A novel recessive splicing mutation in the POU1F1 gene causing combined pituitary hormone deficiency.

BACKGROUND: Mutations in the gene encoding the pituitary transcription factor POU1F1 (Pit-1, pituitary transcription factor-1) have been described in combined pituitary hormone deficiency (CPHD). AIM: The aim of this study was the characterisation of the molecular defect causing CPHD in a patient born to consanguineous parents. SUBJECT AND METHODS: The case of a 12.5-yr-old girl presenting with severe growth failure at diagnosis (-3 SD score at 3 months) and deficiency of GH, PRL, and TSH was investigated for the presence of POU1F1 gene mutations by denaturing high performance liquid chromatography analysis. RESULTS: A novel mutation adjacent to the IVS2 splicing acceptor site (IVS2-3insA) was identified in the patient at the homozygous state. Analysis of patient's lymphocyte mRNA and an in vitro splicing assay revealed the presence of 2 aberrant splicing products: a) deletion of the first 71 nucleotides of exon 3, altering the open reading frame and generating a premature stop codon, b) total exon 3 skipping resulting in an in frame deleted mRNA encoding a putative protein lacking part of the transactivation domain and of the POUspecific homeodomain. Notably, the patient's relatives heterozygous for the mutation had PRL levels under the normal range with no evident clinical symptoms. CONCLUSIONS: The IVS2- 3insAmutation, responsible for CPHD at the homozygous state, causes the presence of 2 aberrant splicing products encoding non-functional products. In the heterozygotes one normal allele might not guarantee a complete pituitary function.

Hypothalamus-pituitary-adrenal axis evaluation in patients with hypothalamo-pituitary disorders: comparison of different provocative tests.

BACKGROUND: The insulin tolerance test (ITT) is the gold standard test to evaluate hypothalamic-pituitary-adrenal (HPA) axis in suspected ACTH insufficiency. When contraindicated, alternative tests have been proposed such as metyrapone and ACTH stimulation test. 250 microg ACTH is a supramaximal dose and unreliable in this setting. The diagnostic reliability of 1.0 microg ACTH test is controversial and very low doses have been proposed. DESIGN: In 31 patients with hypothalamo-pituitary disorders and normal basal cortisol, we compared the diagnostic sensitivity, specificity and accuracy of metyrapone [metyrapone test (MET) 30 mg/kg p.o.], high (HDT, 250 microg i.v.), low (LDT, 1.0 microg i.v.) and very-low (VLDT, 0.06 microg i.v.) dose ACTH tests. Receiver operator curve (ROC) analysis was applied with ITT as reference test. RESULTS: MET approached the best pairs of values for highest sensitivity (71.4% and 64.3%) and highest specificity (100% and 82.4%) using ACTH and 11-deoxycortisol (11-DOC) cut-off of 17.3 pmol/l and 144.3 nmol/l. Either HDT or LDT sensitivity approached 71.4% with a specificity of 82.4% or 73.3% with a specificity of 80% for cortisol cut-off of 582.1 or 477.3 nmol/l. VLDT approached the highest sensitivity (57.1%) and highest specificity (88.2%) for a cortisol cut-off of 364.2 nmol/l. CONCLUSION: Neither MET nor ACTH test can be considered completely reliable for the diagnosis of secondary hypoadrenalism, when compared with ITT that remains the best test. Either MET or ACTH stimulation test, at both high and low dose, show an overall similar reliability, provided that appropriated cut-off values were considered; testing with very low ACTH doses seems to be misleading.

Diagnosis of adult GH deficiency.

The current guidelines for the diagnosis of adult GHD are mainly based on the statements from the GH Research Society Consensus from Port Stevens in 1997. It is stated that diagnosis of adult GHD must be shown biochemically by provocative tests within the appropriate clinical context. The insulin tolerance test (ITT) was indicated as that of choice and severe GHD defined by a GH peak lower than 3 microg/L. The need to rely on provocative tests is based on evidence that that the measurement of IGF-I as well as of IGFBP-3 levels does not distinguish between normal and GHD subjects. Hypoglycemia may be contraindicated; thus, alternative provocative tests were considered, provided they are used with appropriate cut-off limits. Among classical provocative tests, arginine and glucagon alone were indicated as alternative tests, although less discriminatory than ITT. Testing with the combined administration of GHRH plus arginine was recommended as an alternative to ITT, mostly taking into account its marked specificity. Based on data in the literature in the last decade, the GRS Consensus Statements should be appropriately amended. Regarding the appropriate clinical context for the suspicion of adult GHD, one should evaluate patients with hypothalamic or pituitary disease or a history of cranial irradiation, as well as those with childhood-onset GHD are at obvious risk as adults for severe GHD. Brain injuries (trauma, subarachnoid hemorrage, tumours of the central nervous system) very often cause acquired hypopituitarism, including severe GHD. Given the epidemiology of brain injuries, the important role of the endocrinologist in providing major clinical benefit to brain injured patients who are still undiagnosed should be underscored. From the biochemical point of view, although normal IGF-I levels do not rule out severe GHD, very low IGF-I levels in patients highly suspected for GHD (i.e. patients with childhood-onset, severe GHD or with multiple hypopituitarism acquired in adulthood) can be considered as definitive evidence for severe GHD; thus, these patients would skip provocative tests. Patients suspected for adult GHD with normal IGF-I levels must be investigated by provocative tests. ITT remains a test of reference but it should be recognized that other tests are as reliable as ITT. Glucagon as classical test and, particularly, new maximal tests such as GHRH in combination with arginine or GH secretagogues (GHS) (i.e. GHRP-6) have well defined cut-off limits, are reproducible, able to distinguish between normal and GHD subjects. Overweight and obesity have confounding effect on the interpretation of the GH response to provocative tests. In adults cut-off levels of GH response below which severe GHD is demonstrated must be appropriate to lean, overweight and obese subjects to avoid false positive diagnosis in obese adults and false negative diagnosis in lean GHD patients. Finally, normative values of GH response to provocative tests may depend on age, particularly in the transitional age; the normative cut-off levels of GH response to ITT in this phase of life are now available.

