Effects of exogenous melatonin on pituitary hormones in humans.

The effects of melatonin on physiological function remain unclear, although the therapeutic potential of melatonin is being increasingly recognized. The aim of the present study is to investigate the effects of exogenous melatonin on the spontaneous release of pituitary hormone in humans. A double blind placebo-controlled protocol was designed to examine 12 adult healthy volunteers and 12 sleep disorder patients who have been treating with low doses of melatonin for 1 year. Either exogenous melatonin or placebo of 1 mg was given at 09:00 hours, followed by the collection of blood samples every 20 min for 4 h. Each blood sample was examined for levels of serum melatonin, PRL, LH, FSH, GH and TSH. LH levels were higher in sleep disorder patients compared with the healthy volunteers. In other pituitary hormones, there were no significant difference between healthy adults and sleep disorder patients. In all subjects, PRL levels were stimulated by acute administration of 1 mg of exogenous melatonin, while the levels of other pituitary hormones were not affected. These results suggested that exogenous melatonin can affect the spontaneous release of LH and PRL in humans. In addition, we demonstrated that 1-year oral melatonin treatment did not affect the responses to the acute administration of melatonin.

Prevalence of GH-1 gene deletion in patients with isolated growth hormone deficiency in Japan. GH Gene Study Group.

A multicenter study was carried out to investigate the prevalence of growth hormone (GH-1) gene deletions among patients with isolated growth hormone deficiency (IGHD) and extremely short stature in Japan, using PCR method. Genomic DNA was extracted from the whole blood samples of 48 patients (34 males and 14 females) at 20 hospitals. All the patients fulfilled the inclusion criteria as follows: (1) IGHD patients whose every peak serum GH level in more than two tests <5 ng/ml and (2) pretreatment height < -- 3SD, regardless of family history and facial feature characteristic of GH-1 gene deletion. The subjects were screened for deletions in GH-1 gene, using a PCR method that could identify deletions of 6.7, 7.0 and 7.6 kbp. Three (6.25%) out of 48 subjects were found to have such deletion fragments. The first case was a boy homozygous for deletion of 6.7 kbp fragments. The second case was a girl heterozygous for 6.7 kbp deletion. A direct sequence analysis revealed a 2-bp deletion in exon 3 on the remaining allele that created a stop codon in exon 4. The third case was a boy also heterozygous for 6.7 kbp deletion. By direct sequencing analysis, three point mutations were detected in the promoter region on the opposite allele together with a four-base addition at base 250. One of the mutations was in the area of Pit-1 binding site (at base - 123). The latter two cases apparently represent new types of compound heterozygote of GH-1 gene deletion. Our results suggest that GH-1 gene mutation is not so rare in extremely short IGHD children in Japan.

Deletion of NKX2.1 gene encoding thyroid transcription factor-1 in two siblings with hypothyroidism and respiratory failure.

Thyroid transcription factor-1 encoded by the NKX2.1 gene is a candidate regulator of thyroid and lung morphogenesis and function in humans. We report 2 female siblings with congenital thyroid dysfunction and recurrent acute respiratory distress carrying a heterozygous deletion of chromosome 14q12-13.3, resulting in haploinsufficiency for the NKX2.1 gene. This observation further supports a physiologic role for thyroid transcription factor-1 in early human thyroid and pulmonary function.

Growth hormone advances spermatogenesis in premature rats treated with gonadotropin-releasing hormone agonist.

To clarify the effect of GH on the development of seminiferous tubules in premature male rats, we investigated whether GH accelerates spermatogenesis under the condition of gonadotropin deprivation. Male Wistar rats aged three weeks were divided into three groups and subjected to administration of either long-acting GnRH agonist (GnRHa) or a combination of GnRHa and rat GH, with normal saline solution as control. After the 4-week treatment, sperm density and motility in the right epididymis were measured and seminiferous tubules of right testes were histologically examined. Sperm density and motility were significantly higher in GnRHa+GH-treated rats than in GnRHa-treated rats. In histological examination, the numbers of germ cells in various stages were increased in GnRHa+GH-treated rats compared with GnRHa-treated rats, with the number of mature spermatid being noticeably higher in GnRHa+GH-treated rats. These results suggest that administration of GH decreases loss of germ cells at various stages of spermatogenesis under the condition of gonadotropin withdrawal.

