Effect of acute elevation of IGF-I on circulating GH, TSH, insulin, IGF-II and IGFBP-3 levels in non-endocrine short stature (NESS).

It is not clear whether acute and slight elevation of serum IGF-I, which does not affect blood glucose levels, modulates circulating GH levels. To clarify this, small doses of recombinant human IGF-I (rhIGF-I, 5 microg/kg, i.v.) were administered as a bolus to 10 children with non-endocrine short stature (NESS) (5 males and 5 females, 11.2+/-0.7 yr old) after an overnight fast. Physiological saline was administered intravenously to sex- and age-matched NESS controls (5 males and 5 females, 10.9+/-0.7 yr old). The changes of serum GH, TSH, PRL, IGF-I, IGF-II, IGFBP-3, T4, T3 and plasma glucose levels after the administration were compared to those of the control subjects. Serum IGF-I levels increased significantly from 15 to 150 min after injection compared to those in the control group. The peak value was observed at 15 min (delta increment, 74.6+/-11.8 microg/l). At 15 min after the injection, serum insulin was suppressed significantly (p<0.05), although plasma glucose levels were not modified significantly. Serum TSH showed a significant decrease by rhIGF-I at 15 min and 60 min, whereas serum T4 and T3 levels were not modified. Serum GH was also significantly suppressed at 60 min (p<0.02) and showed a rebound increase at 120 min (p<0.05). Serum IGFBP-3 levels after rhIGF-I were higher than controls at 90 min and 150 min. No significant changes of serum PRL, IGF-II, (IGF-I plus IGF-II)/IGFBP-3 ratios were observed after the IGF-I injection compared to controls. These results indicate that circulating IGF-I is a physiological regulator of GH secretion in normal children, since the changes of IGF-I after the small doses of rhlGF-I administration were within physiological ranges and did not affect plasma glucose levels.

Isochromosome consisting of terminal short arm and proximal long arm X in a girl with short stature.

A 16-year-old girl with short stature, short neck, shield chest, and cubitus valgus was studied. FISH analyses of her structurally altered X chromosome showed a der(X)- (wcpX+,TelXp/Yp++,SHOX++,STS++,KAL-, 37A12-,DXZ1+,XIST++,97L7++,300O13-,404F- 18-,417G15-,404F18-,140A-,TelXq/Yq-). These results, together with the high-resolution banding analysis, indicated her karyotype to be 46,X,der(X)(Xpter-->Xp22.3::Xq22.3--> cen-->Xq22.3::Xp22.3-->Xpter). The der(X) was an isochromosome, consisting of duplicated terminal short arms and duplicated proximal long arms. This in turn suggested that the chromosome was formed through pericentric inversion of an X chromosome, followed by isochromosome formation through sister chromatid exchange at Xp, close to the centromere. Replication R-banding analysis showed that the abnormal X chromosome was late replicating. Analysis of digestion patterns with a methylation-sensitive restriction endonuclease of the phosphoglycerate kinase 1 gene, located in Xq13.3, indicated that its inactivation patterns were completely skewed.

Association of intrauterine growth retardation with monosomy of the terminal segment of the short arm of the X chromosome in patients with Turner's syndrome.

Short stature, which may be a result of intrauterine growth retardation (IUGR), is a characteristic of Turner's syndrome. However, the loci responsible for IUGR have not been well studied. We reviewed the birth records of 74 patients with Turner's syndrome: 20 with pure X monosomy, 44 with X-mosaicisms, and 10 with X-structural abnormalities. The overall incidence of IUGR was 39.2% (29 of 74 patients). The SHOX gene is encoded in a terminal segment of the short arm of the X chromosome. In 39 patients where two copies of the SHOX gene were absent, the incidence of IUGR was 46.2% (18 of 39 patients). In 14 patients with two copies of the SHOX gene, the incidence of IUGR was significantly lower at 7.1% (1 of 14 patients). Our results suggest that SHOX influences in utero growth in Turner's syndrome.

Clinical and molecular studies in 15 females with ring X chromosomes: implications for r(X) formation and mental development.

