptide of type I collagen, respectively.
Results: CAH patients were shorter than controls (women –6.8 and men –13.3 cm). Therefore, several methods were used to account for the effect of this difference on bone measurements. Whole-body BMD measurements were significantly lower, compared with controls (P < 0.03), after controlling for height (on average –2.5% in females and –9.3% in male patients). No differences were found in lumbar spine measurements. Bone-specific alkaline phosphatase and C-terminal telopeptide of type I collagen serum concentrations were higher in CAH patients than control subjects (P < 0.04). BMD measurements and bone metabolism markers did not correlate with the actual glucocorticoid dose or mean dose over the previous 7 yr.
Conclusions: Young adult patients with the classical form of CAH have decreased bone density values, compared with healthy controls. This may put them at risk of developing osteoporosis early in life.
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Introduction |
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Treatment of CAH patients consists of long-term glucocorticoid therapy (2). Glucocorticoid both replaces the deficient cortisol and reduces ACTH overproduction and overstimulation of the adrenal cortex, thereby decreasing adrenal androgen secretion. Glucocorticoid must be dosed carefully to avoid excessive or insufficient adrenal suppression (3).
Glucocorticoid therapy is the most frequent cause of drug-induced osteoporosis (4). Bone loss is generally rapid during the first 6 months of glucocorticoid treatment, with an average decrease of 5% over the first year of long-term treatment (5, 6). Thereafter bone loss is 1–2%/yr (5). Recent studies indicate that fracture risk is increased, even at low doses of glucocorticoids (7), but it is most rapid and extensive at prednisone doses 7.5 mg or more per day or equivalent (8). Prolonged steroid therapy, even in substitution doses, may lead to a reduced bone mineral density (BMD) (9), but it is uncertain whether glucocorticoid replacement therapy affects bone mass in patients with CAH. Previous reports in CAH patients showed increased (10, 11), decreased (12, 13, 14, 15, 16, 17, 18), or normal (19, 20, 21, 22, 23) BMD.
Similarly, studies on bone metabolism in patients with CAH led to discordant results (14, 19). A study on adult patients (19) showed decreased serum concentrations of osteocalcin and bone alkaline phosphatase and lower urine levels of cross-linked N-telopeptide of type I collagen, compared with healthy controls. Another study (14) reported normal concentrations of calciotropic hormones and markers of bone metabolism in a group of children with CAH.
The objective of the present study was to measure bone mass and bone metabolism markers in young adult patients with a narrow age range with the classical form of CAH who have been treated since diagnosis with glucocorticoid and compare the results with a large group of healthy individuals. We also assessed the presence of a relationship among glucocorticoid dose, hormonal control, and bone mass and bone metabolism measurements.
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Subjects and Methods |
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Eligible for the study were Caucasian CAH patients with a confirmed diagnosis of 21-OHD who completed their sexual development and who were younger than 30 yr. Excluded were patients with other causes of CAH and those of non-Caucasian origin. Thirty-three patients agreed to participate in the study. Three patients were excluded from the analyses because of the presence of risk factors for reduced bone mass (familial osteoporosis, prematurity, delayed puberty). The presence of other known causes of low bone mass was excluded in the remaining CAH patients. We ended up with 15 women and 15 men, aged 16.4–29.7 yr. Of all the patients, 24 subjects (12 women, 12 men) had the classical SW form, and six subjects (three women, three men) had the SV form. Twenty-seven were diagnosed within the first month of life, whereas three patients (all the men with the SV form) have been diagnosed within the first 6 yr of life. Diagnosis was made on the basis of clinical evidence and elevated basal serum concentrations of 17-OHP or increased urinary excretion of 17-ketosteroids and pregnatriol; the presence of ambiguous genitalia of another origin was excluded in girls by karyogram analysis. Further molecular analysis of the CYP21 gene has been performed in all patients, confirming the diagnosis in all cases. All patients had been treated from the time of diagnosis. Patients with the SW form received glucocorticoid and mineralcorticoid, whereas patients with the SV form were treated with glucocorticoid alone. Treatment at the time of the study consisted of hydrocortisone given two or three times daily or dexamethasone once daily. SW patients were treated with 9
-fludrocortisone. Age at menarche was 12.8 ± 1.6 yr. All female patients had regular menses. One young patient was taking a contraceptive pill at the time of the study.