Growth hormone/insulin-like growth factor I axis, glucose metabolism, and lypolisis but not leptin show some degree of refractoriness to short-term fasting in acromegaly.

Starvation exerts critical influence on somatotroph and leptin secretion. Fasting enhances GH levels in normal subjects, but not in GH hyposecretory states, while it always inhibits leptin secretion. We aimed to clarify the GH/IGF-I and metabolic response to short-term fasting in a GH hypersecretory state such as acromegaly. To this goal, in 8 active acromegalic (ACRO) and in 7 normal women (NS) we evaluated mean GH (mGHc), leptin (mLEPc), insulin (mINSc), glucose (mGLUc) concentrations as well as IGF-I, IGF binding protein (IGFBP)-3, IGFBP-1, and free fatty acid (FFA) levels before and after 36-h fasting. Before fasting, mGHc, IGF-I, mINSc, mGLUc, and FFA levels in ACRO were higher (p<0.01) than in NS. IGFBP-3, IGFBP-1, and mLEPc were similar in ACRO and in NS. Fasting clearly (p<0.02) increased mGHc in NS only. After 36-h fasting, significant IGF-I reduction was recorded in NS only (p<0.03). IGFBP-3 did not change both in ACRO and NS. IGFBP-1 significantly increased (p<0.05) after fasting in both groups but in ACRO were lower (p<0.03) than in NS. Fasting decreased (p<0.03) mLEPc, mGLUc, and mINSc in ACRO as well as in NS; mINSc and mGLUc after fasting in ACRO persisted higher (p<0.005) than in NS. FFA levels were increased by fasting in NS (p<0.02), but not in ACRO. This study shows that GH/IGF-I axis, glucose metabolism, and lypolisis but not leptin display some degree of refractoriness to short-term fasting in acromegaly. The lack of any GH response to fasting in acromegaly would likely reflect neuroendocrine alterations secondary to the GH hypersecretory state. On the other hand, the lack of somatotropic response and the peculiarly blunted metabolic reaction to short-term fasting would partially reflect the delayed adaptation of insulin resistance to starvation.

Unreliability of classic provocative tests for the diagnosis of growth hormone deficiency.

In this study we investigated 9 prepubertal children with blunted GH response to classic pharmacological stimuli in contrast with normal auxological evaluation. The children were followed to evaluate their growth velocity for a longer period before starting replacement GH therapy. To evaluate the pituitary reserve a supraphysiologic stimulus such as GHRH plus arginine was used. Serum GH levels were measured by a time-resolved immunofluorimetric assay before and after 1 microg/kg body weight iv injection of GHRH, while serum PRL, IGF-I, and insulin were evaluated only in basal conditions using an automatic immunometric assay. Out of 9 studied subjects, 7 underwent GHRH plus arginine administration and showed a normal GH response; the parents of the remaining 2 children refused the test. Normal serum levels of PRL, IGF-I, insulin, and a normal insulin sensitivity were observed in all children. After 1 yr, the growth rate in each patient was further improved and reached almost normal values. Our results further confirm that the decision to start replacement GH therapy should be based on both auxological parameters and laboratory findings. The GHRH plus arginine test appears to be useful to identify false GH deficiency in children showing a blunted GH response to classic stimuli in contrast with normal growth rate.

Acute ghrelin response to intravenous dexamethasone administration in idiopathic short stature or isolated idiopathic growth hormone-deficient children.

Acylated ghrelin has been originally described for its potent GH-releasing activity mediated by the activation of the GH secretagogue receptor type 1a. More recently, ghrelin has been reported to exert several other GH-independent biological actions, among which in the modulation of metabolic functions. Glucocorticoids are well known to exert important metabolic functions but also to modulate GH secretion, although through mechanisms that have not been fully clarified so far. Interestingly, the existence of a feedback link between glucocorticoids and ghrelin system has already been reported. The aim of our study was to evaluate the acute GH and ghrelin responses to dexamethasone (DEX) administration in children with idiopathic short stature (ISS) or isolated idiopathic GH deficiency (GHD). Eight children with ISS (age: 9.5+/-1.2 yr) and 7 with GHD (12.1+/-1.4 yr) underwent iv DEX administration (0.3 mg/body surface area at 0 min). IGF-I, GH, and ghrelin levels were assayed at baseline and every 30 min from 120 up to 240 min after DEX. Compared to baseline levels DEX decreased ghrelin in ISS at 120 min and 240 min (p<0.04). On the other hand DEX did not modify ghrelin levels in GHD. After DEX, ghrelin was reduced in ISS compared to GHD (p<0.02). DEX increased GH in ISS but not in GHD (peak: 11.1+/-1.2 vs 7.6+/-0.9 microg/l). Basal, as well as after-DEX ghrelin levels negatively correlated with IGF-I in GHD (p<0.03) and with height SD score (HSDS) in ISS (p<0.02). Acute DEX administration is able to decrease ghrelin in ISS, but not in GHD children. Both basal and after-DEX ghrelin levels negatively correlate with IGF-I and HSDS. All these data suggest the existence of a feedback link among ghrelin, glucocorticoids and the GH/IGF-I axis.

