Development of a microplate duplex immunoassay to simplify detection of growth hormone doping: Proof of concept

The World Anti-Doping Agency (WADA) prohibits athletes from using recombinant growth hormone (GH). The validated method used in antidoping laboratories for the direct detection of exogenous GH in serum requires two immunoluminometric assays (ILMAs): The first mainly measures the concentration of the full-length (22 kDa) form of GH (recGH), and the second measures concentrations of multiple GH fragments produced by the pituitary gland (22 kDa, 20 kDa and other forms) (pitGH). The tube-by-tube analysis is laborious. A recent development opened new possibilities to simplify the detection of recGH in serum: multiplexed immunoassays that detect multiple targets in a single well of a 96-well plate using an ELISA-like procedure with high sensitivity. Our aim was to evaluate this technology by developing a customized assay for GH detection. One pair of antibodies with specificities similar to those of the recGH assay and one pair of antibodies compatible with pitGH detection were selected for a single duplex assay. Forty-eight serum samples (negative athlete samples and positive samples following GH administration) were analyzed using the two methods. The microplate duplex assay discriminated between the negative athlete samples and the positive controls, although the rec/pit ratios from the duplex assay were lower than those obtained with the ILMAs. This new assay would offer a modern alternative to ILMAs, with fewer analytical steps and a smaller sample volume. However, an adaptation of the decision limits seems mandatory.     

Cognitive, emotional, physical and social effects of growth hormone treatment in adults with Prader–Willi syndrome

Background Prader–Willi syndrome (PWS) is a multisystem genetic disorder characterized by short stature, muscular hypotonia, hyperphagia, obesity, maladaptive behaviour, hypogonadism and partial growth hormone (GH) deficiency (GHD). Severe GHD of other aetiologies has been shown to affect mood and quality of life negatively, and there are reports of improvements with GH replacement. We have studied cognitive, emotional, physical and social parameters in PWS adults at baseline, during and after GH treatment. Patients and methods Nineteen patients, 9 females and 10 males, median age 25 years, mean BMI 35 kg/m² participated in this study. Approximately half of the group had GHD. All patients fulfilled the clinical criteria for PWS and 13 had a positive genotype. The patients were randomized to 6 months of treatment with either GH [1.6 IU/day (0.53 mg/day)] or placebo, followed by 12 months of active GH treatment. Treatment was then stopped, and the patients were followed for an additional period of 6 months. A test battery for general cognitive evaluation and a computer-based measurement of reaction time, motor speed and fluency were employed at baseline, after 6 months and at the end of GH treatment. At the same time intervals, a self-evaluation questionnaire was answered at the end of each test session. Other questionnaires reflecting the patients’ cognitive, emotional, physical and social status were answered by relatives/caretakers at baseline and at 3 and 6 months following cessation of GH treatment. Results Baseline cognitive level was estimated to be moderately to mildly impaired; IQ range was 40–90. The results from some of the cognitive and the motor performance tests improved significantly after 6 and 18 months of GH treatment. According to the questionnaires, both the patients and the relatives/caretakers evaluated physical status rather negatively at baseline, but still, impairments in both physical and social status and overall functioning were observed when GH treatment was discontinued. The self-evaluation did not change in any aspect during GH treatment. Conclusions In this pilot study of an adult PWS cohort, we were able to document beneficial effects in mental speed and flexibility and in motor performance during GH treatment. Impairment was seen in physical and social status as well as overall functioning, when GH treatment stopped. Studies of larger cohorts are needed to further elucidate the role of GH treatment in this group of patients.     

Membrane-impermeable estrogen is involved in regulation of calbindin-D9k expression via non-genomic pathways in a rat pituitary cell line,GH3 cells

Estrogen (E2) has been shown to regulate various functions for many pituitary hormones. Recently, the potential roles of non-genomic pathways in E2-induced actions have been proposed in the previous studies, however, the effects of E2 remain to be elucidated in regard to non-genomic induction of cytosolic protein calbindin-D9k (CaBP-9k). To gain a better understanding of the molecular events underlying E2-induced expression of CaBP-9k, rat pituitary tumor cells (GH3 cells) were treated with E-BSA (membrane impermeable E2-conjugated with BSA). Non-genomic induction of CaBP-9k by E-BSA was determined using RT-PCR and western blot analysis. The significant increase in CaBP-9k mRNA level was observed as early as 15 min following treatment with a high concentration of E-BSA (10⁻⁶ M), whereas rapid and significant induction of CaBP-9k protein was noted at 5, 15 and 30 min after E-BSA exposure (p < 0.05). In order to determine the potential involvement of different signaling pathways, several inhibitors were employed, i.e., ICI 182,780 for the estrogen receptor (ER) pathway, pertussis toxin (PTX) for the G-protein-coupled signaling pathway, U0126 (U) for the ERK (extracellular regulated kinase) and wortmannin (W) for the Akt (protein kinase B). Co-treatment with ICI 182, 780 and PTX reversed an E-BSA-induced increase in CaBP-9k mRNA and protein. Although neither U nor W alone attenuated E-BSA-induced effects, these inhibitors together abolished E-BSA-induced CaBP-9k expression, suggesting their involvement in its regulation. Taken together, these results demonstrate the involvement of various signaling pathways in E2-induced regulation of CaBP-9k. In addition, ER and G-protein-coupled signaling pathways may play central roles in the non-genomic activities of E2 and that downstream signaling via ERK and Akt are required to evoke ER-mediated induction of CaBP-9k in vitro.     