As a control group, we studied 138 white volunteers (84 women) aged 16.0–30.0 yr and recruited from the same geographical area. All subjects were healthy and appropriately physically active for their age; none was involved in competitive sport activities. Candidates were excluded if they had a history of chronic illness; they had one or more fractures; or they had taken any medication, hormone, vitamin preparation, or calcium supplements regularly.
Informed consent was obtained from all patients and volunteers. Informed consent of minor subjects was also obtained from their parents or legal guardians. The study was made in accordance to the principles of the Declaration of Helsinki.
Methods
All candidates for this study underwent physical examination to obtain anthropometric measures and assess pubertal development, when needed. Body weight was measured to the nearest 0.1 kg on a balance beam scale (Seca, Hamburg, Germany), and height was measured to the nearest millimeter using a wall-mounted stadiometer (Holtain Ltd., Crosswell, UK). Body mass index (BMI) was computed as weight/height2.
Glucocorticoid treatment. Glucocorticoid doses were expressed as dose per body surface per day (milligrams per square meter per day). A mean dose was calculated also over the 7 yr preceding the investigation. Doses of glucocorticoids were converted to hydrocortisone growth retarding equivalents using the following formula: 30 mg hydrocortisone = 37.5 mg cortisone acetate = 6.0 mg prednisone = 0.375 mg dexamethasone.
Hormonal control.
Hormonal control was assessed by collecting all results of serum concentration of 17-OHP, 
4-androstenedione (4-A), and testosterone from the patients’ records in the preceding 7 yr. The mean levels of circulating steroids were used to calculate the correlations between hormonal control and bone mineral measurements. The serum measurements of the above mentioned hormones obtained the day of the examination were used to calculate the correlations with the markers of bone metabolism.
Serum levels of 17-OHP were measured by RIA (17-OH progesterone; Diagnostic System Laboratories Inc., Webster, TX). Intra- and interassay variations were less than 6% and less than 8%, respectively. Sensitivity of the assay was 0.06 nmol/liter (0.02 ng/ml).
Concentrations of 4-A were detected by RIA (active androstenedione, Diagnostic System Laboratories). Intraassay variation was less than 6%; interassay variation was less than 10%. Sensitivity of the assay was 0.1 nmol/liter (0.03 ng/ml).
Testosterone was measured by RIA (CoTube testosterone; Bio-Rad, Hercules, CA) in serum samples. Sensitivity of the assay was 0.27 nmol/liter (0.08 ng/ml). Within-run variation was less than 12%; between-run variation was less than 13%.
Plasma renin activity was measured by RIA (GammaCoat plasma renin activity; DiaSorin S.p.A., Saluggia, Italy). Sensitivity of the assay was 0.018 ng/tube. Within- and between-assay variabilities were less than 13%.
Bone mineral measurements. Bone mineral measurements were made with a dual-energy x-ray absorptiometer (DPX-L; GE-Lunar Corp., Madison, WI). The instrument was calibrated on a daily basis according to the manufacturer’s instructions. Reproducibility was calculated as coefficient of variation (CV) obtained by weekly measurements of a standard phantom on the instrument and repeated measurements obtained in three subjects of different ages. The CV of our instrument is 0.6% with the standard phantom; in vivo we calculated a CV of 1.4% for the lumbar spine and 1.5% for the whole skeleton. The effective radiation dose for each scan was about 0.3 µSv for the lumbar spine and less than 0.03 µSv for the whole-body scans (24). The data were analyzed by the same operator (S.M.), using the same software (version 1.5 h). BMD was measured at the L2-L4 vertebrae level and in the whole skeleton. BMD measurements (grams per square centimeter) were converted to Z-scores by subtracting the reference population (age and sex matched) mean from the raw measurement, and dividing this by the SD, using the GE-Lunar database.
Biochemical measurements. Blood samples were taken in a fasting state at around 0800–0900 h in all subjects. Blood was collected before the morning dose of glucocorticoid in CAH patients. Blood was allowed to clot immediately after venipuncture; serum was separated by centrifugation, and it was stored at –30 C until analysis. Serum levels of PTH, bone-specific alkaline phosphatase (BALP), and carboxy-terminal telopeptide of type I collagen (CTX) were measured in all subjects.