High-dose growth hormone (GH) treatment in prepubertal GH-deficient children.

Two clinical studies were conducted to determine the effect of different doses of growth hormone (GH) on prepubertal growth in GH-deficient boys. In one study, GH doses of 1.0 and 1.5 IU/kg/week (0.33 and 0.5 mg/kg/week) were given to groups of five children and compared with a conventional Japanese dose of 0.5 IU/kg/week (0.17 mg/kg/week) in 15 children. A significant dose-dependent increase in height velocity occurred in the first year of treatment, but differences between doses were not significant thereafter. In a second study, GH was administered to ten boys at a dose of 0.5 IU/kg/week for the first year, 0.75 IU/kg/week for the second year, 1.0 IU/kg/week for the third year and 0.5 IU/kg/week for the fourth and subsequent years (0.17, 0.25, 0.33 and 0.17 mg/kg/week, respectively). During the second and third years of GH treatment, these boys had significantly higher growth rates than controls, who were given GH at 0.5 IU/kg/week (0.17 mg/kg/week) throughout, indicating successful reduction in 'waning' of the treatment effect. At the end of the fourth year, the different protocols from the two studies had both resulted in a greater height SDS than the controls, and did not advance bone maturation. In conclusion, these protocols may be effective in increasing prepubertal height gain in children with GH deficiency.

Isolated partial growth hormone deficient short stature with syringomyelia not associated with birth injury.

A 59-year-old female with 20-year history of slowly progressing muscle atrophy and sensory disturbance of upper extremities showed short stature, scoliosis, hunger type of sensory dissociation of the upper extremities and pyramidal tract sign of the lower extremities. Magnetic resonance imaging (MRI) clarified hypoplasia of the anterior pituitary lobe, Arnold-Chiari malformation and cervical syringomyelia. Insulin and arginine stimulating tests revealed partial type of isolated growth hormone (GH) deficiency but GH gene analysis detected no defects of GH genes. It was considered to be a rare case of non-hereditary hypopituitarism with Chiari malformation and syringomyelia not associated with perinatal injury, namely a midline anomaly syndrome.

Glucoregulatory disorders in school refusal students.

OBJECTIVES: Our previous studies demonstrated autonomic nervous system disorders and cerebral blood hypoperfusion in school refusal students with underlying emotional distress due to fear or anxiety associated with school attendance. Because severe stress is known to affect glucoregulatory metabolism, this study used the oral glucose tolerance test (OGTT) to measure glucose metabolism in school refusal students. DESIGN: A three-hour OGTT was performed. In preparation for the test, students fasted overnight. After a fasting blood sample was drawn, students were given solutions containing a predetermined amount of glucose based on their body weight (1.75 g/kg to a maximum 75 g). After glucose ingestion, blood samples were drawn at 30, 60, 90, 120, 150, and 180 mm to measure blood glucose (BG), immunoreactive insulin (IRI), pancreatic glucagon (IRG) and growth hormone (GH) levels. BG levels, IRI response, cumulative BG (sigma BG), cumulative IRI (sigma IRI), insulin/glucose ratio (delta IRI/delta BG), and insulinogenic index (sigma IRI/sigma BG) were then compared to previously reported normal control data. As an index of emotional difficulties, the self-rating depressive scale (SDS) was carried out. PATIENTS: Eighty-one school refusal students (40 males and 41 females), 11-19 years of age (14.8 +/- 2.1), were studied. Their school refusal periods ranged from one month to eight years. All students were within -15 to +20% (-0.04 +/- 8.6) of ideal body weight. MEASUREMENTS: BG levels were determined using a glucose oxidase reaction method. Serum hormones were measured by radioimmunoassay. RESULTS: BG levels at all OGTT time intervals and sigma BG were significantly higher in school refusal students than the normal control data (sigma BG: 39.5 +/- 4.4 vs 33.3 +/- 3.4 mmol/l P < 0.001). Although the insulin response was abnormally low relative to the prevailing hyperglycaemia (sigma IRI/ sigma BG: subjects vs control = 232 +/- 129 vs 375 +/- 271, P < 0.01), normal beta cell secretory ability was speculated (sigma IRI: subjects vs controls = 2805 +/- 1274 vs 2523 +/- 1219 pmol/l). This suggests a relative suppression of insulin secretion. A paradoxical increase of GH was observed in 19 students after glucose ingestion. CONCLUSIONS: Glucoregulatory disorders observed in school refusal students may be caused by emotional distress. Multiple factors including autonomic nervous system disorders, derangement of neuropeptides in the hypothalamus, and hormonal imbalances may also affect glucoregulatory metabolism, predisposing these students to hyperglycaemia. We speculate that the glucoregulatory system compensates for decreased blood flow to the brain by increasing blood glucose concentrations, thereby providing sufficient glucose as the primary energy source used during normal brain metabolism.