Heterozygous mutation of HESX1 causing hypopituitarism and multiple anatomical malformations without features of septo-optic dysplasia.

Isolated GH deficiency or combined pituitary hormone deficiencies have been associated with mutations in transcription factors encoding genes that control organogenesis or cell differentiation. Among these factors, Hesx1 is essential for the development of the optic nerve and regulates some of the earliest stages in pituitary development and is intimately involved in orchestrating the expression of other factors involved in pituitary organogenesis. Mutations in HESX1 are reported in patients with hypopituitarism either with typical septo-optic dysplasia (SOD) or with neuromorphological abnormalities not included in classical SOD. The present report describes clinical features, biochemical parameters, and characterization of a missense mutation (Gln6His) in exon1 of HESX1 in a pre-pubertal child who progressively developed multiple hypopituitarism, firstly GH and, afterwards, TSH and ACTH deficiencies, in a pluri-malformative syndrome characterized by short stature and anatomical malformations not associated with a classical SOD phenotype. This finding further supports the necessity to stay alert in evaluating a gene that plays a minor role in the pathogenesis of sporadic hypopituitarism, such as HESX1 gene even when the phenotype does not fit in with a classical SOD syndrome.

Normal age-dependent values of serum insulin growth factor-I: results from a healthy Italian population.

Serum IGF-I levels were measured in 547 non-hypopituitaric, non-acromegalic healthy subjects of both sexes in Italy to develop reference values in relation to age and sex. Participant subjects were stratified in three age classes (25- 39, 40-59 and >or=60 yr) and IGF-I assay was carried out by double-antibody radio immunoassay. Pearson's correlation coefficient between age and IGF-I values was calculated by sex and predefined age ranges. IGF-I levels significantly decreased with age (p<0.001, Kruskal-Wallis test) while sex was not a significant factor. The median IGF-I levels were 206 ng/ml in the 25-39 yr range, 147 ng/ml in the 40-59 yr range and 103 ng/ml in the >or=60 yr range. Pearson's correlation coefficient confirmed the negative correlation between age and IGF-I levels in the total sample of subjects (r=-0.529). The r coefficient between age and IGF-I levels did not differ between sexes (r=-0.570 in males and r=-0.529 in females), thus reflecting no sex-effect on IGF-I levels decline over years. No correlations were found in the 25-39 yr range (r=-0.036) or in the 40-59 yr range (r=-0.080) either, while in subjects aged >60 yr, IGF-I levels tended to further decrease with increased age (r=0.389). Ranges of normal values set at the 2.5th-97.5th percentile in the three age ranges were 95.6-366.7 ng/ml between 25 and 39 yr, 60.8-297.7 ng/ml between 40 and 59 yr and 34.5-219.8 ng/ml in subjects aged >or=60 yr. This study may contribute to the development of age-specific reference ranges for IGF-I determination in serum of normal subjects of both sexes in Italy.

Growth hormone receptor gene mutations in two Italian patients with Laron Syndrome.

Laron Syndrome (LS) represents a condition characterized by GH insensitivity caused by molecular defects in the GH receptor (GHR) gene or in the post-receptor signalling pathway. We report the molecular characterization of two unrelated Italian girls from Sicily diagnosed with LS. The DNA sequencing of the GHR gene revealed the presence of different nonsense mutations, occurring in the same background haplotype. The molecular defects occurred in the extracellular domain of the GHR leading to a premature termination signal and to a truncated non-functional receptor. In one patient, a homozygous G to T transversion, in exon 6, led to the mutation GAA to TAA at codon 180 (E180X), while in the second patient a homozygous C to T transition in exon 7 was detected, causing the CGA to TAA substitution at codon 217 (R217X). Both probands presented the polymorphisms Gly168Gly and Ile544Leu in a homozygous state in exons 6 and 10, respectively. The E180X represents a novel defect of the GHR gene, while the R217X mutation has been previously reported in several patients from different ethnic backgrounds but all from countries located in the Mediterranean and Middle Eastern region.

Pituitary imaging abnormalities in patients with and without hypopituitarism after traumatic brain injury.

Recent evidence suggests that patients with traumatic brain injury (TBI) are at substantial risk of hypopituitarism. The pathomechanisms, however, are not completely understood yet. Little is known about the association of morphological changes in the sella region with pituitary function in TBI. In this study, we assessed morphological abnormalities of the sella region in patients with TBI and their relation to endocrine function. We studied magnetic resonance (MR) or computed tomography (CT) scans of 22 patients with TBI [17 men, 5 women, age (mean+/-SD) 43.5+/-10.6 yr, time after trauma 17.4 +/-15.0 yr]. Of these, 15 patients had some degree of hypopituitarism. We found abnormalities of the sella region in 80% of the patients with hypopituitarism and 29% of those without hypopituitarism (Fisher's exact test, p=0.032). The most common abnormality was loss of volume or empty sella, followed by native signal inhomogeneities, perfusion deficit, and lack of neurohypophyseal signal. Our results indicate that pituitary imaging abnormalities are more common in TBI patients with hypopituitarism than those without. Both immediate trauma-induced pathology as necrosis and hemorrhage as well as multifactorial mid- to long-term changes may underlie these abnormalities.