We report clinical and molecular findings in 15 Japanese mosaic females with r(X) chromosomes, 45,X/46,X,r(X), confirmed by fluorescence in situ hybridization (FISH) analysis for DXZ1 and whole X chromosome painting. Cases 1-3, 5-7, and 11-13 had mental retardation (MR), the remaining cases being free from MR. FISH analysis showed that XIST was absent from the r(X) chromosomes in cases 1-4 and was present on the r(X) chromosomes in cases 5-15. X-inactivation analysis for the methylation status of the AR gene indicated that, of eight cases with XIST-positive r(X) chromosomes in more than 10% (23%-62%) of lymphocytes (cases 5-12), cases 5-10 had selective X-inactivation, whereas cases 11 and 12 had active X disomy. Microsatellite analysis for multiple loci on the pericentromeric region revealed that, of 11 cases with r(X) chromosomes in more than 10% (13%-62%) of lymphocytes (cases 1, 2, and 4-12), cases 1, 2, and 5-10 had heterozygous alleles for at least one locus, whereas cases 4, 11, and 12 had single alleles for all the loci examined. The results suggest that the r(X) and normal X chromosomes could be of biparental or uniparental origin, and that mental status in females with r(X) chromosomes is determined by multiple factors, including the presence or absence of XIST on the r(X) chromosomes and the size and frequency of active r(X) chromosomes, in addition to co-incidental genetic and environmental factors.

Recent status in the occurrence of leukemia in growth hormone-treated patients in Japan. GH Treatment Study Committee of the Foundation for Growth Science, Japan.

The Foundation for Growth Science in Japan has monitored the safety and efficacy of GH treatment in GH-deficient patients since 1975. Data were collected from more than 32,000 patients up to December 31, 1997. New leukemia was observed in 14 patients and myelodysplastic syndrome (MDS) in one patient. The types of leukemia were acute lymphocytic leukemia (n = 6; 40%), acute myelocytic leukemia or MDS (n = 7; 47%), and chronic myelocytic leukemia (n = 2; 13%). Leukemia developed in 9 patients during GH treatment and in 6 after the cessation of GH treatment. Six patients had known risk factors for leukemia, such as Fanconi's anemia and previous radiation or chemotherapy. Patient-years of GH therapy was defined as the time from the first dose of GH to the date of the last visit during GH therapy, and patient-years of risk was defined as the time from the first dose of GH to December 31, 1997. The incidence of leukemia of patient-years of GH therapy and patient-years of risk in GH-treated patients without risk factors was 3.0/100,000 and 3.9/100,000, respectively, a figure similar to the incidence in the general population aged 0-15 yr. We conclude that the incidence of leukemia in GH-treated patients without risk factors is not greater than that in the general population aged 0-15 yr, and a possible increased occurrence of leukemia with GH treatment appears to be limited to patients with risk factors.

Height responses in complete idiopathic growth hormone deficient children less than three years of age during growth hormone therapy. Executive Committee of the International Cooperative Growth Study in Japan.

To know the effect of the age factor on growth response to growth hormone (GH) therapy in patients less than three years of age with complete idiopathic growth hormone deficiency (GHD). One hundred thirty-seven prepubertal children with complete idiopathic GHD from the database of the International Growth Cooperative Study (ICGS) of Japan for more than two years were analyzed. The patients were divided into four groups according to age at the start of GH therapy; group 1, < or = three years, group 2, three to < or = six years, group 3, six to < or = nine years, group 4, >nine years. In group 1 the mean birth weight SDS of -0.17 was not the lowest. Patients in group 1 had the lowest HSDS (-4.53) at the start of the therapy (P<0.01). During two years of the treatment, the mean HSDS improved from -4.53 to -2.15 with an increase in mean SD score of 2.12 in group 1, but in group 4 it was only 0.92 (P<0.01). In group 1, the mean HSDS declined from -0.17 at birth to -4.53 progressively. Delta height SDS during the first year of treatment in Group 1 was 1.59 SD, which is the highest. The decrease in %overweight during the first year showed that in group 1, the reduction in adiposity was the greatest among the groups (P<0.05). Our data showed that in children with complete GHD less than three years of age, there was severe growth failure, and the onset was not in the prenatal period, but in the postnatal period. Early detection of GHD and therapy with GH is important because normalization of height may be achieved earlier than in older age groups.

Secretory mechanisms of growth hormone (GH)-releasing peptide-, GH-releasing hormone-, and thyrotropin-releasing hormone-induced GH release in patients with acromegaly.

