L Cinquanta

Effects of long-term treatment with growth-hormone on bone and mineral metabolism in children with growth-hormone deficiency

The effects of growth hormone (GH) deficiency and recombinant human GH replacement (0.6 IU/kg per week) on bone and mineral metabolism in 26 GH-deficient children were studied for 12 months. Before therapy, all children had significantly reduced serum levels of osteocalcin, carboxyl-terminal propeptide of procollagen type I, and 1,25-dihydroxyvitamin D, whereas serum ionized calcium, phosphate, intact parathyroid hormone, calcitonin, and 25-hydroxyvitamin D concentrations were in the normal range. All children had significant reduction of bone density for their chronologic, statural, and bone ages. During therapy with recombinant human GH, a decrease of serum ionized calcium levels and increases of phosphate, osteocalcin, carboxyl-terminal propeptide of procollagen type I, and intact serum levels of parathyroid hormone were found. A significant increase of serum levels of 1,25-dihydroxyvitamin D was found at 12 months. The urinary phosphate/urinary creatinine ratio decreased, whereas values for nephrogenous cyclic adenosine monophosphate and the ratio of the maximum rate of renal tubular reabsorption of phosphate to the glomerular filtration rate increased. Bone density significantly improved at 12 months, with a complete recovery in 12 children (46.2%). Significant relationships were found among growth velocity, bone density, maximum tubular reabsorption/glomerular filtration rate ratio, and serum levels of carboxyl-terminal propeptide of type I procollagen. The changes in serum levels of this propeptide during the first week of recombinant human GH treatment were positively related to growth velocity at 6 and 12 months and to bone density at 12 months of treatment, whereas the changes in osteocalcin levels were not. We conclude that recombinant human GH treatment caused significant modifications of mineral metabolism and significantly increased bone density, and that measurement of serum levels of the propeptide during the first week of recombinant human GH administration may be a useful tool in predicting improved growth velocity and bone density during long-term recombinant human GH replacement.

Volumetric Bone Mineral Density in Young Women with Turner’s Syndrome Treated with Estrogens or Estrogens plus Growth Hormone

To explore the effects of estrogen replacement therapy (ERT) and recombinant growth hormone (GH) treatment on bone mineral density (BMD) in Turner’s syndrome, we assessed volumetric BMD (vBMD), which is less dependent on body and bone sizes, in these patients at final height. The areal BMD (aBMD) was measured in 26 young women with Turner’s syndrome (age range 17.5–25.0 years) by dual-energy X-ray absorptiometry, and vBMD was calculated. Patients were subdivided as group 1 (n = 12; ERT alone) and group 2 (n = 14; GH + ERT). Years of estrogen exposure were not different between the groups (group 1: 6.4 ± 1.5 years; group 2: 5.3 ± 1.7 years); in group 2, GH therapy was 5.3 ± 1.4 years. Final heights were significantly higher in group 2 than in group 1 (148.1 ± 3.0 vs. 142.0 ± 2.8 cm; p < 0.0001) as well as aBMD (1.073 ± 0.118 vs. 0.968 ± 0.122 g/cm2; p < 0.04). vBMD was higher in group 2 but not significantly different from group 1 (0.374 ± 0.030 vs. 0.358 ± 0.027 g/cm3; p = 0.169). aBMD was reduced with respect to the normative values in both groups (group 1: –1.97 ± 1.04 SDS, p < 0.0001 vs. 0; group 2: –0.93 ± 1.01 SDS, p < 0.005 vs. 0), whereas vBMD was not (group 1: –0.07 ± 0.79 SDS; group 2: 0.42 ± 0.82 SDS). Our data suggest that: in Turner’s syndrome GH administration improves final height and aBMD, but it does not significantly increase vBMD; aBMD reduction in Turner’s syndrome is likely due to the impaired growth and reduced bone size; Turner’s patients on ERT from adolescence show vBMD values in the normal range in young adulthood.