Endocrine dysfunction in patients operated on for non-pituitary intracranial tumors.

OBJECTIVE: Hypopituitarism frequently follows pituitary neurosurgery (NS) and/or irradiation. However, the frequency of hypothalamic-pituitary dysfunction after NS of non-pituitary intracranial tumors is unclear. The aim of this study was to assess the presence of endocrine alterations in patients operated on for intracranial tumors. DESIGN: This is a retrospective study. METHODS: We studied 68 consecutive adult patients (28 female, 40 male, age 45.0 +/- 1.8 years; body mass index (BMI): 26.5 +/- 0.6) with intracranial tumors who underwent NS only (n = 17) or in combination with radiotherapy (RT) and/or chemotherapy (CT) (n = 51). In all subjects, basal endocrine parameters and the GH response to GHRH + arginine test (using BMI-dependent cut offs) were evaluated. RESULTS: In 20.6% of the patients, peripheral endocrinopathy related to CT and/or RT was present. Hypopituitarism was found in 38.2% of the patients. Total pituitary hormone, multiple pituitary hormone, and isolated pituitary hormone deficits were present in 16.2, 5.8, and 16.2% respectively. The most common pituitary deficits were, in decreasing order: LH/FSH 29.4%, GH 27.9%, ACTH 19.1%, TSH 17.7%, and diabetes insipidus 4.4%. Hyperprolactinemia was present in 13.2%. The prevalence of hypopituitarism was higher in patients who underwent NS only and with tumors located closely to the sella turcica, but a substantial proportion of patients with tumors not directly neighboring the sella also showed hypopituitarism. CONCLUSIONS: Hypopituitarism frequently occurs after NS for intracranial tumors. Also, exposure of these patients to CT and/or RT is frequently associated with peripheral endocrinopathies. Thus, endocrine evaluation and follow-up of patients treated for intracranial tumors should be performed on a regular basis.

Low dose hexarelin and growth hormone (GH)-releasing hormone as a diagnostic tool for the diagnosis of GH deficiency in adults: comparison with insulin-induced hypoglycemia test.

GH deficiency (GHD) in adults must be shown by provocative testing of GH secretion. Insulin-induced hypoglycemia (ITT) is the test of choice, and severe GHD, treated with recombinant human GH replacement, is defined by a GH peak response to ITT of less than 3 microg/L. GHRH plus arginine (ARG) is a more provocative test and is as sensitive as ITT provided that appropriate cut-off limits are assumed. GH secretagogues are a family of peptidyl and nonpeptidyl GH-releasing molecules that strongly stimulate GH secretion and, even at low doses, truly synergize with GHRH. Our aim was to verify the diagnostic reliability of the hexarelin (HEX; 0.25 microg/kg, iv) and GHRH (1 microg/kg, iv) test for the diagnosis of adult GHD. To this goal, in the present study we 1) defined the normal ranges of the GH response to GHRH+HEX in a group of normal young adult volunteers (NS; n = 25; 18 men and 7 women; age, 28.5+/-0.6 yr) and in 11 of them verified its reproducibility in a second session, and 2) compared the GH response to GHRH+HEXwith that to ITT in a group of normal subjects (n = 33; 12 men and 21 women; age, 34.1+/-1.5 yr) and hypopituitaric adults with GHD (n = 19; 10 men and 9 women; age, 39.9+/-2.2 yr; GH peak <5 microg/L after ITT). The GH response to GHRH+ARG was also evaluated in all GHD and in 77 normal subjects (40 men and 37 women; age, 28.1+/-0.6 yr). The mean GH peak after GHRH+HEX in NS was 83.6+/-4.5 microg/L; the third and first percentile limits of the normal GH response were 55.5 and 51.2 microg/L, respectively). The GH response to GHRH+HEX in NS showed good intraindividual reproducibility. In GHD the mean GH peak after GHRH+HEX (2.6+/-0.7 microg/L) was similar to that after GHRH+ARG (3.6+/-1.0 microg/L), and both were higher (P < 0.001) than that after ITT (0.6+/-0.1 microg/L); the GH responses to GHRH+HEX were positively associated with those to ITT and GHRH+ARG. Analyzing individual GH responses, 100% had severe GHD after ITT (GH peak, <3 microg/L). After GHRH+HEX all GHD had GH peaks below the third percentile limit of normality appropriate for this test (i.e. 55.5 microg/L). Thirteen of 19 (68.4%) GHD subjects had GH peaks below 3 microg/L after GHRH+HEX but all 19 (100%) had GH peaks below the first percentile limit of normality (i.e. 51.2 microg/L). The GH responses to GHRH+HEX were highly concordant with those after GHRH+ARG. In conclusion, the present results define normal limits of the GH response to stimulation with low dose HEX+GHRH in normal adults and show that this test is as sensitive as ITT for the diagnosis of adult GHD provided that appropriate cut-off limits are considered.