The GH secretory mechanism of GH-releasing hexapeptide (GHRP-6), GHRH, and TRH were studied in vivo and in vitro in seven patients with acromegaly. In an in vivo study, these patients showed clear GH responses to single administration of GHRP (four of four patients), GHRH (seven of seven patients), and TRH (seven of seven patients) and enhanced responses to GHRP plus GHRH (two of four patients) or TRH plus GHRH (six of six patients). In an in vitro dispersed cell study, the majority of patients examined also showed clear GH responses to GHRP (four of four patients), GHRH (six of six patients), and TRH (four of four patients) and an enhanced response to GHRP plus GHRH (three of three patients) or TRH plus GHRH (three of four patients). In one patient (no. 3), GHRP plus forskolin (adenylate cyclase activator), but not GHRP plus phorbol 12-myristate 13-acetate (protein kinase C activator), additively enhanced the GH response. Nordihydroguaiaretic acid (NDGA; inhibitor of arachidonic cascade) inhibited GH release induced by GHRP, TRH, GHRH, TRH plus GHRH, or GHRP plus GHRH, but did not inhibit basal GH secretion. In contrast, NDGA distinctly elevated intracellular cAMP levels in another patient (no. 7) when coadministered with GHRP, GHRH, or GHRP plus GHRH, whereas cAMP levels were not modified by single administration of GHRP and NDGA. The GH response to the combined administration of GHRP and GHRH was synergistic in this patient, but was additive in the other two patients. It is concluded that GHRP, TRH, and GHRH directly stimulate in vivo and in vitro GH release from human somatotropinomas, and GHRP and TRH mainly exert their action through activation of the phosphatidylinositol-protein kinase C pathway, whereas GHRH exerts its action through the adenylate cyclase-protein kinase A pathway. These three agents seem to release GH via the arachidonic cascade.

Plasma GH responses to GHRH, arginine, L-dopa, pyridostigmine, sequential administrations of GHRH and combined administration of PD and GHRH in Turner's syndrome.

To investigate GH secretory capacities in patients with Turner's syndrome, GHRH, arginine, L-dopa and pyridostigmine (PD) were administered singly and GHRH was administered sequentially for 3 days. In addition, plasma GH and TSH responses to GHRH and TRH after pretreatment with PD were analyzed to investigate whether the hypothalamic cholinergic somatostatinergic system functioned normally. The maximal GH responses to GHRH, L-dopa and PD were significantly smaller in Turner's syndrome (no.=14) than in normal short children (NSC, no.=14). However, there was no difference in plasma GH responses to arginine between the two groups. In ten patients with Turner's syndrome, the plasma GH response to GHRH did not improve even after the sequential 3-day administrations. Although plasma GH and TSH responses to GHRH and TRH were significantly enhanced by the pretreatment of PD in NSC (no.=12), these responses were not enhanced in Turner's syndrome. Plasma GH response to GHRH in Turner's syndrome with normal body fat was still significantly lower than in NSC. It is therefore concluded that somatotroph sensitivity to GHRH is decreased in Turner's syndrome and that this may be due to the primary defects of the somatotrophs rather than to the increased body fat. In addition, the network of cholinergic-somatostatinergic systems seemed to be impaired in these patients, while the activity of hypothalamic somatostatin neurons was thought to be maintained.

Registration system for growth hormone (GH) treatment with standardized immunoreactive GH values in Japan.

The Foundation for Growth Science has been controlling the use of GH by its registration system, which includes a scoring system for the eligibility for GH treatment according to the diagnostic criteria for GH deficiency (GHD) established by the Study Group for Hypothalamo-pituitary Disorder of the Ministry of Health and Welfare. Until 1995, 28,876 patients with GHD (19,432 boys and 9,444 girls) had been registered as eligible for GH treatment. The number of patients registered in a year increased gradually till 1990 due to the unlimited hGH supply by recombinant techniques and the change in the criteria for GH treatment and the number registered became stable after 1990. The frequency of GH-treated patients is calculated to be 55.2/100,000 persons (72.2/100,000 in boys and 37.1/100,000 in girls) in patients born between 1960 and 1990. The highest frequency was 148.4/100,000 persons (191.7/ 100,000 boys and 103.7/100,000 girls) in 1981, when 2,278 patients (1,508 boys and 770 girls) were born. Eligibility for GH treatment is assessed according to the scoring system which is basically dependent on peak GH values in provocation tests so that standardization of GH values measured with the various commercial GH kits is required to avoid inequality of patients' access to the treatment. In samples obtained by GRF test in 10 normal volunteers, hGH was measured with seven human GH (hGH) kits at a laboratory center. Since the RIA value has been used historically for the diagnosis of GHD, the mean of two RIA measurements was selected as the basis for the standardization procedure and the linear regression formula was used for each hGH kit. After the freely available supply of hGH obtained by recombinant DNA techniques, the role of the Foundation for Growth Science has changed to avoid hGH abuse. Even with this regulation, the frequency of registered patients may indicate a tendency to GH overuse.