The growth hormone receptor gene mutation of a Japanese patient with Laron syndrome.

Deletions and point mutations of the growth hormone (GH) receptor gene (GHR) have been identified in patients with Laron syndrome. We report the first detection of the GHR mutation among Japanese patients with Laron syndrome. Using the Japanese female patient's genomic DNA as a template, all exons and flanking portions of introns of GHR were amplified by polymerase chain reaction (PCR). Sequencing of the PCR products showed that the patient was homozygous for a G to A substitution at the first position of intron 4. This substitution was same as that detected in a Spanish patient and a north European patient. The base change occurred at the 5' splice consensus sequence of intron 4, resulting in the abolition of a BanI restriction site. Since this substitution was not detected by a BanI restriction analysis in 85 control individuals, it is more likely a disease-related splice mutation than a polymorphism. The mutation in our patient was predicted to destroy the original 5' splice site of intron 4 of GHR and to produce a new cryptic splice site, leading to abnormal mRNA processing and a lack of GH binding activity of GH-binding protein (GHBP).

A case of isolated growth hormone (GH) deficiency with compound heterozygous abnormality at the GH-1 gene locus.

We report a Japanese boy with IGHD who is a compound heterozygote at the GH-1 gene locus. The patient and his mother were heterozygous for a 6.7 kb deletion of the GH-1 gene. A T-->C transition at position -123, an A-->G transition at position -6 and an A-->T transition at position -1 in the GH-1 promoter region and the addition of AGAA at base 250 in intron I were observed in one allele of the patient and his father. These results demonstrate that familial IGHD is a heterogeneous disease that perturbs different steps in the expression of the GH-1 gene.

Recurrent dislocation of the patella in Turner's syndrome.

We report two patients with Turner's syndrome who presented with recurrent dislocation of the patella (RDP). Both had a positive family history of patellar instability. The association of RDP with Turner's syndrome has not been reported previously. A subsequent study in 14 patients with Turner's syndrome demonstrated radiographical patellofemoral incongruency in eight patients, but no clinical manifestation of patellar instability. Although the present study suggests a possible link between Turner's syndrome and patellofemoral incongruency, it could not ascertain whether or not Turner's syndrome predisposes a patient to RDP.

Pulsatile LH-RH administration induces puberty in hypogonadotropic GH-deficient patients.