Comparison between insulin-induced hypoglycemia and growth hormone (GH)-releasing hormone + arginine as provocative tests for the diagnosis of GH deficiency in adults.

There is now wide consensus that, within an appropriate clinical context, GH deficiency (GHD) in adults must be shown biochemically by provocative testing of GH secretion and that appropriate cut-off limits have to be defined for each provocative test. Insulin-induced hypoglycemia (ITT) is indicated as the test of choice, and severe GHD, to be treated with recombinant human GH replacement, is defined by a GH peak response to ITT of less than 3 micrograms/L. GHRH + arginine (GHRH + ARG) is one of the most promising tests in alternative to ITT. In fact, it has been reported as a potent, reproducible, and age-independent test and that it is able to distinguish between GHD and normal adults. The aim of the present study was to compare the GH response to ITT and GHRH + ARG in a large group of hypopituitary adults (n = 40; 29 male and 11 female; age: 36.4 +/- 2.1 yr). The third centile limit of the peak GH response to ITT has been reported as 5 micrograms/L, whereas in our lab, that to GHRH + ARG is 16.5 micrograms/L. In hypopituitary adults, the mean peak GH response to ITT (1.5 +/- 0.2 micrograms/L, range: 0.1-8.5 micrograms/L) was lower (P < 0.001) than that to GHRH + ARG (3.0 +/- 0.4 micrograms/L, range 0.1-12.0 micrograms/L), though there was positive correlation (r = 0.61, P < 0.001) between the GH responses to the 2 tests. The peak GH response to GHRH + ARG, but not that to ITT, was positively (though weakly) associated with insulin-like growth factor-I levels (r = 0.35, P < 0.03). Childhood and adult onset GHD patients, as well as patients with single and multiple pituitary insufficiencies, had similar peak GH responses to ITT or GHRH + ARG. Analyzing individual GH responses, 4/40 (10%) of the hypopituitary patients had GH peaks higher than 5 micrograms/L after ITT; moreover, 3 other patients (7%) had GH peaks, after ITT, higher than 3 micrograms/L. On the other hand, after GHRH + ARG, all patients had GH peaks lower than 16.5 micrograms/L, whereas 21/40 (52.5%) had GH peaks higher than 3 micrograms/L. Because 3 micrograms/L is the arbitrary cut-off for ITT, the third centile limit of which is 5 micrograms/L, we arbitrarily considered 9 micrograms/L as the cut-off point for GHRH + ARG. It is noteworthy that 37/40 (92.5%) patients had a GH peak, after GHRH + ARG, below this limit. In conclusion, our present results confirm that the ITT test is a reliable provocative test for the diagnosis of adult GHD, whereas they show that the GHRH + ARG test is, at least, as sensitive as the ITT test (provided that appropriate cut-off limits are considered). Note that even the arbitrary cut-off point below which severe GHD is demonstrated has to be appropriate to the potency of the test.

Retesting young adults with childhood-onset growth hormone (GH) deficiency with GH-releasing-hormone-plus-arginine test.

Within an appropriate clinical context, severe GH deficiency (GHD) in adults has to be defined biochemically by provocative testing of GH secretion. Patients with childhood-onset GHD need retesting in late adolescence or young adulthood to verify whether they have to continue recombinant human GH treatment. GHRH + arginine (GHRH+ARG) is the most reliable alternative to the insulin-induced hypoglycemia test (ITT) as a provocative test for the diagnosis of GHD in adulthood, provided that appropriate cut-off limits are assumed (normal limits, 16.5 microg/L as 3rd and 9.0 microg/L as 1st centile). We studied the GH response to a single GHRH (1 microg/kg iv) + ARG (0.5 g/kg iv) test in 62 young patients who had undergone GH replacement in childhood, based on the following diagnosis: 1) organic hypopituitarism with GHD (oGHD) In = 18: 15 male (M), 3 female (F); age, 26.8+/-2.2 yr; GH peak < 10 microg/L after two classical tests]; 2) idiopathic isolated GHD (iGHD) [n = 23 (15 M, 8 F); age, 23.0+/-1.5 yr; GH peak < 10 microg/L after two classical tests]; and 3) GH neurosecretory dysfunction (GHNSD) [n = 21 (10 M, 11 F); age, 25.1+/-1.6 yr; GH peak > 10 microg/L after classical test but mGHc < 3 microg/L]. The GH responses to GHRH+ARG in these groups were also compared with that recorded in a group of age-matched normal subjects (NS) [n = 48 (20 M, 28 F); age, 27.7+/-0.8 yr]. Insulin-like growth factor I levels in oGHD subjects (61.5+/-13.7 microg/L) were lower (P < 0.001) than those in iGHD subjects (117.2+/-13.1 microg/L); the latter were lower than those in GHNSD subjects (210.2+/-12.9 microg/L), which, in turn, were similar to those in NS (220.9+/-7.1 microg/L). The mean GH peak after GHRH+ARG in oGHD (2.8+/-0.8 microg/L) was lower (P < 0.001) than that in iGHD (18.6+/-4.7 microg/L), which, in turn, was clearly lower (P < 0.001) than that in GHNSD (31.3+/-1.6 microg/L). The GH response in GHNSD was lower than that in NS (65.9+/-5.5 microg/L), but this difference did not attain statistical significance. With respect to the 3rd centile limit of GH response in young adults (i.e. 16.5 microg/L), retesting confirmed GHD in all oGHD, in 65.2% of iGHD, and in none of the GHNSD subjects. With respect to the 1st centile limit of GH response (i.e. 9.0 microg/L), retesting demonstrated severe GHD in 94% oGHD and in 52.1% of iGHD. All oGHD and iGHD with GH peak after GHRH+ARG lower than 9 microg/L had also GH peak lower than 3 microg/L after ITT. In the patients in whom GHD was confirmed by retesting, the mean GH peak after GHRH+ARG was higher than that after ITT (3.4+/-0.5 vs. 1.9+/-0.4). In conclusion, given appropriate cut-off limits, GHRH+ARG is as reliable as ITT for retesting patients who had undergone GH treatment in childhood. Among these patients, severe GHD in adulthood is generally confirmed in oGHD, is frequent in iGHD, but never occurs in GHNSD.