Twenty-four-Hour Osteocalcin, Carboxyterminal Propeptide of Type I Procollagen, and Aminoterminal Propeptide of Type III Procollagen Rhythms in Normal and Growth-Retarded Children

ABSTRACT: The relationships between spontaneous variations in serum 24-h osteocalcin (OC), carboxyterminal propeptide of type I procollagen (PICP), and aminoterminal propeptide of type III procollagen (PIIINP) concentrations and GH secretion, measured as GH response to provocative pharmacologic stimuli and spontaneous GH secretion during 24 h, were evaluated in prepubertal normal children and in GH-deficient and GH-secreting short normal children (SNC). All the subjects showed a circadian rhythm in smoothed 24-h OC and PICP mean data with higher nocturnal values in comparison with diurnal values. Conversely, serum PIIINP concentrations did not vary throughout the day. In children with classic GH deficiency and nonclassic GH deficiency, mean 24-h serum levels and smoothed 24-h mean data for OC, PICP, and PIIINP were significantly reduced (p < 0.001) with respect to age-matched controls. SNC showed mean 24-h OC concentrations similar (p = NS) to those we found in age-matched controls, but they had significantly lower (p < 0.001) diurnal 12-h mean data in comparison with controls. SNC also showed both 24-h PICP and PIIINP mean data and smoothed 24-h PICP and PIIINP mean data significantly lower (from p < 0.02 to p < 0.001) at all the time points of measurement in comparison with controls. Twenty-four-hour PICP and PIIINP mean data were positively related to spontaneous 24-h GH concentrations (r = 0.77, p < 0.005 and r = 0.69, p < 0.005, respectively) and growth velocity (r = 0.85, p < 0.005, and r = 0.70, p < 0.005, respectively), whereas 24-h OC mean data were not. Our study suggests that circadian serum PICP and PIIINP concentrations show GH dependency in children with classic GH deficiency and those with nonclassic GH deficiency, but this was less evident in SNC. Serum PICP and PIIINP concentrations may reflect somatic growth in children with short stature that is or is not related to GH deficiency.

In vitro effects of growth hormone and other hormones on chondrocytes and osteoblast-like cells

The influence of growth hormone (GH), insulin‐like growth factor I (IGF‐I), parathyroid hormone(1–34) (PTH(1–34)), 1,25‐dihydroxycholecalciferol (1,25(OH),D3) and 17P‐oestradiol on proliferation and on production of cytokines, such as interleukin‐lp (IL‐1β), IL‐6, IL‐8 and transforming growth factor‐β (TGF‐β), was studied in chondrocytes obtained from the growing cartilage of the iliac crest and in the osteoblast‐like cell clone SaOS‐2. GH and IGF‐I were mitogenic for chondrocytes and SaOS‐2 cells, as indicated by the dose‐related increase in uptake of [3H]thymidine. PTH(1–34) was also mitogenic, while 1,25(OH)2D3 inhibited the proliferation of both chondrocytes and SaOS‐2 cells in a dose‐dependent manner. 17β‐oestradiol was stimulatory in SaOS‐2 cells, but gave a biphasic pattern in chondrocytes; it was stimulatory at low concentrations (0.1 nmol/1) and inhibitory at supraphysiological doses (10 nmol/l). Using the cDNA polymerase chain reaction, specific mRNAs for IL‐1β, IL‐6, IL‐8 and TGF‐β were found in chondrocytes, while SaOS‐2 cells had a positive signal only for TGF‐β. Specific enzyme immunoassays revealed detectable levels of IL‐1β, IL‐6 and IL‐8 only in chondrocytes. IL‐6 was increased by GH and IGF‐I, and lowered by 1,25(OH)2D3 and supraphysiological doses of 17β‐oestradiol, while PTH(1–34) had no effects. IL‐8 was not influenced by GH or IGF‐I, was slightly but not significantly increased by PTH(1–34) and was reduced by 1,25(OH)2D3 and 17β‐oestradiol at supraphysiological doses. No detectable amounts of TGF‐β were found either in chondrocytes or in SaOS‐2 cells using an enzyme immunoassay specific for TGF‐β2; it is likely that the cells produce TGF‐β in a latent form that needs to be activated. These results show that hormones involved in growth, sexual maturation and calcium metabolism possess in vitro stimulatory or inhibitory effects on cell proliferation. Furthermore, these hormones may regulate certain cytokines that are thought to influence some chondrocyte and osteoblast‐like cell functions, but the roles of these in bone growth need to be further elucidated.

Effect of celiac disease and gluten-free diet on growth hormone-binding protein, insulin-like growth factor-I, and insulin-like growth factor-binding proteins.