Intact PTH was measured by an immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The intraassay variance was less than 4%, whereas interassay variation was less than 7%. Sensitivity of the assay was 1 pg/ml.
BALP, a marker of bone formation, was measured in serum, using a commercial immunoassay (Alkphase-B; Metra Biosystems, Inc., Mountain View, CA). Intraassay reproducibility was less than 4%, and interassay variation was less than 7%. Sensitivity was 0.7 U/liter.
CTX, a marker of bone resorption, was measured in serum by ELISA (Serum CrossLaps; Osteometer Biootech A/S, Herlev, Denmark). Detection limit of the assay was 0.094 ng/liter. Intraassay variability was less than 5.4%, and interassay variability was less than 8.1%.
Statistical analysis. Descriptive statistics were calculated for all the variables, and data are expressed as the mean ± SEM or median (range), unless otherwise stated. Distribution of the variables was checked using the Shapiro-Wilk W test. Variables that were not normally distributed were log transformed for statistical analyses. All statistical analyses were conducted at the alpha = 0.05 level and were two tailed. The statistical software JMP IN (SAS Institute, Inc., Cary, NC) was used for the analyses.
Comparisons between groups (male vs. females, patients vs. healthy subjects, etc.) were performed by Student’s t test or multiple regression analyses, when correction for the effect of confounding variables was needed.
Simple correlation analyses were performed to assess the relationship between variables.
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Results |
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. Height was significantly lower in patients, compared with controls (women: t = –3.69, P = 0.0004; men: t = –6.25, P < 0.0001). On average, CAH female patients were 6.8 cm shorter than healthy young women, and male patients were 13.3 cm shorter than healthy young men. No differences in body weight were noted. Consequently, BMI values were significantly higher in patients than control subjects (women: t = 3.83, P = 0.0002; men: t = 2.83, P = 0.0063). Glucocorticoid actual dose and mean dose of the preceding 7 yr are shown in Table 2. The mean serum concentrations of the preceding 7 yr, measured before taking the morning glucocorticoid dose, are shown in the same table. No significant differences between female and male patients were observed, except for testosterone concentration (P < 0.0001). Testosterone concentrations of male patients were within the normal range for adult subjects.
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Results of bone mass measurements are shown in Table 3. Dual-energy X-ray absorptiometry (DXA) measurements are greatly influenced by bone size (25). Because CAH patients were shorter than healthy controls, we tried several approaches to correct for the differences in body dimensions. First, we analyzed the raw data using multiple regression analyses to adjust for anthropometric differences, as proposed by Prentice et al. (26). We then calculated the bone mineral apparent density of the vertebrae using the formula proposed by Carter et al. (27): bone mineral apparent density = bone mineral content (BMC)/bone area1.5. Recently, Fewtrell et al. (28) demonstrated that the correction of BMC values by height elevated at the third power is a valid method to account for anthropometric differences in lumbar spine measurements. We therefore used the proposed formula: BMCh = BMC/h3. Finally, we applied the formula proposed by Katzman et al. (29) to whole-body BMC values [BMD = BMC/(BA2/h)]. Lumbar spine BMD values of female patients were not different from those of control subjects after correction for confounding variables by multiple regression analysis (P = 0.31), with the method of Carter et al. (27) (P = 0.47) and Fewtrell et al. (28) (P = 0.18). Similarly, spine BMD measurements of male patients were not significantly different from those of healthy controls with the multivariate method (P = 0.07), the method of Carter et al. (27) (P = 0.75), and the method of Fewtrell et al. (28) (P = 0.14). Female patients showed whole-body bone mineral measurements significantly lower than control subjects with both the multivariate method (P = 0.0026) and the method of Katzman et al. (29) (P = 0.0011). Similarly, male CAH patients had total body BMD measurements that were significantly lower than those of healthy subjects by both methods (multivariate: P = 0.0037; Katzman: P = 0.0075).
