Growth hormone replacement therapy in growth hormone deficient children and adults: Effects on hemochrome

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1 J. Endocrinol. Invest. 29: , Growth hormone replacement therapy in growth hormone deficient children and adults: Effects on hemochrome S. Bergamaschi 1, C. Giavoli 1, E. Ferrante 1, A. Lania 1, R. Rusconi 2, A. Spada 1, and P. Beck-Peccoz 1 1 Institute of Endocrine Sciences; 2 Department of Pediatrics, University of Milan, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Fondazione IRCCS, Milan, Italy ABSTRACT. Several lines of evidence have suggested a role of the GH/IGF-I axis in the regulation of hemochrome. Many studies have been carried out in GH deficient children and adults about this topic, reporting predominantly a positive effect of recombinant human GH (rhgh) on red series, with no action on serum leucocytes and platelets counts. The aim of this study was to assess the impact of GH deficiency (GHD) and of rhgh replacement on blood cells count in 17 pre-pubertal children with idiopathic isolated GHD (11 males and 6 females, aged 9.1±0.8 yr) and in 18 patients with adult-onset GHD (12 males and 6 females, aged 47.9±3.0 yr). Evaluation of absolute and SD score (SDS) values of red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets and white blood cells was performed at baseline and after 12 months of rhgh treatment (0.045±0.001 mg/kg bw/day and 4.2±0.5 µg/kg bw/day for children and adults, respectively). At baseline, all patients showed low IGF-I levels. Effectiveness of rhgh therapy was documented by significant increase in height SDS, height velocity and serum IGF-I levels in children. In adults, adequacy of rhgh was demonstrated by significant increase in serum IGF-I and significant decrease in body fat. At baseline, about 25% of patients (4 of 17 children and 4 of 18 adults) showed normochromic normocytic anemia, while the other indices were normal. In 7 of the 8 anemic patients, normal levels of hemoglobin were restored on rhgh, while no change in all the other indices was observed. In conclusion, rhgh therapy at physiological doses has no effect on erythropoiesis in GHD children and adults with normal blood cells count, while in patients with normochromic normocytic anemia rhgh is able to restore normal hemoglobin levels. (J. Endocrinol. Invest. 29: , 2006) INTRODUCTION Several reports suggest a role of the GH/IGF-I axis in the regulation of hematopoietic system. In vitro studies reported that both GH and IGF-I stimulate the growth of erythroid precursor cells formed by bone marrow and peripheral blood erythroid cells in the presence of erythropoietin (1-3). Moreover, multiple lines of evidence demonstrated that GH and IGF-I stimulate hematopoiesis in vivo. Accordingly, Key-words: Growth hormone, hemochrome, children. Correspondence: P. Beck-Peccoz, MD, PhD, Institute of Endocrine Sciences, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Fondazione IRCCS, Via F. Sforza 35, Milano, Italy. paolo.beckpeccoz@unimi.it Accepted January 19, growth arrest in hypophysectomized rats is associated to erythropoiesis reduction (4), and GH and IGF-I are able to restore erythropoietic function directly or by enhancing erythropoietin production (5). Few studies in GH deficient children reported the normalization of low pre-treatment hemoglobin levels after recombinant human GH (rhgh) replacement (6-9), thus confirming the stimulatory effect of GH on erythropoiesis. However, these studies were carried out in an inhomogeneous population, frequently with different or not specified pubertal stage. Similarly, the few studies reporting a positive effect of rhgh therapy on red series in GH deficient adults (10-12) were performed with doses (12 µg/kg bw/day) higher than those commonly used in recent years (13), making it difficult to differentiate a physiological from a pharmacological effect of rhgh. 399

2 S. Bergamaschi, C. Giavoli, E. Ferrante, et al. J. Endocrinol. Invest. 