Bone loss is correlated to the severity of growth hormone deficiency in adult patients with hypopituitarism.

Reduced bone mineral density (BMD) has been reported in patients with isolated GH deficiency (GHD) or with multiple pituitary hormone deficiencies (MPHD). To investigate whether the severity of GHD was correlated with the degree of bone mass and turnover impairment, we evaluated BMD at the lumbar spine and femoral neck; circulating insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), and osteocalcin levels, and urinary cross-linked N-telopeptides of type I collagen (Ntx) levels in 101 adult hypopituitary patients and 35 sex- and age-matched healthy subjects. On the basis of the GH response to arginine plus GHRH (ARG+/-GHRH), patients were subdivided into 4 groups: group 1 included 41 patients with a GH peak below 3 microg/L (0.9 +/- 0.08 microg/L), defined as very severe GHD; group 2 included 25 patients with a GH peak between 3.1-9 microg/L (4.7 +/- 0.4 microg/L), defined as severe GHD; group 3 included 18 patients with a GH peak between 9.1-16.5 microg/L (11.0 +/- 0.3 microg/L), defined as partial GHD; and group 4 included 17 patients with a GH peak above 16.5 microg/L (28.3 +/- 4.3 microg/L), defined as non-GHD. In all 35 controls (group 5), the GH response after ARG+/-GHRH was above 16.5 microg/L (40.7 +/- 2.2 microg/L). In patients in group 1, circulating IGF-I (P < 0.001), IGFBP-3 (P < 0.05), osteocalcin (P < 0.001), and urinary Ntx levels (P < 0.001) were lower than those in group 3-5, which were not different from each other; the t score at the lumbar spine (-1.99 +/- 0.2) and that at the femoral neck (-1.86 +/- 0.3) were lower than those in groups 3 (-0.5 +/- 0.7, P < 0.01 and -0.3 +/- 0.7, P < 0.01, respectively), 4 (-0.5 +/- 0.2, P < 0.01 and -0.3 +/- 0.7, P < 0.01, respectively), and 5 (-0.5 +/- 0.2, P < 0.001 and 0.0 +/- 0.02, P < 0.001, respectively). In patients in group 2, circulating IGF-I and IGFBP-3 levels were not different from those in group 1, whereas the t scores at the lumbar spine (-1.22 +/- 0.3) and femoral neck (-0.9 +/- 0.3) were significantly higher and lower, respectively, than those in groups 1 and 5 (P < 0.05) but not those in groups 3 and 4, and serum osteocalcin and urinary Ntx levels were significant higher than those in group 1 and lower than those in groups 3-5 (P < 0.001). To evaluate the effect of isolated GHD vs. MPHD, patients were subdivided according to the number of their hormonal deficits, such as panhypopituitarism with (10 patients) or without (31 patients) diabetes insipidus, GHD with 1 or more additional pituitary deficit(s) (36 patients), isolated GHD (7 patients), 1-2 pituitary hormone deficit(s) without GHD (10 patients), and normal anterior pituitary function (7 patients). The t score at the lumbar spine and femoral neck and the biochemical parameters of bone turnover were not significantly different among the different subgroups with similar GH secretions. A significant correlation was found between the GH peak after ARG+GHRH and IGF-I, osteocalcin, urinary Ntx levels, and the t score at the lumbar spine, but not that at the femoral neck level. A significant correlation was also found between plasma IGF-I levels and the t score at the lumbar spine and femoral neck, serum osteocalcin, and urinary Ntx. Multiple correlation analysis revealed that the t score at the lumbar spine, but not that at the femoral neck, was more strongly predicted by plasma IGF-I levels (t = 3.376; P < 0.005) than by the GH peak after ARG+GHRH (t = -0.968; P = 0.338). In conclusion, a significant reduction of BMD associated with abnormalities of bone turnover parameters was found only in patients with very severe or severe GHD, whereas normal BMD values were found in non-GHD hypopituitary patients. These abnormalities were consistently present in all patients with GHD regardless of the presence of additional hormone deficits, suggesting that GHD plays a central role in the development of osteopenia in hypopituitary patients.

The growth hormone (GH) response to the arginine plus GH-releasing hormone test is correlated to the severity of lipid profile abnormalities in adult patients with GH deficiency.

