Bone, Vol. 16, No. I January 1995:57-59

Osteocalcin in Congenital Adrenal Hyperplasia L. LIsA,1 M. NERADILovA,2 N, TOMAsovA,3 M. SOUTOROvA,2 and J. ZIMAK2

I First Department of Paediatrics, Second Medical School, Charles Unil'ersity, Czech Republic 2 Institute of Endocrinology, Prague, Czech Republic 3 Department of Biochemistry, Prague, Czech Republic

Osteocalcin in the serum reflects bone turnover. It is known that prolonged therapy with glucocorticoids inhibits bone turnonr. The aim of this study was to evaluate the osteocal­cin le\'el in children with congenital adrenal hyperplasia treated by glucocorticoids and mineralocorticoids and to as­sess the influence of 1,25(OHhD3' The subjects were 75 chil­dren with congenital adrenal hyperplasia, aged 1-18 years, treated with glucocorticoids and mineralocorticoids in sub~ stitution doses from birth. These children demonstrated low le\'els of osteocalcin and alkaline phosphatase, whereas cal­cium and phosphate were in the normal ranges. Despite these abnormalities, no osteoporosis was detectable and a normal growth rate was confirmed, most probably because of higher leYels of androgens; 17-0H progesterone averaged 11.8 nmol/l. After treatment with 1,25(OHhD3 , the osteocalcin levels increased, followed later by increases of alkaline phos­phatase and bone isoenzyme. (Bone 16:57-59; 1995)

Key Words: Osteocalcin; Alkaline phosphatase; Congenital adrenal hyperplasia; 1,25(OHhD3 •

Osteocalcin (OC), a noncollagenous protein, is synthesized by osteoblasts. Its serum levels reflect bone turnover (Burkhardt 1984; Neradilova 1990; Price et aI. 1980), as proved by the correlation of OC levels and histomorphology of osseous tissue (Delmas et al. 1986; Garcia-Carasco et aI. 1988; lowell et aI. 1987; Price et aI. 1980; Vermeulen et aI. 1989). In normal chil­dren, the level of OC depends on the intensity of bone metabo­lism as well as on the growth rate with diurnal variations. Data on OC levels in different pathological conditions can be found in the literature (Berensdorf et aI. 1984; Buckhardt 1984; Canalis 1983; Delmas et aI. 1986; Goldschalf et aI. 1988; lowsey & Riggs 1979; Nielsen et aI. 1988; Peretz et aI. 1989; Reid et aI. 1986).

We studied a group of pati~nts with congenital adrenal hy­perplasia (CAH) treated with glucocorticoids to establish the changes in OC levels due to this treatment.

Materials and Methods

Patients

Fifteen healthy children (eight boys and seven girls), aged 10-12 years, were studied to confirm the data of normal values for

Address for correspondence and reprints: L. Lisa, First Department of Paediatrics, Second Medical School, Charles University. V uvalu 84, 150 18 Prague 5-Matal. Czech Republic.

© 1995 by Elsevier Science Inc. 57

children given by the producer of the commercial radioimmuno­assay (RIA) kit (OSTK-PR, CIS, Oris, France).

The subjects were 75 children (35 boys and 40 girls), aged 1-18 years at the time of the study, treated for'protracted periods with glucocorticoids for proven CAH. They received hydrocorti­sone, 15-20 mg/m2 per day, and fludrocortisone, 0.025-0.1 mg! day. Plasma levels of 17-0H progesterone were estimated to confirm adequate substitution treatment; levels lower than 12 nmoUI were considered proof of successful treatment. This value is slightly higher than that in normal children, in whom levels of 17-0H progesterone do not exceed 7 nmol/I.

In 12 CAH patients, 1,25(OHhD3' 0.25 v.g/48 h, was ad­ministered for 3 months. Levels of OC, calcium, phosphate, and alkaline phosphatase were followed for 3 months after the onset of the 1,25(OHhD3 therapy.

Methods

Blood samples were drawn in the morning after fasting. Sera were separated by centrifugation 30-40 minutes after venipunc­ture and then stored at -40°C for 4 weeks until analysis.

The serum OC concentration was assessed in duplicate by the commercially available RIA kit mentioned above. The principle of the assay is based on cross-reaction of the human peptide with rabbit antiserum against bovine OC. The standard and the tracer e25I) were of bovine origin. Intra- and inter-assay coefficients of variations of the method were 6% and to%, respectively.

Determination of the catalytic concentration of alkaline phos­phatase in serum was based on enzymatic cleavage of the sub­strate disodium 4-nitrophenyl-phosphate (15 mmoUI) in N-meth­yl-glucamine buffer, pH to.1 (0.35 molll). After electrophoretic separation in acetate cellulose trips were densitometry record (Helena Lab., TX) quantified in the activities of alkaline phos­phatase isoenzyme.

Calcium and phosphate levels were measured by Autoanalyser.

Statistics

Results are expressed as mean ± standard deviation. Student's t-test was uscd.

Results

The serum levels of OC in patients with CAH were significantly lower (1.63 ± 0.96 v.g!I) than those in healthy children (11.38 ±

8756·3282195IS9.50 8756-3282(9.t)OOOO8-N

58 L. Lisa et al. Congenital adrenal hyperplasia

01: 11 ---.. ---i-----------r ------,

''lI/'"' I I 12 - .... -

10 _ •••

._i _____ _

·-···-----I-~~--·--­~.-.------- . ~---

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Figure 1. Levels ofOC in children with CAli before and aftertreatment with 1,25(OHhDj.