Three growth hormone (GH) deficient males with hypogonadotropic hypogonadism were treated with pulsatile luteinizing hormone-releasing hormone (LH-RH) administration. In two of them, the GH deficiency was idiopathic, but in the other it was secondary, caused by suprasellar germinoma. In response to LH-RH therapy, the serum testosterone (T), testicular volume, and body height increased in all three patients, and normal serum T levels and spermatogenesis were achieved in two patients. Gonadotropin responses to an LH-RH test preceding therapy did not seem to be an accurate predictor of the success of LH-RH therapy. We conclude that GH-deficient patients with hypogonadotropic hypogonadism can be expected to achieve normal pubertal development and spermatogenesis with pulsatile LH-RH administration.

The majority of the marker chromosomes in Japanese patients with stigmata of Turner syndrome are derived from Y chromosomes.

DNA analyses of 41 individuals with stigmata of Turner syndrome and a 45,X/46,X+mar or 46,X+mar karyotype were carried out. Southern-blot analysis employing 17 Y-specific probes were used to determine whether the marker chromosome was Y-chromosomal in origin. Of the 41 DNA samples from these patients, 23 contained detectable Y-chromosomal DNA. Points of chromosome breakage were distributed over the entire length of the Y long arm. Three individuals, who carry different portions of the Y chromosome, had developed gonadoblastoma. GBY (the gonadoblastoma locus on the Y chromosome) is mapped proximal to DYS132, midway between the 13 Yq loci that we have studied. We also used a polymerase chain reaction technique that could detect 7 loci over the length of the Y chromosome. This technique may be useful for the rapid assessment of marker chromosomes, especially for evaluating the risk of gonadoblastoma.

A child with pituitary gigantism and precocious adrenarche: does GH and/or PRL advance the onset of adrenarche?

We describe a female child with pituitary gigantism and precocious adrenarche. From two years of age she showed unusual overgrowth, and at 5 years old she was 133.5 cm (+ 5.5 SD) tall and weighed 40.5 kg. Her precocious manifestations were public hair, acne vulgaris, hirsutism, and advanced bone age. Endocrinological examination revealed markedly increased serum growth hormone (GH) and prolactin (PRL), which responded paradoxically to a TRH test. In addition, the concentrations of serum dehydroepiandrosterone (DHA) and its sulfate (DHAS) were increased to adult levels, moving in accordance with changes in ACTH, which suggested that these androgens were secreted from the adrenal glands functionally. These androgens seemed to be responsible for her partial precocity. Prior reports have suggested that GH and/or PRL overproduction might have played a role in the induction of adrenarche. Also, in previous reports of 9 gigantism patients under 10 years old, the manifestation of precocious adrenarche was suggested in 8. Further investigation of the influence of GH and PRL on adrenal androgen production in children with pituitary gigantism is required. On the other hand, in short children with normal GH secretion, attention should be paid to whether or not the GH therapy in early childhood induces precocious adrenarche.

Endocrinological evaluation of GH deficient patient with acromegaloidism showing excessive growth.

In this report we describe the first case of a girl with acromegaloidism in Japan. She had large and coarse facial features with acral enlargement accompanying height overgrowth; these resemble the manifestations of acromegaly and gigantism due to growth hormone (GH) overproduction. However, pituitary function studies revealed a dysfunction of her GH secretion. Moreover, markedly decreased serum somatomedin C (SM-C) levels also indicated impairment of GH secretion. Therefore, GH and SM-C cannot have been responsible for promoting somatic growth. However, serum alkaline-phosphatase (Al-P) and osteocalcin, were increased, indicating that stimulation of bone metabolism was increased without GH and SM-C effects. The patient is a typical case showing growth without GH, and these data suggest the existence of an unidentified growth promoting factor that is independent of GH and SM-C.

Prenatal DNA analysis in four embryos/fetuses at risk of 21-hydroxylase deficiency.