The aim of the present study was to correlate the degree of the GH response to the combined arginine and GHRH (ARG+GHRH) test with clinical status in 157 adult hypopituitary patients and 35 healthy controls. On the basis of the GH response to ARG+GHRH, the 192 subjects were subdivided into 5 groups: group 1, very severe GH deficiency (GHD; 65 patients with GH peak <3 microg/L); group 2, severe GHD (37 patients with GH peak between 3.1-9 microg/L); group 3, partial GHD (25 patients with GH peak between 9.1-16.5 microg/L); group 4, non-GHD (30 patients with GH peak >16.5 microg/L); and group 5 (35 controls with GH peak >16.5 microg/L). Plasma insulin-like growth factor I (IGF-I) concentrations were lower (P < 0.001) in patients of group 1 (74.4 +/- 6.7 microg/L) and group 2 (81.4 +/- 6.8 microg/L) than in those of group 3, 4, and 5 (163.6 +/- 40.6, 185.9 +/- 21, and 188.8 +/- 11.1 microg/L, respectively). Plasma IGF-binding protein-3 concentrations were lower (P < 0.01) in group 1 (2.1 +/- 0.2 mg/L) and group 2 (2.0 +/- 0.2 mg/L) than in group 3 (3.4 +/- 0.7 mg/L) and group 5 (3.8 +/- 0.2 mg/L). In patients of group 1, total cholesterol (228.3 +/- 5.7 mg/dL) and triglycerides levels (187.4 +/- 15.3 mg/dL) were higher than those in group 3 (196.6 +/- 9.6 and 115.8 +/- 10.1 mg/dL, respectively), group 4 (176.8 +/- 11.3 and 101.4 +/- 12.5 mg/dL, respectively), and group 5 (160 +/- 6.9 and 99.3 +/- 5.4 mg/dL, respectively). High density lipoprotein cholesterol levels were lower in patients of group 1 (45.2 +/- 2.4 mg/dL) than in those of group 4 (54.7 +/- 3.5 mg/dL; P < 0.05) and group 5 (53.6 +/- 2 mg/dL; P < 0.001), whereas low density lipoprotein cholesterol levels were higher in patients of group 1 (127.3 +/- 7.9 mg/dL), group 2 (129.2 +/- 9.5 mg/dL), and 3 (133 +/- 9 mg/dL) than in those of group 5 (102.4 +/- 7.4 mg/dL; P < 0.05). Patients of group 2 had total cholesterol, high density lipoprotein cholesterol, and triglycerides levels at an intermediate level with respect to those in groups 1, 3, and 4. Among the five groups, no difference was found in fasting glucose concentrations, heart rate, or systolic and diastolic blood pressures. A significant increase in fat body mass and a decrease in lean body mass and total body water were found in all patients compared to controls. Disease duration was significantly shorter in patients of group 4 than in those of the remaining three groups (P < 0.001). A significant correlation was found between the GH peak after ARG+GHRH and disease duration (r = -0.401; P < 0.001), plasma IGF-I (r = 0.434; P < 0.001), total cholesterol (r = -0.324; P < 0.001), and triglycerides levels (r = -0.219; P < 0.05). A significant multiple linear regression coefficient was found between the GH peak after ARG+GHRH and plasma IGF-I levels (t = 2.947; P < 0.005), total cholesterol levels (t = -2.746; P < 0.01), and disease duration (t = -2.397; P < 0.05). In conclusion, the results of the present study indicate that the degree of the GH response to ARG+GHRH is correlated with the severity of lipid profile abnormalities and substantiate the reliability of the ARG+GHRH test for the diagnosis of GHD in adults. Because at present GH treatment is recommended only in adult patients with severe GHD, patients with a GH response below 9 microg/L to the ARG+GHRH test should be treated with GH, as should patients with a peak GH response to an insulin tolerance test below 3 microg/L.

Diagnosis of GH deficiency in adults.

Within an appropriate clinical context, growth hormone deficiency (GHD) in adults must be demonstrated biochemically. The assays of insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3) per se do not establish the diagnosis of adult GHD. Similarly, the evaluation of spontaneous growth hormone (GH) secretion over 24 h has no diagnostic value in adulthood even when an ultra-sensitive GH assay is used. The diagnosis of adult GHD is established by provocative testing of GH secretion, and insulin-induced hypoglycaemia using the insulin tolerance test (ITT) is indicated as the test of choice. Alternative provocative tests of GH secretion have been proposed and have to be used with appropriate cut-off limits. Testing with GH releasing hormone (GHRH) alone has no diagnostic value, but when GHRH is given in combination with arginine or pyridostigmine it becomes the most potent and reproducible provocative test to evaluate the maximal secretory capacity of somatotrope cells. The potentiating effect of arginine on the GHRH-induced GH response is fully preserved while the stimulatory effect of GHRH + pyridostigmine is reduced in ageing. The GHRH + arginine test is well tolerated and reproducibly distinguishes between normal and GHD adult and elderly subjects. Thus, the GHRH + arginine test is the most promising alternative to the ITT provided that cut-off limits appropriate to its potent stimulatory effect are considered.

Occurrence of GH deficiency in adult patients who underwent neurosurgery in the hypothalamus-pituitary area for non-functioning tumour masses.