Failure to thrive is common in children with celiac disease. As alterations in the growth hormone-insulin-like growth factor I (GH-IGF-I) growth axis have been reported in these patients, we studied the behavior of growth hormone-binding proteins (GH-BPs I and II), IGF-I and its binding proteins in 14 children with celiac disease, either before or after a 6-month gluten-free diet. GH-BP II levels were significantly lower in patients during the active phase of the disease than after the diet or in comparison with control subjects, appropriate for age and sex. There was no difference in the GH-BP-I levels of patients and controls, nor did they change after the diet. Blood levels of IGF-I and IGFBP-3 were reduced before the diet in all patients while ligand blotting showed that IGFBP-2 and 1 were increased. All of these parameters normalized after the gluten-free diet. IGFBP-4 was not greatly influenced by the disease. Furthermore, we found a significant, positive correlation between GH-BP II and IGF-I or IGFBP-3 levels. The height standard deviation scores and body mass indices of the patients improved significantly after the diet. The body mass index significantly and positively correlated with GH-BP II, IGF-I or IGFBP-3 levels. In conclusion, our data show that celiac children had multiple alterations in the growth axis during the active phase of the disease which disappeared during the gluten-free diet.

Bone loss during gonadotropin-releasing hormone agonist treatment in girls with true precocious puberty is not due to an impairment of calcitonin secretion

Gonadal steroids drive the significant bone mineral increase that occurs at puberty, while estrogen deprivation in postmenopausal women results in bone mass reduction. We looked for bone mineralization in girls with true precocious puberty (TPP) before and after six months of LH-RH analogs treatment. Calcitonin secretion in these girls were studied too. Bone mineral content (BMC) and BMC/BW ratio (single photon absorptiometry) were measured in seven girls (aged 4.3 to 8.7 years) with TPP before LH-RH agonist therapy (long acting D-Trp6 -LH-RH 60 µg/kg im every 28 days) was started; the patients were reevaluated after six months of therapy. Before therapy, BMC and BMC/BW were increased for chronological age but appropriate for bone age according to our mineralization normative data. After six months of LH-RH analog administration, 17β-estradiol and LH levels were suppressed and BMC and BMC/BW showed a small but significant decrease (respectively — 5.4 %, p < 0.02 and −6.3 %, p < 0.02). Basal and calcium stimulated calcitonin levels (total and extractable) did not significantly change during the study period. We conclude that in girls with TPP bone mineralization was increased for chronological age but normal for bone age. The estrogen withdrawal secondary to LH-RH analog therapy caused a reduction in bone mass. Such a bone loss is not due to an impairment of calcitonin secretion.

Plasma growth hormone-binding protein activity, insulin-like growth factor I, and its binding protein levels in patients with Turner's syndrome: effect of short- and long-term recombinant human growth hormone administration.

ABSTRACT: Plasma growth hormone-binding protein (GH-BP) activity and the levels of IGF-I and its binding proteins (IGFBP) were studied in eight girls with Turners syndrome before and during recombinant-hGH (r-hGH) administration. Growth hormone and GH-BP activity were assayed at baseline and hourly, over a 12-h period, after an intramuscular bolus of 0.09 mg/kg of the hormone. After 7 d, each patient received r-hGH at 0.33 mg/kg/weekly s.c. every day at nighttime; plasma growth hormone-binding protein activity, blood IGF-I, and IGFBP were evaluated before and on d 7, 30, 180, and 360. Baseline reference values were obtained from 10 bone age-matched healthy girls. Basal GH-BP activity, IGF-I, and IGFBP levels were similar in patients and controls. Four h after the intramuscular injection, GH-BP activity maximally increased and returned to baseline 6–7 h later; during long-term r-hGH administration GH-BP activity peaked at +180 d but declined to pretreatment at +360 d. IGF-I, IGFBP-3, and IGFBP-4 increased under r-hGH and, in contrast to GH-BP activity, remained high throughout the study. In conclusion, in girls with Turners syndrome, GH-BP activity, IGF-I, IGFBP-3, and IGFBP-4 are induced by r-hGH. However, the increase of IGF-I and IGFBP-3 does not require an increased level of the cellular growth hormone receptors, as suggested by the unchanged +360 d values of plasma GH-BP activity compared with baseline. The absence of an association among any of the biochemical parameters studied and the growth of the patients taking r-hGH suggests that a peripheral defect may affect their growth.

