0031-399819513706-0731$03.0010 PEDIATRIC RESEARCH Vol. 37, No. 6, 1995 Copyright O 1995 International Pediatric Research Foundation, Inc. Printed in U.S.A. Progressive Normalization of Growth Hormone-Binding Protein and IGF-I Levels in Treated Growth Hormone-Deficient Children* JULIANE LEGER, MICHELE NOEL, PAUL CZERNICHOW, AND MARIE-CATHERINE POSTEL-VINAY Department of Pediatric Endocrinology and Diabetes, Hopital Robert Debre' [J.L., M.N., P.C.] and INSERM Unite' 344, Molecular Endocrinology, Hopital Necker-Enfants Malades, Paris, France [M.-C.P.-V.] 'ACT The short- and long-term effects of hGH treatment on growth increase in serum IGF-I level was observed as soon as 1 wk of hormone (GH)-binding protein (GHBP) were examined in 18 hGH therapy, and after 3 mo, the mean IGF-I value was normal. prepubertal children, aged 1.5-10 y, with isolated idiopathic GH No correlation was found between the increase in GHBP, IGF-I deficiency. The patients were studied before and at regular levels, and the growth velocity at 12 and 24 mo of treatment. intervals during 24 mo of hGH therapy (0.6 IU/kg/wk, given These findings support the role of GH in the regulation of daily). Pretreatment GHBP values were low: 14.6 2 1.2% of GHBPIreceptor in man. The time course of the GH effect appears radioactivity (p < 0.0001 versus normal prepubertal children). to be progressive and variable. (Pediatr Res 37: 731-735,1995) After the first hGH injection, GHBP levels fell significantly at 6 h (8.2 2 1.3% of radioactivity) and then remained at basal level during the first week. Under hGH therapy, an increase in GHBP Abbreviations was observed, but it occurred at different times of treatment, from GH, growth hormone 1 to 12 mo, and the mean GHBP value became significantly GHBP, growth hormone-binding protein higher than the value before treatment after 12 mo of therapy. An BMI, body mass index Identification of the serum GHBP and demonstration that the the plasma GH binding activity. On one hand, GH is able to amino acid sequence of GHBP is identical to that of the increase GHBP levels, as was shown in children with GH extracellular domain of the membrane GH receptor have pro- deficiency (5, 6) and also in children with idiopathic short vided new tools to study GH action in man (1, 2). In clinical stature (7). On the other hand, absence of GH effect on GHBP research, measurement of serum GHBP is the only approach to has been reported in treated GH-deficient children (8). estimate the GH receptor because there are no accessible cells In this work, we have examined short-term and long-term in which GH receptors can be measured. In man, GHBP effects of hGH treatment in prepubertal children with isolated probably results from proteolytic cleavage of the membrane GH deficiency. GHBP was measured longitudinally before receptor as no specific mRNA for the short form of the GH treatment and during 24 mo after the beginning of GH therapy. receptor is detected in human tissues (3). The plasma GHBP Short-term effects of hGH may represent an important issue as level is believed to reflect the concentration of tissue GH they could be used to better predict response to hGH therapy in receptors. In animal models, GH receptors have been shown to physiopathologic situations. be modified in several pathologic situations (4). As membrane GH receptors are regulated by many factors, the level of METHODS plasma GHBP is expected to be under a multifactorial control. Among many factors, GH has been shown to play an Subjects and protocol. Eighteen prepubertal children (11 important role in the regulation of its receptors. However, a boys and 7 girls), aged 1.5-10 y (mean age: 5.6 + 2.9 y), with controversy has recently arisen concerning the effect of GH on isolated idiopathic GH deficiency, were studied before treat- ment and at regular intervals during the first 2 y of hGH therapy. GH deficiency was defined as subnormal GH re- Received September 8, 1994; accepted December 20, 1994. sponses to at least two provocative tests (peak GH < 10 p,g/L), Correspondence: Dr. M-C Postel-Vinay, INSERM Unit6 344, Faculti de Mtdecine which was chosen by the French Pituitary Agency for provid- Necker, 156 Rue de Vaugirard, 75730 Paris Cedex 15, France. * This work was presented in part at the Joint Meeting of LWPESESPE, San Francisco, ing hGH therapy. Recombinant hGH (Saizen, Serono Labora- June 1993 (Abstract 356). tories and Genotonorm, Kabi Pharmacia Laboratories) was given s.c. daily, at the dosage of 0.60 IU/kg/wk. All but four >6 pg/L, as previously reported (5); this was the case for all subjects were followed longitudinally