29: , The aim of this study was to assess the impact of GH deficiency (GHD) and rhgh replacement therapy on blood cells count in a homogenous population of pre-pubertal children with idiopathic isolated GH deficiency and in a group of GH deficient adults treated with physiological doses of rhgh. MATERIALS AND METHODS Informed consent was obtained from all participants and the study was approved by the local Ethics Committee. In all the patients, children and adults, other causes of anemia were ruled out at the beginning of the study. In particular, celiac disease was excluded by determination of anti-gliadin and -tissue transglutaminase antibodies (AGA and tga), that were negative in all patients; thyroid function was normal at baseline and after rhgh. Adult patients with central hypothyroidism were adequately replaced. Pediatric patients Seventeen children (11 males and 6 females, mean chronological age 9.1±0.8, range yr) were studied. All the children had idiopathic isolated GHD [auxological parameters: height 2.4±0.2 SD score (SDS), target height 0.8±0.2 SDS, height velocity 3.7±0.3 cm/yr], diagnosed by GH peaks <10 or 20 µg/l on at least two provocative GH stimulation tests (arginine, clonidine and GHRH and piridostigmin, respectively), or mean spontaneous nocturnal GH secretion <3 µg/l. Magnetic resonance imaging of the sellar region was normal in all patients. At baseline mean bone age, estimated according to the method of Tanner and Whitehouse (14), was 6.9±0.8 yr (range yr) and the ideal body weight for height was 101±3%. All patients were pre-pubertal and remained pre-pubertal during the follow-up. Subcutaneous rhgh was given as daily injections, at a mean dose of 0.045±0.001 mg/kg bw/day. The effectiveness of the first year of rhgh therapy was assessed by the evaluation of height SDS, height velocity, serum IGF-I levels and IGF-I SDS. None of the patients was taking any other medication. Main hormonal and auxological baseline findings are detailed in Table 1. Adult patients Table 1 - Clinical findings of children with isolated idiopathic GH deficiency (GHD). Subject (no.) Gender (M/F) CA (yr) BA (yr) Ht (SDS) Eighteen adults (12 males and 6 females, mean chronological age 47.9±3.0, range yr) were studied. The clinical characteristics of these patients are described in Table 2. All these patients had multiple hormone deficiency and were receiving conventional replacement therapy at stable doses for more than 6 months before treatment with rhgh. Severe GHD was defined as a peak response of serum GH <3 µg/l to insulin tolerance test or <9 µg/l to arginine+ghrh, as previously reported (15). No patient was treated with rhgh before the beginning of the study. Subcutaneous rhgh was given as daily injections, at a mean dose of 4.2±0.5 µg/kg bw/day (0.33±0.05 mg/day). The effectiveness of the first year of rhgh therapy was assessed by the evaluation of body fat mass and serum IGF-I levels. mghc (NV >3 µg/l) GH peak (NV >10 µg/l) C A I 1 M M M M F F M F M F M F M M M F M M: male; F: female; CA: chronological age; BA: bone age; Ht: height; C: clonidine; A: arginine; I: insulin tolerance test. 400

3 J. Endocrinol. Invest. 29: , rhgh therapy and hemochrome Table 2 - Clinical findings of adult patients with GH deficiency (GHD). Subject (no.) Gender Age (yr) Diagnosis Treatment Pituitary deficiencies 1 M 67 NFPA TNS+RT T, A, G 2 M 66 NFPA TNS T, A, G 3 M 44 NFPA TNS+RT T, A, G, D 4 M 51 NFPA TNS A 5 M 49 NFPA TNS+RT T, A, G 6 M 38 NFPA TNS A 7 M 59 NFPA TNS T, A, G 8 M 57 NFPA TNS T, A, G 9 M 56 Macroprolactinoma TNS+RT T, A, G 10 M 55 Macroprolactinoma TNS T, A, G 11 M 50 Macroprolactinoma TNS T, A 12 M 43 Craniopharyngioma TNS T, A, G, D 13 F 47 NFPA RT T, D 14 F 30 NFPA TNS+RT T, A, G 15 F 52 Craniopharyngioma TNS T, A 16 F 35 Traumatic hypopituitarism / T, A, G 17 F 18 Empty sella / T, A, G 18 F 35 Empty sella / T, A, G, D M: male; F: female; TNS: transphenoidal surgery; R: radiotherapy; T: hypothyroidism; A: hypoadrenalism; G: hypogonadism; D: insipidus diabetes. Methods Red blood cell (RBC) indices [hemoglobin (Hb); hematocrit (Ht); RBC count, mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC), number of platelets (Plt) and white blood cells (WBC)] were determined at baseline and after 12 months of rhgh therapy. Because of physiological age-related variations of RBC indices, in order to compare data from children of different ages, the SDS for Hb, Ht and MCV were calculated for each pediatric patient according to the reported reference values (16). Anemia was defined as a blood Hb concentration 2 SDS below the age and sex related mean for pediatric patients and according to the World Health Organization (WHO) criteria for adult patients (Hb <13 g/dl in men and <12 g/dl in women) (17). Serum IGF-I levels were measured by a commercial RIA kit from Mediagnost (Tübingen, Germany) with separation of IGF-I from binding proteins obtained by acidification in IGF-II excess. The intra- and inter-assay coefficients of variation were 3.2 and 8.9%, respectively, and IGF-II cross-reactivity <0.05%. IGF-I levels were compared with their age-appropriate absolute values normal range and were expressed both as absolute values and as SDS. Body composition was evaluated by whole body bioelectrical impedance analysis (BIA), using a portable impedance analyser (RJL Systems, Detroit, MI), following the instruction given by the