Plasma chromogranin A in pheochromocytoma, primary hyperparathyroidism and pituitary adenoma in comparison with catecholamine, parathyroid hormone and pituitary hormones.

Plasma levels of chromogranin A (CgA) were measured by ELISA in 22 patients with pheochromocytoma (18 non-metastatic, 3 metastatic, and 1 mixed neuroendocrine-neural tumor), 9 patients with primary hyperparathyroidism, and 9 patients with pituitary adenoma. The plasma levels of CgA were compared with norepinephrine, epinephrine, parathyroid hormone and pituitary hormones, i.e., growth hormone and prolactin. In pheochromocytoma, CgA in preoperative plasma of the patients without metastasis was 228 +/- 38 U/L (mean +/- SEM) and significantly higher than healthy controls (30 +/- 11 U/L, n = 40). Plasma CgA was decreased after removal of the tumors (28 +/- 6.0 U/L), except in three patients with metastatic pheochromocytoma and a mixed neuroendocrine neural tumor. The concentration of CgA in the patients with non-metastatic pheochromocytoma was significantly correlated with that of plasma norepinephrine (P < 0.005, r = 0.68) and urinary norepinephrine (P < 0.05, r = 0.65), but not with that of epinephrine. There was an exceptional case in which CgA was extremely high, but the CA level was normal. This tumor was a highly malignant pheochromocytoma with extensive metastases composed of small tumor cells which were occasionally positive for tyrosine hydroxylase immunohistochemically. These cells were considered to be poorly differentiated tumor cells and synthesized a very small amount of norepinephrine. Plasma levels of the patients with primary hyperparathyroidism and the patients with pituitary adenoma were 44 +/- 4 U/L and 48 +/- 8 U/L, respectively. Only one patient with a growth hormone-producing pituitary adenoma had a high level of CgA. Plasma CgA is a useful tumor marker for pheochromocytoma, even for malignant pheochromocytoma without elevated CA level, but not for hyperparathyroidism, or pituitary adenoma.

Growth response to growth hormone therapy in patients with different degrees of growth hormone deficiency.

Growth response to GH therapy in prepubertal patients with idiopathic GH deficiency (GHD) was analyzed in terms of the chronological age at the start of GH treatment and the GH secretory capacity, by using the large database provided by the International Cooperative Growth Study (ICGS) Japan. 1192 patients, aged from 3 to 10 years were divided into three groups with the following maximum GH values in GH stimulation tests: Group A: both < or = 5 ng/ml, group B: both 5-10 ng/ml, group C: one > 10 ng/ml. Analysis of age-related growth response using with delta height SDS (delta height SDS) as a response variable revealed that the group A patients responded better to GH, while there was no differences between the other groups. Simple and multiple regression analysis showed that IGF-I and chronological age (CA) negatively correlated with growth response, and target height SDS-height SDS positively correlated. These three most important predictors accounted for 49% of the variation in the growth response in group A, whereas six variables such as CA, frequency of GH injection, % overweight, GH dose, target height-height SDS, and pretreatment height velocity SDS accounted for only 28% of those in groups of B and C. These results lead us to conclude that growth response to GH is related to the degree of GH impairment with its cut-off level of 5 ng/ml. From these findings it might be suggested that treatment regimen should be tailored to individual requirements according to the degree of GHD.

The inhibitory effects of growth hormone-releasing hormone (GHRH)-antagonist on GHRH, L-dopa, and clonidine-induced GH secretion in normal subjects.

The relative inhibitory potency of GHRH-Antagonist (GHRH-Ant) to GHRH(1-44)NH2 and mechanism of L-dopa- or clonidine-induced GH release were studied in seven normal subjects using GHRH-Ant. One hundred micrograms of GHRH-Ant (iv for 75 min) did not inhibit plasma GH responses to bolus injection of 100 micrograms and 10 micrograms GHRH or simultaneous infusion of 5 micrograms GHRH (iv for 75 min). However, 200 micrograms GHRH-Ant (iv for 75 min) significantly inhibited GH release, which was induced by simultaneous infusion of 5 micrograms GHRH. Although 100 micrograms GHRH-Ant could not significantly inhibit L-dopa-induced GH release, 200 micrograms GHRH-Ant almost completely inhibited the response. Similarly, the same dose of GHRH-Ant markedly inhibited the GH-releasing activity of clonidine. It is concluded that the inhibitory potency of GHRH-Ant on GHRH(1-44)NH2 is relatively weak (about 1/60 in molar base), and that L-dopa- or clonidine-induced GH release seems to be mediated by the release of hypothalamic GHRH.