3.03 I-lgII) (p < 0.001). Calcium and phosphate levels were within the normal ranges. Total alkaline phosphatase and bone isoenzyme levels were on average lower in children with CAH (Table 1).

In the children with CAH, OC levels increased after treatment with 1,25(OHhD3. Calcium and phosphate values remained in the normal ranges. Total alkaline phosphatase and bone isoen­zyme increased (Figures 1 and 2).

Discussion

Osteocalcin is the main noncollagenous protein of the bone, and its expression is stimulated by 1,25(OHhD3 and inhibited by glucocorticoids (Berensdorf et al. 1984; Jowell et al. 1987). Marked reduction of OC levels during protracted and even dur­ing short-term glucocorticoid treatment has been confirmed re­peatedly (Berensdorf et al. 1984; Canalis 1983; Goldschalf et al. 1988; Jowsey & Riggs 1979; Nielsen et al. 1988; Peretz et al. 1989; Reid et al. 1986). The drop in the OC level in patients treated with glucocorticoids confirmed observations made in vitro (Canalis 1983; Jowell et al. 1987; Vermeulen et al. 1989): The effect of glucocorticoids is mediated by specific receptors (Mader et al. 1989). In our study, we looked for changes of OC levels during substitution corticoid treatment of children with CAH. No osteoporosis was found on x-ray, and the growth rate was in the normal range for the Czech population. Higher levels of the androgen 17-0H progesterone (up to 12 nmolll) might be of some importance, as steroid receptors were detected in cul­tured human osteoblast-like cells (Johansen et al. 1988), indi­cating that sex hormones directly affect bone growth. Johansen et al. (1988) found relatively low serum testosterone levels at the

II

n

u --

Bone, Vol. 16, No. I January 1995:57-59

. -- ---- --.. -j -1-------f ......... ··············· __ ··········1- - ............ ----.. -.

i J i ....... ~-=- --i . -." ..... -...... --... ---. 1 ._._----- .--- I i~:.. .. -. o---·~·.:·~:~::::=·_·-----.-:.:::··I······ , ,

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Figure 2. Levels of ALP in children with CAH before and after treat­ment with 1,25(OHhDj .

time of maximal increase in serum OC, and increased serum testosterone levels at the time of low OC. By their direct effects on osteoblasts, androgens are promoters of bone formation, whereas corticosteroids are inhibitors. Our results-low serum OC levels and normal bone structure-show that in patients with CAH, conflicting effects of glucocorticoids and of androgens may be the cause of normal bone development. According to Aurbach et al. (1992), no major defects occur in bone structure when OC synthesis is blocked. !

1,25(OHhD3 plays a part in bone metabolism and the regu­lation of bone homeostasis. It also influences cellular functions and in some tissues may affect cellular replication via the bond with the steroid receptor (Baran et al. 1992; Eisman et al. 1989; Eisman 1979; Goldschalf et al. 1988; Greig et al. 1989; Gund­berg et al. 1983; Mader et al. 1989). 1,25(OHhD3 improves the serum level of OC during glucocorticoid treatment in patients with CAH; in contrast to alkaline phosphatase, OC reacts rap- . idly. The evidence for the dependence of OC levels on 1,25(OHhD3 was given first by Lajeunesse et a!. (1990). 1,25(OHhD3 affects differentiation of the cell clone MG-63 into osteoblast-like cells. These cells have the phenotype of an os­teoblast, synthesize OC, and response to the stimulus of para­thyroid hormone or prostaglandin E2 via cyclic adenosine mono­phosphate. Osteocalcin levels rise as a result of some hormones e.g., growth hormone, but always in the presence of 1,25(OHhD3·

In conclusion, changes in the serum levels of OC, a marker of bone turnover, after glucocorticoid treatment of CAH were con­firmed. The absence of osteoporosis in the presence of low OC levels may be due to compensation by higher androgen produc­tion. 1,25(OHhD3 is able to increase plasma OC levels.

Table I. Osteocalcin, calcium, phosphate, total alkaline phosphatase, and bone isoenzyme

Osteocalcin (l1g!l) Calcium (mmoUI) Phosphate (mmoUI) Total alkaline phosphatase (I-lkatll) Bone isoenzyme (I-lkatll)

l __ _

Controls (n = 15)

11.38 ::t 3.03 2.48 ::t 0.23 1.61 ::t 0.43 8.46 ::t 4.16 6.04 ::t 0.27

Congenital adrenal hyperplasia (n = 75)

1.63 ± 0.96 2.61 ::t 1.14 1.32 ± 0.23 3.38 ::t 2.47 1.87 ± 0.89

After 1,25(OHhDj (n = 12)

6.60 ± 2.56 2.58 ::t 1.08 1.34 ::t 0.25 6.55 ::t 1.83 3.70 ::t 1.60

Bone, Vol. 16, No. I January 1995:57-59

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L. Lisa et al. 59 Congenital adrenal hyperplasia

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Dale Received: June 18. 1993 Date Rel'ised: June 23. 1994 Date Accepted: July 25. 1994