Hypothalamus-pituitary tumours and their treatments (neurosurgery and/or radiotherapy) are major causes of acquired hypopituitarism. Scientific and clinical evidences show the positive effect of GH replacement therapy in severe adult GH deficiency (GHD) pointed toward the need of diagnostic screening of conditions at high risk for GHD. We screened 152 adults (82 males, 70 females; age: 52.3+/-1.2 years, age-range: 20-80 years, BMI: 26.4+/-0.8 kg/m(2)) in order to disclose the presence of GHD after neurosurgery for hypothalamus-pituitary tumours. The whole group (studied at least 3 months after neurosurgery) included: 111 non-functioning pituitary adenomas and 41 peri-pituitary tumours (24 craniopharyngiomas, 7 meningiomas, 5 cysts, 2 chondrosarcomas, 1 colesteatoma, 1 germinoma and 1 hemangiopericitoma). In 14 patients who underwent both neurosurgery and radiotherapy due to a tumour remnant, the somatotroph function was evaluated again 6 months after the end of radiotherapy. GHD was assumed to be shown by GH peak <5 microg/L (severe <3 microg/L) after Insulin Tolerance Test (ITT) or <16.5 microg/L (severe <9 microg/L) after GH-releasing hormone+arginine test (GHRH+ARG) (3rd and 1st centile limits of normality, respectively), two widely accepted provocative tests. Before neurosurgery GHD was present in 97/152 (63.8%) and resulted severe in 66/152 (43.4%) patients. After neurosurgery GHD was present in 122/152 (80.2%) and severe in 106/152 (69.7%). While 26 patients developed severe GHD (GHD) as consequence of neurosurgery, only one patient who had been classified as GHD before neurosurgery showed normal GH response after surgery. After neurosurgery, 91.0% (81/89) of the pan-hypopituitaric patients showed severe GHD. Considering the 14 patients who underwent also radiotherapy after neurosurgery, 7/14 had GHD before neurosurgery while 12/14 became severe GHD after radiotherapy in a context of pan-hypopituitarism. IGF-I levels below the 3rd age-related normal limits were present in 39.0% of patients in whom severe GHD was showed by provocative tests. In conclusion, this study shows that the occurrence of acquired severe GHD is extremely common in adult patients bearing non-functioning tumour masses in the hypothalamus-pituitary area and further increases after neurosurgery. All patients bearing non-functioning hypothalamus-pituitary tumours should undergo evaluation of their somatotroph function before and after neurosurgery that represents a condition at obvious more than high risk for hypopituitarism.

IGF-I levels in prepubertal and pubertal children with Down syndrome.

BACKGROUND: Growth retardation is a main feature of Down syndrome but it is still unclear whether an alteration of the GH/IGF-I axis is present in this condition. Concerning IGF-I levels, they have been found reduced by some authors but normal by others. METHODS: On these bases, IGF-I levels have been assessed from prepubertal to late pubertal stages of gonadal maturation in a large group of children and adolescents with Down syndrome (DS, 68 M, 45 F, 12.5 +/- 0.6 yr; prepubertal n = 39, pubertal n = 74) with those in a group of normal children and adolescents (NS, 75 M, 87 F; 11.1 +/- 0.4 yr; prepubertal n = 94, pubertal n = 68). RESULTS: Within each group, IGF-I levels were gender-independent while showed age-related variations with positive association with pubertal stage--peaking up in pubertal stage IV--(DS: r = 0.6, NS: r = 0.4, both p < 0.0001) and testosterone (DS: r = 0.6, NS: r = 0.5, p < 0.001) or estradiol (DS: r = 0.6, NS: r = 0.5, p < 0.001) levels. Considering whole groups, mean IGF-I levels in DS were slightly but significantly lower than those in NS (257.9 +/- 12.5 vs 310.8 +/- 12.6 micrograms/l, p < 0.02). Analyzing individual IGF-I levels in DS with respect to normal ranges per pubertal stage, more than 85% of IGF-I levels resulted within the normal limits. These results demonstrate that IGF-I levels in DS patients are generally within the normal range--though a slight reduction of mean IGF-I levels is present--and follow normal age-related variations with clear cut increase at puberty and positive association with gonadal steroid levels. CONCLUSIONS: This evidence points toward the need to clarify the GH/IGF-I axis function and activity in DS patients.

Growth hormone deficiency in the transition adolescent: should treatment be continued in adult life?

Adults with growth hormone (GH) deficiency (GHD) have impaired health, which improves with GH replacement. GHD in adulthood leads to impairment in body composition and structure functions as well as to deranged lipoprotein and carbohydrate metabolism leading to increased cardiovascular morbidity. Therefore the transition adolescent in whom severe GHD is confirmed has to continue GH replacement with an appropriate age-related dosage. All short children who have been treated with rhGH for classical and non-classical GHD should be suspected as potentially GHD in adulthood though only in classical organic and idiopathic forms is severe GHD likely to be confirmed. GHD must be shown biochemically by single provocative testing. Insulin-induced hypoglycemia (ITT) and GHRH + arginine are the tests of choice provided that appropriate cutoff limits are assumed; these tests show good specificity and sensitivity. Testing with GHRH + GH secretagogues is another reliable alternative. Low IGF-I levels can be definitive evidence of persistent severe GHD in patients with genetic GHD or panhypopituitarism, but normal IGF-I levels do not rule out severe GHD. Individual titration of the rhGH dose is recommended and measurement of IGF-I levels is needed for monitoring the adequacy of replacement. The mean GH dose for replacement in the transition adolescent, however, is still higher than in adulthood; after puberty the rhGH dose should be progressively decreased in the following years (probably up to 25 years old) in order to obtain optimal peak bone mass.