manufacturer. Body fat percentage (BF%) was calculated using Segal s regression equation (18) and the results were compared with those reported by Pichard et al. (19) in normal subjects matched for age and sex. Statistical analysis The data are expressed as mean±se. After checking the Gaussian distribution of the data, differences between before- and aftertherapy evaluations were assessed by the two-tailed Student s t-test for paired observations. A p value <0.05 was accepted as significant. RESULTS Pediatric patients At baseline, 3 boys and 1 girl were affected with normochromic normocytic anemia (Hb 11.7±0.2 g/dl, 2.8±0.3 SDS; MCV 79.2±2.4 fl; MCH 26.3±0.4 pg). In all the other children, the hemochrome parameters were normal, as compared with the age- and sex-adjusted reference ranges. The effectiveness of rhgh therapy was demonstrated by significant increase (p<0.001) in height SDS (from 2.4±0.2 to 1.8±0.2), height velocity (from 3.7±0.3 to 9.2±0.5 cm/yr), serum IGF-I levels (from 401

4 S. Bergamaschi, C. Giavoli, E. Ferrante, et al. J. Endocrinol. Invest. 29: , ±2.6 to 39.8±7.1 nmol/l) and IGF-I SDS (from 2.0±0.3 to 0.4±0.4). After 12 months of rhgh therapy, no significant differences were seen between basal and post-treatment values of hemochrome parameters (Table 3). In 3 of the 4 children affected with normochromic normocytic anemia, Hb returned to the normal values (Fig. 1). Adult patients At baseline, 3 men and 1 woman were affected with normochromic normocytic anemia (men: Hb 12.7±0.1 g/dl, MCV 87.5±2.2 fl, MCH 31.0±0.6 pg; woman: Hb 11.7 g/dl, MCV 88.4 fl, MCH 29.5 pg). In all the other patients, the hemochrome parameters were in the normal range. The effectiveness of rhgh therapy was demonstrated by significant decrease (p<0.015) in BF% (from 30.0±4.3 to 26.1±4.1%) and significant increase (p<0.0001) in serum IGF-I levels (from 10.0±1.4 to 27.1±2.2 nmol/l). After the 12-month period of rhgh therapy, no significant differences were seen between basal and post-treatment values of hemochrome parameters (Table 4). In the 4 patients affected with normochromic normocytic anemia before rhgh treatment, Hb returned to the normal values. DISCUSSION The main result of the present study is that rhgh therapy does not influence hemochrome both in children and in adults with GHD. In particular, blood cell indices were not affected by rhgh even at the high doses used to allow a normal pubertal spurt in peripubertal children. Noteworthy is also the fact that about 25% of the evaluated patients (4 of 17 children and 4 of 18 adults) showed normochromic normocytic anemia at baseline. Due to the small number of subjects, it is difficult to interpret this data as a consequence of GHD. However, other factors causing anemia had been ruled out (ie celiac disease or hypothyroidism). Therefore, it is tempting to hypothesize a possible involvement of GHD in this disorder, since in 7 of the 8 patients normal levels of hemoglobin were restored after 12 months of rhgh therapy. Indeed, a beneficial effect of rhgh replacement in anemic patients with adult GHD has already been reported (20). Multiple evidence deriving from in vitro and in vivo studies suggest that either GH or IGF-I are able to exert stimulatory effects on erythropoiesis via an indirect and direct way, respectively. In fact, GH potentiates the action of erythropoietin by acting on its receptor. Conversely, IGF-I has been shown to have an Epo-like activity, acting via an Epo-independent pathway on normal human adult erythropoiesis. IGF-I enhances erythroid maturation and proliferation, while Epo prevents apoptosis and maintains erythroid cell viability and development (2, 5, 10). Accordingly, IGF-I receptors have been demonstrated on erythroid precursor cells (21). Hb (SDS) Table 3 - Hematopoietic parameters (mean±se) of pediatric patients at baseline and after 12 months of rhgh. Parameters Basal 12 months p Hb (SDS) 0.4± ±0.5 ns Ht (SDS) 0.9± ±0.5 ns RBC (x10 12 /l) 4.7± ±0.1 ns MCV (SDS) 0.0± ±0.5 ns MCH (pg) 26.3± ±0.5 ns MCHC (g/dl) 34.0± ±0.4 ns WBC (x10 9 /l) 6.9± ±0.6 ns Plt (x10 9 /l) 302±20 321±16 ns Hb: hemoglobin; Ht: hematocrit; RBC: red blood cells; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; WBC: white blood cells; Plt: platelets. Hb (g/dl) Fig. 1- Hemoglobin (Hb) SDS and Hb levels in children and adults, respectively. Variations observed in the mean of normal patients ( ) and in males ( ) and female ( ) with normochromic normocytic anemia. 402

