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Low Growth Hormone-Binding Protein in Infants with Congenital Hypothyroidism

Department of Pediatrics, University of Bologna (A.C., E.C., A.B., C.C., A.P., G.P.S., M.L., P.P.); Central Laboratory (R.D.I.); and the Unit of Clinical Pharmacology, St. Orsola Hospital (S.B.), Bologna, Italy

Address all correspondence and requests for reprints to: Prof. Emanuele Cacciari, Clinica Pediatrica I, Via Massarenti 11, 40138 Bologna, Italy. E-mail ped1{at}alma.unibo.it

	Abstract

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We evaluated the circulating levels of GH, insulin-like growth factor I (IGF-I), GH-binding protein (GHBP), and IGF-binding protein-3 (IGFBP-3) before L-T₄ therapy in 19 infants with congenital hypothyroidism (CH), aged 12–29 days, diagnosed by neonatal screening and in a group of age- and sex-matched control infants. The same parameters were reevaluated after several months of treatment. Serum GHBP was measured by the high performance liquid chromatography-gel filtration method; serum GH, IGF-I, and IGFBP-3 levels were determined by commercial kits.

The hypothyroid patients, before beginning therapy, presented significantly lower GHBP values than controls (P < 0.0001); during treatment, these values increased significantly; however, after 6 months they were still significantly lower than control values (P < 0.01). The pretreatment levels of GH were not significantly different from control values; after 1 month of treatment, GH did not show the decrease observed in controls and, therefore, was significantly higher (P < 0.01). The pretreatment levels of IGF-I were not significantly different from control values, but were lower in patients with severe than in those with mild hypothyroidism. They decreased at about 4 months of life and became significantly lower than control values at about 7 months of age (P < 0.05).

In conclusion, it may be hypothesized that the condition of CH induces a change in GHBP expression, perhaps beginning in fetal life. The intrauterine production of IGF-I seems to be independent of the levels of GHBP and partially affected by fetal thyroid function.

	Introduction

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SHORT STATURE is one of the most typical features of untreated congenital hypothyroidism (CH), although the mechanism by which thyroid hormones play their role is still controversial (1, 2, 3). In recent years the discovery of insulin-like growth factors (IGFs) as main GH-mediated growth factors and of specific binding proteins able to modulate the bioavailability of the IGFs at the cellular level has helped to clarify some of the mechanisms of growth (4, 5, 6, 7, 8, 9, 10). Furthermore, in 1986, Herington and Baumann identified a specific, high affinity, low capacity binding protein for GH, corresponding to the extracellular domain of the membrane receptor for GH, the serum concentration of which is thought to be a good marker of tissue concentration for GH receptors (11, 12, 13, 14). Recent studies on the effect of thyroid hormones on the GH-IGF axis and circulating levels of IGF-binding proteins (IGFBPs) and GH-binding proteins (GHBP) have provided conflicting results (3, 15, 16, 17); in only a few studies, moreover, have children affected by CH been taken into account (18, 19).

We therefore evaluated the circulating levels of GH, IGF-I, and their binding proteins, GHBP and IGFBP-3, before replacement therapy in 19 CH infants, aged 12–29 days, diagnosed by neonatal screening and in a group of age- and sex-matched control infants. The same parameters were reevaluated after several months of treatment with L-T₄.

	Subjects and Methods

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We examined with parental consent 19 consecutive CH infants diagnosed at our center between August 1, 1994 and December 31, 1995 by neonatal screening (20). Table 1 reports neonatal parameters, thyroid hormone levels, bone age at diagnosis (epiphyseal surface of the knee) (21), and etiology of the congenital defects (thyroid scan with^99mTcO₄^-) (22) in the CH patients. The subjects were examined in a longitudinal study for the first time at 19 ± 6 days of age (range, 12–29 days) before starting L-T₄ therapy (8 µg/kg·day) and then after 1 (38–67 days), 3 (86–150 days), and 6 (168–240 days) months from the beginning of treatment. The dosage of L-T₄ was adjusted in an attempt to keep the serum free T₄ (fT₄) and TSH concentrations within the normal range. Reference serum hormone concentrations in our laboratory for normal infants aged 1–6 months were: fT₄, 10.3–21.5 pmol/L; and TSH, 0.5–5 mU/L. At each examination, when specimens for TSH and thyroid hormones were obtained, blood samples were taken to determine serum levels of GH, IGF-I, GHBP, and IGFBP-3. The control group (obtained cross-sectionally) for these growth factors was 36 healthy infants (8–240 days of age) subdivided into 3 groups (12, 6, 8, and 10 subjects, respectively) age and sex matched with CH infants at each examination; the blood samples from controls were incidental to venipuncture for other purposes (e.g. screening examinations or normal humoral controls after an acute disease).