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Biochemical markers of bone metabolism
PTH serum concentrations of female patients did not differ significantly (t = 1.6; P = 0.11) from those of healthy subjects (Table 4). The serum levels of BALP of female patients were higher than those of control subjects, but the difference did not reach statistical significance (t = 1.56; P = 0.12). Bone resorption rate, as expressed as CTX serum concentrations, was significantly higher in women with CAH than healthy women (t = 2.73; P = 0.0086). Because the hormones contained in the contraceptive pill influence bone metabolism, the statistical analyses were repeated excluding the patient on the birth control pill. The median PTH concentration after the exclusion of the patient was 21 (12–36) pg/ml, not significantly different from healthy subjects (t = –1.5; P = 0.14). BALP serum levels were 25.3 (15.8–89.4) U/liter, significantly higher than healthy controls (t = 2.04; P = 0.048). The difference in CTX serum levels between patients and controls remained significant (t = 4.19; P = 0.0002), and the median concentration was 0.90 (0.33–1.69) ng/ml.
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). BALP and CTX levels were significantly higher in male patients than control subjects (t = 2.36; P = 0.023, and t = 2.19; P = 0.034, respectively). CAH patients with lumbar spine BMD Z-scores less than –1 had BALP serum concentrations of 37.6 (29.0–38.6) U/liter and CTX serum concentrations of 0.90 (0.33–1.33) ng/ml. BALP serum levels of CAH patients with normal BMD Z-scores were 32.9 (15.6–41.7) U/liter, and CTX levels were 0.82 (0.12–1.48) ng/ml. The differences between the two groups of patients were not statistically significant.
Glucocorticoid treatment, hormonal control, and bone mineral measurements
No significant correlations were found among the actual glucocorticoid dose, the mean dose of the previous 7 yr, and the BMD measurements or the BMD Z-scores. Moreover, bone mineral measurements did not correlate with 17-OHP, 4-A, testosterone, or renin concentrations.
Glucocorticoid treatment, hormonal control, and bone metabolism markers
We did not find relationships between the actual dose of glucocorticoids and BALP (r = 26; P = 0.15) or CTX (r = 0.03; P = 0.81). BALP serum levels correlated directly with 17-OHP (r = 0.52; P = 0.0025), 4-A (r = 0.39; P = 0.034), testosterone (r = 0.47; P = 0.0064), and renin activity (r = 0.37; P = 0.036). No correlations were found among CTX serum concentrations, 17OHP, 4-A, testosterone, and renin.
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Discussion |
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We also paid attention to the interpretation of DXA bone mineral measurements. The patients participating in the current study were much shorter than healthy control subjects, and they had higher BMI values. A major limitation of DXA technique is the dependence of bone mineral measurements on bone size (25). The larger the bone, the higher the measurement. It is therefore crucial for a correct interpretation of DXA results to account for size-related differences. Several approaches have been proposed to account for this limitation when comparing subjects with different body size (25). We used different methods to correct for the height differences (26, 27, 28, 29). The results of the different approaches were similar, indicating that the lower bone mass measurements observed in CAH patients are real and not size dependent. The results of the current study are apparently in contrast with a previous report of our group (20). The divergent results could, however, be explained by the age differences and the presence of a group of patients with the nonclassical form of CAH in the previous study. Lumbar spine bone mineral measurements were not different from those of healthy controls. However, the approach of Carter et al. (27) highlights the reduced bone volume of the vertebrae, being the BMC low and the bone mineral apparent density normal. The combination of reduced BMC and bone volume is a known risk factor for fracture (30) because smaller bones are less resistant to mechanical stress than bigger bones.