for 2 y of treatment. blood samples collected 6 h after the first GH injection and for Patient 14 could not be followed after 6 mo of therapy, and one sample collected at 12 h. three patients (nos. 1, 8, and 11) were studied for 1 y only. None of the patients of the study group developed detectable Three patients (nos. 13, 17, and 18) entered puberty between GH antibodies as observed on the elution profiles from HPLC- 18 and 24 mo of the treatment; for that reason and for that time gel filtration of lZ51-h~~incubated with serum. The GH period, results for these patients were not included. Height and concentration was measured by immunoradiometric assay us- weight were measured before treatment and every 3 mo on ing polyclonal antibodies (Pharmacia, Saint-Quentin en Yve- hGH treatment. Height was expressed as the SD score (SDS) lines, France). IGF-I was measured by RIA, after acid gel for chronologic age (9). Weight for height was expressed as filtration (12). IGF-I antibody was kindly donated by Dr. P. body mass index [BMI = weight (kg)/height (m2)] in SD for Chatelain, Lyon, France. chronologic age (10). Growth velocities were calculated 12 mo Statistics. Results are expressed as mean + SEM. Statistical after beginning hGH therapy by subtracting the height at differences between groups were assessed using variance anal- baseline from the height at 12 mo. The clinical characteristics ysis. If overall significant differences were present, specific of patients are presented in Table 1. differences between two time periods were sought by the The 18 patients were investigated for biologic parameters Wilcoxon signed rank test (with a Bonferroni correction for before and 6, 12, and 24 mo after the onset of hGH treatment. simultaneity). The Mann-Whitney U test was used to compare The short-term response to the hGH treatment was evaluated in data in two different groups. Correlation between variables was 10 of them, and blood samples were collected before and at 6, evaluated by linear regression analysis. 12, 24, and 48 h, 8 d, and 1 and 3 mo after the beginning of treatment. For technical reasons, some blood samples could not RESULTS be obtained for all measurements. After the first 48 h of treatment, sampling was performed between 14 and 20 h after In 18 children with isolated GH deficiency, basal GHBP the last hormone injection. values were lower (14.6 + 1.2% of radioactivity, p < 0.001) Informed consent was obtained from the parents. than in normal prepubertal children (mean value in a group of Procedures. Blood samples were collected during the study 15 prepubertal normal children: 24.8 + 1.7%) (11). As shown period, and sera were stored at -20°C until assayed. Periodic in Table 2, values ranged from 7.0 to 25.4% of radioactivity. In samples were measured in the same assay. this group of prepubertal children, no relationship was found GHPB was measured by HPLC-gel filtration, as previously between GHBP levels and chronologic or bone age. However, described (11). Briefly, 100 pL of serum were incubated with a positive correlation was found between GHBP levels and 100 pL of potassium phosphate, 0.1 M, pH 7.0, containing BMI before treatment, as shown in Fig. 1 (r = 0.63, p = '"I-~GH (1 X lo5 cpm). After 20 h at 4OC, the incubation was 0.005). injected onto HPLC Protein Pak 300 sw column (Waters, GHBP and GH levels for 48 h after the first hGH injection Milford, MA). Elution was performed isocratically using de- are presented in Fig. 2. In the group of 10 children studied for gassed buffer pumped at a rate of 0.5 mWmin. Radioactivity the short-term hGH response, the GH peak at 6 h was associ- was recorded on line using a Berthold LB 504 y detector ated with a significant decrease in GHBP (p < 0.005). Both connected to a computer. To evaluate nonspecific binding, 2 GH and GHBP returned to baseline 24 h after the first hGH pg of hGH were added to the incubation. The binding of injection (Fig. 2 and Table 3). Under hGH treatment, an 125~-h~~is expressed as the percentage of total radioactivity increase in GHBP was observed in all patients but two (patients (radioactivity in the individual peak divided by total radioac- 10 and 14); however, in one patient GHBP could not be tivity in the peaks). The interassay coefficient of variation was assayed after 6 mo of treatment and in the other one, GHBP 8% (11). In some serum samples, the GH concentration was was normal before hGH therapy. Increase in GHBP occurred elevated, and the binding of 12'1-h~~to GHBP had to be slowly and at different times of GH therapy: in the group of 10 corrected for occupancy of the receptor by GH.