Serum GH was measured by a commercial liso-solid phase RIA assay (Technogenetics, Milan, Italy). The intra- and interassay coefficients of variation were 5.8% and 9.7%, respectively, at a level of 1.2 µg/L, and 5.3% and 8.9% at a level of 12.5 µg/L. The sensitivity of the assay was 0.1 µg/L, as determined by the mean ± 2 SD of the zero standard.

Serum IGF-I values were determined by commercial kit (Nichols Institute Diagnostics, San Juan Capistrano, CA) that included an acid-ethanol extraction. The sensitivity of the method was 0.06 ng/mL, and intra- and interassay coefficients of variation were 2.6% and 9.5%, respectively.

Serum IGFBP-3 levels were measured by commercial RIA kit (Diagnostic Systems Laboratories, Inc., Webster, TX). The sensitivity of the method was 1.1 ng/mL at the 95% confidence limit. Intra- and interassay coefficients of variation were 5.2% and 6.9%, respectively.

Free thyroid hormone and TSH serum levels were measured by commercial kits, as previously described (20).

All statistical analyses were performed using the computer program Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL). For normally distributed data, statistical significance was assessed using Student’s t test, ANOVA for repeated measurements, and Pearson’s correlation index. Nonparametric statistical analysis (Mann-Whitney test, Friedman’s test, and Pearson’s correlation index computed on the ranks) was also used when considered suitable for nonnormal distribution and/or low number of data.

	Results

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Thyroid hormone values and auxological evaluation

In the CH infants, TSH and fT₄ values were already within the normal range after 1 month of treatment; at the 6-month examination, the L-T₄ dose did not seem adequate due to the increase in weight of our patients, and TSH levels increased (Table 2). Thyroid hormone values and L-T₄ dose were not significantly different in patients with mild (T₄ spot value, >25 nmol/L; n = 15 subjects) or severe (T₄ spot value, <25 nmol/L; n = 4 subjects) hypothyroidism at any time during the follow-up.

GHBP

The hypothyroid patients, before beginning therapy, presented significantly lower GHBP values than the controls (P < 0.0001). During treatment, these values increased significantly; however, after 6 months, they were still significantly lower than control values (P < 0.0001, by ANOVA; Table 2). GHBP values were not significantly different in patients with severe or mild congenital hypothyroidism. In the CH infants, there was no significant correlation at any time among GHBP, thyroid hormone, and L-T₄ dose.

GH

In the CH infants, GH values decreased significantly during treatment (P < 0.01, by ANOVA). The pretreatment levels of GH were not significantly different from control values, whereas in the CH infants after 1 month of treatment, GH did not show the decrease observed in controls and, therefore, was significantly higher (P < 0.01); this significant difference disappeared at subsequent examinations (Table 2). GH values were not significantly different in patients with severe or mild hypothyroidism; in the CH infants, there was no significant correlation among GH, thyroid hormone, and L-T₄ dose at any time.

IGF-I

In the CH infants, IGF-I levels were not significantly different from control values both before and after 1 month of therapy. However, pretreatment levels of IGF-I were significantly lower in patients with severe hypothyroidism than in patients with mild hypothyroidism (48.9 ± 12.5 vs. 97.7 ± 35.2 µg/L; P < 0.001). At about 4 months of age, the IGF-I levels in hypothyroid subjects decreased, and they became significantly lower than control values at about 7 months of age (P < 0.05). Furthermore, IGF-I values in both hypothyroid and normal subjects showed a wide range of variability (Table 2). In the CH infants, the levels of IGF-I before starting therapy and 6 months from the beginning of therapy showed a positive significant correlation with fT₄ (P < 0.001) and a negative correlation with TSH (P < 0.01).