Bone metabolism rate of CAH patients was higher than that of healthy control subjects. Both male and female patients had high bone formation and bone resorption activities, as measured by specific biochemical markers. Only one previous study (19) measured bone metabolism indices in CAH patients. BALP and osteocalcin were measured as bone formation markers, whereas bone resorption was assessed by serum measurements of tartrate-resistant acid phosphatase and urinary excretion of N-telopeptide of type I collagen. All markers were decreased in CAH patients, compared with sex- and age-matched controls. The major limitation of the study was the small population considered (11 subjects) and its extreme heterogeneity: the age at diagnosis ranged from 0 to 26 yr, and the patients presented several clinical forms of CAH. Our results, obtained in a larger group of patients, are coherent with the observed bone mass measurements: high bone turnover could in fact be a cause of low bone mass. A possible reason for the high bone turnover rate found in CAH patients is represented by the interplay between exogenous glucocorticoids and endogenous production of androgens. Recent studies showed that replacement glucocorticoid therapy does not lead to decreased BMD or altered bone metabolism in Addison’s disease (31, 32, 33). CAH patients receive glucocorticoid not only to replace what is not produced by the adrenal glands but also to suppress the overproduction of androgens. Although yet to be demonstrated, slightly higher doses of glucocorticoid taken chronically might affect bone metabolism and lead to alterations of bone mass in this condition. In particular, they could increase bone resorption rate. Moreover, low androgen concentration is a known factor for reduced bone mass and altered bone metabolism (34). Suppressed androgen production with high dose of glucocorticoid may thus interfere with normal bone metabolism and lead to reduced bone mineral content. On the other hand, suboptimal glucocorticoid treatment leads to overproduction of androgens, which in turn act on osteoblasts promoting bone formation.
We found a direct correlation between the bone formation index and the serum concentrations of 17OHP, 4-A, testosterone, and renin activity. This finding indicates that the poorer the hormonal control, the higher the bone formation activity, giving thus indirect evidence on the importance of glucocorticoid use on bone metabolism. The equilibrium between exogenous glucocorticoid treatment and endogenous production of androgens is critical for the maintenance of a balanced bone metabolism in CAH patients. The finding of higher bone metabolism indices in our patients may also be the result of the presence of a group of younger patients. Bone metabolism markers are higher in children and adolescents than adults, reflecting the high metabolic activity necessary to increase bone mass and for skeletal growth. We could not ascertain whether that was the case in our series because of the small number of subjects.
Decreased BMD in CAH patients has been attributed to glucocorticoid overdosing (13, 15). However, there is no agreement on the best parameter expressing the glucocorticoid exposure to correlate with bone mass and bone metabolism measurements. Bone mineral measurements have been related to an index of accumulated postmenarcheal exogenous glucocorticoid dose (15); the current and long-term glucocorticoid doses (13); the cumulative glucocorticoid doses of the previous 0.5, 2, and 5 yr (22); or a cortisol index calculated on the basis of the current dose of glucocorticoids and the years of treatment (12). Our patients were followed up regularly in outpatient clinics, and appropriateness of their glucocorticoid dose was checked, in the light of their steroid serum concentration. Based on the experience of previous studies, we felt that the best variables to use were the actual glucocorticoid dose and the mean dose of the preceding 7 yr. Seven years were deemed to be a sufficiently long period to account for bone mineral changes. Both chosen parameters did not correlate with bone mineral measurements in our series. Similar results have been reported by others (12, 22). The lack of correlation between BMD and glucocorticoid dose seems to indicate that other mechanisms may explain the low bone density values observed in our patients. An alternative explanation is offered by the lower stature of CAH patients. This could be due to a premature epiphyseal closure, which limited the duration of bone accretion and reduced the bone mineral content as well. Longitudinal studies could help to elucidate this issue.
In conclusion, young adult patients with the classical form of CAH, receiving glucocorticoids from the first months of life, are significantly shorter and have decreased total body BMD values, compared with healthy controls. Male patients tend to have lower BMD measurements, compared with female patients. High bone turnover rate might be responsible for the altered BMD. Low peak bone mass is an important predictor for the development of osteoporosis later in life (35). Our patients may thus be at risk of reaching early very low BMD values, endangering their bone health. However, the natural history of bone mass changes in CAH patients is not yet known. Longitudinal surveys are needed to study such changes in CAH adult patients.
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Footnotes |
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First Published Online August 22, 2006
Abbreviations: 4-A, 4-Androstenedione; BALP, bone-specific alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; BMI, body mass index; CAH, congenital adrenal hyperplasia; CTX, carboxy-terminal telopeptide of type I collagen; CV, coefficient of variation; DXA, dual-energy X-ray absorptiometry; 21-OHD, 21-hydroxylase deficiency; 17-OHP, 17-hydroxyprogesterone; SV, simple virilizers; SW, salt wasters.
Received December 27, 2005.
Accepted August 10, 2006.
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