Prenatal diagnosis of 21-hydroxylase deficiency (21-OHD) in two unrelated embryos and two fetuses was attempted with the Southern hybridization method using the 21-hydroxylase (21-OHase) complementary DNA as a probe. The two embryos whose genomic DNA was extracted from their chorionic villi both had four TaqI fragments (3.7 kb, 3.2 kb, 2.4 kb and 2.3 kb) identical to those of their respective parents and normal controls, while the DNA from each proband of these two families lacked with the 3.7 kb and the 2.3 kb fragments corresponding to the functional 21-OHase gene (21-OHase B gene). These findings indicated that none of the embryos examined were deletion homozygotes for the 21-OHase B gene. In the two fetuses, only amniotic fluid cells were available for prenatal diagnosis. The results of Southern hybridization analysis were uninformative since all family members, including the probands and fetuses, had all four TaqI fragments. Linkage studies between 21-OHD and human leukocyte antigen (HLA) haplotypes and those between the disease and restriction fragment length polymorphisms of the 4th complement gene revealed that the fetus of one family was normal. The other fetus could not be diagnosed because a recombination between the class I HLA and the 21-OHD loci had occurred in this family.

Treatment of hypopituitarism with recombinant somatropin for 1 year.

Twenty-five hypopituitary patients were treated with recombinant somatropin for 1 year at a dosage of 0.5 IU/kg/week. In previously untreated patients (n = 16), heights increased by between 4.5 and 10.2 cm, with a mean height velocity of 7.7 +/- 1.8 cm/year (mean +/- SD). In previously treated patients (n = 9), heights increased by between 3.9 and 7.6 cm, with a mean height velocity of 5.8 +/- 1.0 cm/year, similar to that observed during previous treatment with pituitary GH. Anti-GH antibodies were observed in two patients at a low titre. The antibodies disappeared in one patient during the treatment. These data indicate that recombinant somatropin has a growth promoting effect and low immunogenicity.

Treatment of pituitary dwarfism with authentic recombinant human growth hormone (SM-9500).

Twenty-one patients with pituitary dwarfism were treated with methionine-free hGH (r-hGH) for 6 months with a dosage of 0.5 IU/kg/week. The height of newly treated patients (N = 14), increased from 2.4 to 5.0 cm during treatment, which corresponded to from 4.8 to 10.0 cm with a mean of 8.1 +/- 0.5 cm/year. In switched patients (N = 7), height increased from 2.2 to 3.8 cm during the treatment, which corresponded to 4.4-7.6 cm with a mean of 6.1 +/- 0.5 cm/year, which was similar to that observed in previous treatment with pituitary extracted hGH (p-hGH). Anti-hGH antibody was observed in two patients (9.5%) at the end of 6 months of treatment with a titer of 10. These data indicate that r-hGH has a growth promoting effect and low antigenicity.

Clinical trial with authentic recombinant somatropin in Japan.

Recombinant somatropin, produced by recombinant DNA technology, was administered by injection in daily doses of 8 IU to six healthy young volunteers. Daily injection for 4 days did not cause any significant change in the results of physical examination, blood count or urinalysis. Non-esterified fatty acid levels increased significantly from 0.45 +/- 0.16 to 1.08 +/- 0.12 mEq/litre (mean +/- SEM) at 4 hours after the first injection (p less than 0.001). Plasma IGF-1 levels increased from 0.80 +/- 0.14 units/ml to 1.72 +/- 0.50, 3.22 +/- 1.02, 3.17 +/- 1.20 and 3.63 +/- 0.78 units/ml at 24 hours after each daily injection for 4 days (p less than 0.001). Plasma hGH reached peak levels at 3 hours after intramuscular injection of recombinant somatropin, 4 IU, and this peak value was 57.3 +/- 2.8 ng/ml. A total of 21 patients with pituitary dwarfism were also treated with recombinant somatropin for 6 months at a dose of 0.5 IU/kg/week. Their heights increased by 2.2-5.0 cm during the 6 months of treatment, which was calculated to be equivalent to 4.4-10.0 cm/year with a mean growth rate of 7.4 +/- 0.4 cm/year. Anti-hGH antibody with a titre of 10 was observed in two patients at the end of 6 months of treatment.