IGFBP-3

In the CH infants, IGFBP-3 levels decreased significantly during treatment (P < 0.01, by ANOVA). No significant differences were found between CH infants and controls at each examination up to 4 months of age; after 6 months of therapy, IGFBP-3 levels became significantly lower than control values (P < 0.05; Table 2). In the CH infants, the levels of IGFBP-3 before starting therapy and 6 months from the beginning of therapy showed a positive significant correlation with fT₄ (P < 0.001).

	Discussion

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In a wide range of subjects, various researchers have observed that from the low levels of GHBP in intrauterine life, there is a gradual postnatal increase that is particularly significant during the first months of life, supporting the idea that the receptor-dependent actions of endogenous GH are limited in the fetus and increase during the neonatal period through the gradual maturation of GH tissue receptors (11, 12, 24).

The results of the present study, based on the examination of a group of CH subjects, provide us with new data on the development of the GH-GH receptor axis. In the CH infants, serum concentrations of GHBP assessed at birth, in the absence of replacement therapy, are significantly lower than control values and similar to those reported previously in fetal blood samples (11, 24, 25). After the initiation of therapy, GHBP levels increased significantly, but they remained below normal for as long as 6 months. These results might be consistent with an element of GH resistance; it may be hypothesized that the condition of CH induced a change in GHBP expression, perhaps beginning in fetal life. Alternatively, persistently low GHBP levels may be due to the effects of the thyroid hormone itself not being supplied in adequate amounts. From this point of view, the L-T₄ dose we used at the beginning of treatment was lower than others used previously (26), especially in patients with severe hypothyroidism. It should be pointed out that the patients receiving this treatment dose had a quick normalization of TSH and fT₄ levels, without significant differences between mild and severe cases of hypothyroidism. It was only after 6 months of treatment that L-T₄ did not seem to be supplied in adequate amounts due to the typically rapid weight increase at this age.

With regard to IGF-I levels, the similar pretreatment values in normal and hypothyroid subjects indicate intrauterine production of IGF-I independent of the levels of GHBP and, therefore, of the GH-GH receptor axis typical in extrauterine life. However, fetal IGF-I levels appeared to be partially affected by fetal thyroid function. In fact, in the first days of life, IGF-I levels, even within the normal range, were significantly lower in patients with severe hypothyroidism than in patients with mild hypothyroidism. Furthermore, the wide individual variations in IGF-I levels in both normal and hypothyroid subjects may be partially explained by the hypothesis that the intrauterine production of IGF-I is regulated by factors other than hormonal, e.g. genetic (10). Our results are in agreement with those of Kandemir et al. (18). In a sample of randomly selected CH infants subdivided according to age at diagnosis and examined before the beginning of therapy, these researchers observed that IGF-I and IGFBP-3 values were the same as control levels in patients examined in the first month of life. After the initiation of therapy, the evolution of IGF-I and IGFBP-3 levels may be due partly to the deficient maturation of GH receptors and partly to the low thyroid hormone values, especially at 7 months of life. These data should be considered preliminary on account of the size of the sample examined (especially the control group), the presence of mild and severe hypothyroidism that may contribute to the difficulty of achieving statistical significance, the impossibility of obtaining fetal blood samples in the pathological subjects, and the lack of longitudinal controls. Only a longer follow-up period of a group of treated CH infants will be able to confirm or disprove the hypothesis of this study.

With regard to IGFBP-3 levels, recent data in the literature have suggested that the major means of their regulation is modulation of the IGFBP protease (27). In our subjects, serum IGFBP-3 concentrations were significantly related to those of IGF-I. The possibility that thyroid hormones may influence protease activity could not be determined in this study, because our RIA method was not able to evaluate the nonfunctioning peptide fragments (27).

In conclusion, it may be hypothesized that the CH-induced change in GHBP expression perhaps begins in fetal life. The intrauterine production of IGF-I seems to be independent of the levels of GHBP and partially affected by fetal thyroid function. Early replacement therapy, made possible by neonatal screening programs, however, seems able to reverse this maturational defect even though a further and more extensive follow-up is needed to identify the size and rapidity of this reversal.

Received December 11, 1997.

Revised April 20, 1998.

Revised June 25, 1998.

Accepted July 1, 1998.

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