Plasma GH responses to human GHRH-antagonist in normal subjects.

The effect of GHRH-antagonist [(N-Ac-Tyr1, D-Arg2) GRF-(1-29)-NH2] on plasma GH morning and evening secretion was evaluated in 14 normal subjects (10 males, 4 females, aged 19-25 years). Plasma GH was determined using a high sensitivity IRMA kit (detection limit, 0.006 micrograms/l). After intravenous infusion of GHRH-antagonist (100 micrograms/100 ml saline over 75 min) in the morning, plasma GH remained constant during the 150 min post-infusion (N = 6). In contrast, when GHRH-antagonist was administered in the evening, plasma GH showed a clear and gradual decrease through the initial 90 min and returned to baseline levels at 150 min. Plasma GH values were also significantly lower from 75 min to 135 min when compared to physiological fluctuations in plasma GH (P < 0.05). Other anterior pituitary hormones remained unaffected by GHRH-antagonist. In conclusion, our data suggest that evening basal GH secretion, but not morning GH secretion, is maintained by endogenous GHRH.

Enhanced plasma GH responses to simultaneous administration of TRH and GHRH in patients with primary hypothyroidism.

The mechanism of aberrant GH responses to TRH was indirectly evaluated in 7 patients with primary hypothyroidism. All patients showed GH response to TRH. When TRH was administered together with GHRH, the plasma GH response was much greater than after a single administration of TRH or GHRH (TRH+GHRH vs. TRH or GHRH: max. delta GH, 16.4 +/- 3.2 vs. 5.4 +/- 1.3 or 6.0 +/- 0.8 microgram/L; AUC, 1282.7 +/- 234.7 vs. 384.0 +/- 95.0 or 441.8 +/- 66.2, both P < 0.01). The combined administration of TRH and GHRH caused an additive, but not a synergistic, GH response. In contrast, 8 normal subjects showed neither any plasma GH response to TRH nor enhancement by TRH of GHRH-induced GH response following combined administration. It is concluded that the sites of action of TRH seemed to be different from GHRH in patients with primary hypothyroidism.

The evaluation of hypothalamic somatostatin tone using pyridostigmine and thyrotropin releasing hormone in patients with acromegaly.

To indirectly evaluate the hypothalamic somatostatin (SS) tone in patients with acromegaly, the effects of pyridostigmine (PD), a cholinesterase inhibitor which can inhibit hypothalamic SS secretion, on TRH-induced TSH secretion and the effects of SMS 201-995 on TSH or GH secretion were studied in acromegalic patients (31-69 yr, n = 10), normal young (21-24 yr, n = 7) and normal old male subjects (62-71 yr, n = 7). After pretreatment with PD (60 mg po, -30 min), normal young subjects showed significantly enhanced TSH responses to TRH (500 micrograms i.v., 0 min) compared to single administration of TRH, whereas normal old and acromegalic patients did not show such enhancement. Plasma TSH response to a single administration of TRH in acromegalic patients was significantly lower than that of normal young and old subjects. Although normal young and old subjects showed significantly enhanced GH responses to GHRH (100 micrograms i.v. at 0 min) after the pretreatment with PD (60 mg, -30 min), no such enhancement was observed in acromegalic patients. In contrast, the decrement in plasma TSH after SMS 201-995 administration was similar between normal subjects (5 young 5 old) and 7 acromegalic patients. Further, the maximal plasma GH decrement after administration was significantly greater in acromegalic patients than in the 5 normal young and 5 old subjects p < 0.01). In conclusion, hypothalamic SS tone does not appear to be elevated in acromegalic patients compared to normal young and probably old subjects.

Enhanced GH responses to combined administration of GHRP and GHRH in patients with acromegaly.

Plasma GH responses to GH-releasing peptide (GHRP) were studied in 11 patients with active acromegaly. Ten of these patients showed plasma GH increases to GHRP (100 micrograms i.v.), whereas every patient showed GH increases to TRH (500 micrograms i.v.) and GHRH (100 micrograms i.v.). When GHRP was administered together with GHRH to the 10 GHRP responders, plasma GH responses were synergistically enhanced. However, combined administration of GHRP and TRH did not enhance the response. The GH response pattern and mean time to the peak value were similar to TRH but were different from GHRH. There was no correlation in the maximum GH increment between GHRP and TRH or GHRP and GHRH. It is concluded that GHRP stimulated GH release in the majority of acromegaly patients. The mode of action of GHRP might be similar to TRH and different from GHRH, although the sites of action of GHRP seemed to be different from those of TRH and GHRH.