ESPE Bulletin Board

1958–1992 Director, Institute of Pediatric and Adolescent Endocrinology, Beilinson Medical Center, Petah Tikva, Sackler Faculty of Medicine, Tel Aviv University (Israel) 1983–1997 Incumbent of the Irene and Nicholas Marsh Chair in Endocrinology and Juvenile Diabetes, Sackler Faculty of Medicine, Tel Aviv University (Israel) 1998 Professor (Emeritus) of Pediatric Endocrinology, Sackler Faculty of Medicine, Tel Aviv University (Israel) 1961 Founding member of the European Society of Pediatric Endocrinology (ESPE) 1966–1967 President, ESPE 1974 Founding member of the International Study Group of Diabetes in Children and Adolescents (ISGF), which became the International Society for Pediatric and Adolescent Diabetes (ISPAD) 1974–1979 Secretary General, ISPAD 1979– President, Israel Growth and Development Association 1988–1989 President, ESPE 1993–1996 President, ISPAD

EFFECT IN VITRO OF r-hGH AND CALCIOTROPIC HORMONES ON HUMAN OSTEOBLAST-LIKE CELLS

Recent in vitro evidences indicate that factors as Parathyroid hormone (PTH) and its related peptide (PTHrp), 1, 25-dihydroxyvitamin D3 and 17β-estradiol (E2) have regulatory effects on mouse- or rat-derived osteoblast-like cells. We studied the effects in vitro of various hormones, i.e. recombinant human growth hormone (r-hGH), PTHrp, 1, 25-dihydroxyvitamin D3 and E2, on type I procollagen (IP) and IGFs binding proteins (IGFBPs) production by human osteosarcoma cells (SaOs).Methods. Cells were cultured in 24-well plates in RPMI-1640 medium. Confluent cultures were added with medium alone (control cultures) or with the various hormones at 10−8 M final concentration in serum-free condition. After 3 days the supernates were recovered and assayed for IP by RIA and IGFBPs by the “ligand blotting” method, using 125I-IGF-I as probe.Results and comment. In the supernates of hormonally-treated cultures IP levels almost doubled the values found in the control ones. The ligand blotting showed the presence of two major bands relative to IGFBP2 and BP4. Such binding proteins had higher levels in the supernates of the hormonally-treated than in the control cultures and, among the hormones used, PTHrp seems the most potent. Thus, our data indicate that PTHrp, r-hGH, 1, 25-dihydroxyvitamin D3 and E2 have important regulatory effects on human osteoblast-like cells not only on bone matrix production but also on proteins that may play an autocrine/paracrine role in bone, as IGFBPs.

GHRH-test in short children with "non classic" GH deficiency. A comparison with "classic" GH deficiency and short normal stature.

In this study GHRH-test has been performed (2 μg/Kg of an iv bolus of GHRH 1–44) sampling for GH measurement every 15 min over 2 hours in three groups of short children. Group 1 consisted of 10 subjects with classic GH deficiency (CGHD): GH response < 10 ng/ml to two conventional tests and 24-h mean GH concentration (MGHC) < 3 ng/ml; group 2 consisted of 16 subjects with non-classic GH deficiency (NCGHD): response > 10 ng/ml to at least one conventional test and MGHC < 3 ng/ml; group 3 consisted of 18 subjects with short normal stature: GH response > 10 ng/ml to at least one conventional test and MGHC >3 ng/ml. GH peak and area under the curve (AUC) values were significantly lower in group 1 than groups 2 and 3 and in group 2 than group 3. GH peak and AUC values statistically correlated with height, height velocity, bone age/chronological age ratio and MGHC. Six children in group 1,14 children in group 2 and all 18 children in group 3 showed after GHRH a GH peak > 10 ng/ml and were considered as ‘responders’. Considering only the responders, GH peak and AUC values were significantly lower in group 1 than groups 2 and 3 and in group 2 than group 3. In conclusion, our data have shown that 87% of children with NCGHD responded to a single bolus of GHRH with an increase in GH levels > 10 ng/ml and that their responses were intermediate compared to those of CGHD and short normal subjects.