5 J. Endocrinol. Invest. 29: , rhgh therapy and hemochrome Table 4 - Hematopoietic parameters (mean±se) of adult patients at baseline and after 12 months of rhgh. Parameters Basal 12 months p normochromic normocytic anemia was present, rhgh was able to restore normal levels of hemoglobin in all but one patient. Hb (g/dl) 13.6± ±0.3 ns Ht (%) 39.4± ±0.8 ns RBC (x10 12 /l) 4.6± ±0.1 ns MCV (fl) 87.1± ±1.3 ns MCH (pg) 30.2± ±0.5 ns MCHC (g/dl) 34.4± ±0.2 ns WBC (x10 9 /l) 6.9± ±0.5 ns Plt (x10 9 /l) 245±14 259±14 ns Hb: hemoglobin; Ht: hematocrit; RBC: red blood cells; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; WBC: white blood cells; Plt: platelets. However, it is difficult to apply these results obtained in vitro and in vivo animal models to human physiology. Although a model that may be helpful to understand the physiological role of the GH-IGF-I system on human hematopoiesis is the condition of GHD, few studies have been performed in GHD children and adults. Low hemoglobin levels have been described in children with isolated idiopathic GHD (6, 7, 9), levels increasing on rhgh (6-8). However, all these reports considered inhomogeneous cohorts of patients, frequently without specified pubertal stage and with chronic pathologies that could impair per se hematopoiesis. The present population of children was homogeneous, only including prepubertal patients throughout the study, with isolated idiopathic GHD. In this series of patients treated for 12 months, rhgh did not induce any effect on the various parameters of blood cells count. As far as adult patients are concerned, previous reports mainly described an impairment of erythropoiesis in GHD and a positive effect of rhgh on red series (10-12). However, all these studies were performed using doses of rhgh 3-folds higher than those presently used, thus making it difficult to distinguish a pharmacological effect from a physiological one. On the basis of our results, we may state that physiological rhgh replacement, both in GHD children and adults, does not affect erythropoiesis. In addition, no effect of rhgh on WBC and platelets has been found, as previously reported (1). All these data confirm the safety and efficacy of rhgh substitution in GHD patients. In conclusion, rhgh replacement therapy at substitutive doses had no additional effect on erythropoiesis in GHD children and adults with normal blood cell count parameters. However, when a condition of ACKNOWLEDGMENTS The authors gratefully thank Dr. G.I. Vaghi and Mrs. A. Maffini for the excellent technical assistance and Mrs. R. Deriu for her irreplaceable nursing assistance. The present work was partially supported by Ospedale Maggiore di Milano IRCCS and MIUR (Rome). REFERENCES 1. Golde DW, Bersch N, Li CH. Growth hormone: species-specific stimulation of erythropoiesis in vitro. Science 1977, 196: Clausters M, Chatelain P, Sultan C. Insulin-like growth factor I stimulates human erythroid colony formation in vitro. J Clin Endocrinol Metab 1987, 65: Merchav S, Silvian Drachsler I, Tatarsky I, Lake M, Skottner A. Comparative studies of the erythroid-potentiating effects of biosynthetic human insulin-like growth factors I and II. J Clin Endocrinol Metab 1992, 74: Nagy E, Berczi I. Pituitary dependence of bone marrow function. Br J haematol 1989, 71: Kurtz A, Zapf J, Eckardt K, Clemons G, Froesch ER, Bauer C. Insulin-like growth factor I stimulates erythropoiesis in hypophysectomized rats. Proc Natl Acad Sci USA 1988, 85: Valerio G, Di Maio S, Salerno M, Argenziano A, Badolato R, Tenore A. Assessment of red blood cell indices in growthhormone-treated children. Horm Res 1997, 47: Ardizzi A, Guzzaloni G, Grugni G, et al. The effect of GH on erythropoiesis in vivo. Minerva Endocrinol 1993, 18: Vihervuori E, Sibila I, Siimes MA. Increases in hemoglobin concentration and iron needs in response to growth hormone treatment. J Pediatr 1994, 125: Eugster EA, Fisch M, Walvoord EC, DiMeglio LA, Pescovitz OH. Low hemoglobin levels in children with in idiopathic growth hormone deficiency. Endocrine 2002, 18: Kotzmann H, Riedl M, Clodi M, et al. The influence of growth hormone substitution therapy on erythroid and myeloid progenitor cells and on peripheral blood cells in adult patients with growth hormone deficiency. Eur J Clin Invest 1996, 26: Ten Have SMTH, van der Lely AJ, Lamberts SWJ. Increase in haemoglobin concentrations in growth hormone deficient adults during human recombinant growth hormone replacement therapy. Clin Endocrinol (Oxf) 1997, 47: Christ ER, Cummings MH, Westwood NB, et al. The importance of growth hormone in the regulation of erythropoiesis, red cell mass, and plasma volume in adults with growth hormone deficiency. J Clin Endocrinol Metab 1997, 82: Sonksen PH, Christiansen JS. Consensus guidelines for the diagnosis and treatment of adults with growth hormone de- 403

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