Effects of subcutaneous sumatriptan on plasma growth hormone concentrations in migraine patients

The purpose of this study was to assess the sensitivity of 5-HT1D receptors in migraine using sumatriptan as a pharmacological probe. The drug stimulates the release of growth hormone (GH) and this effect may be used to explore the function of cerebral serotonergic systems in vivo. We administered sumatriptan and placebo to 15 migraineurs and to 10 controls. Blood samples were collected -15, 0, 15, 30, 45, 60 and 90 min after injection. Placebo had no effect on hormone concentrations. Sumatriptan induced a significant (P<0.01) increase in GH concentrations both in migraine patients and healthy controls. The GH increase was not significantly different in the two groups. Our results suggest that cerebral serotonergic functions mediated by 5-HT1D receptors are not altered in migraine. Sumatriptan overuse could lead to adverse effects mediated by its neuroendocrine activity.     

Two siblings with isolated GH deficiency due to loss-of-function mutation in the GHRHR gene: successful treatment with growth hormone despite late admission and severe growth retardation

Patients with growth hormone releasing hormone receptor (GHRHR) mutations exhibit pronounced dwarfism and are phenotypically and biochemically indistinguishable from other forms of isolated growth hormone deficiency (IGHD). We presented here two siblings with clinical findings of IGHD due to a nonsense mutation in the GHRHR gene who reached their target height in spite of late GH treatment. Two female siblings were admitted to our clinic with severe short stature at the age of 13.8 (patient 1) and 14.8 years (patient 2). On admission, height in patient 1 was 107 cm (-8.6 SD) and 117 cm (-6.7 SD) in patient 2. Bone age was delayed in both patients (6 years and 9 years). Clinical and biochemical analyses revealed a diagnosis of complete IGHD (peak GH levels on stimulation test was 0.06 ng/mL in patient 1 and 0.16 ng/mL in patient 2). Patients were given recombinant human GH treatment. Genetic analysis of the GH and GHRHR genes revealed that both patientscarried the GHRHR gene mutation p.Glu72X (c.214 G>T) in exon 3 in homozygous (or hemizygous) state. After seven years of GH treatment, the patients reached a final height appropriate for their target height. Final height was 151 cm (-1.5 SD) in patient 1 and 153 cm (-1.2 SD) in patient 2. In conclusion, genetic analysis is indicated in IGHD patients with severe growth failure and a positive family history. In spite of the very late diagnosis in these two patients who presented with severe growth deficit due to homozygous loss-of-function mutations in GHRHR, their final heights reached the target height.     

矮小儿童生长激素检测的临床意义分析

目的评价矮小儿童血清中的生长激素（GH）水平,了解生长激素缺乏（GHD）与特发性矮小（ISS）患儿生长激素激发试验后GH峰值分布的差异及临床意义。方法采用化学发光法对80例矮小儿童进行生长激素联合激发试验（可乐定,精氨酸）,检测用药前及用药后30、60、90、120min时的血清GH水平。以测得的GH最高值为峰值,峰值≥10μg/L为GH不缺乏,10μg/L〉峰值≥5μg/L为GH部分缺乏;峰值〈5μg/L为完全缺乏。结果 80例患儿中,GHD占45.0%,其中部分GHD占31.25%;GHD与ISS患儿无性别差异（P〉0.05）。激发后GH峰值出现的时间以0～120分钟为主,占总数的81.25%,激发试验的GH峰值出现时间ISS患儿与GHD患儿无差异（P〉0.05）。结论矮小儿童中由GHD缺乏造成的矮小所占比例较高,且以部分缺乏为主,ISS也是引起矮小的主要原因。生长激素联合激发试验GHD和ISS患儿GH峰值出现的时间段无差异。     

Outcome of surgical intrasellar growth hormone tumor performed by a pituitary specialist surgeon in a developing country

Background

Acromegaly is an excessive GH secretion, which in most cases, is caused by a pituitary GH-secreting adenoma. Traditional treatment of acromegaly consists of surgery, drug therapy, and eventually radiotherapy. The aim of this retrospective study is to evaluate the results of transsphenoidal endoscopic surgery in a group of patients with intrasellar GH adenoma who were operated by a pituitary specialist surgeon. We shall then argue about the economical advantages, for the NHS of a developing country, between surgical and medical treatment.

Methods

We have analyzed data from 33 patients with intrasellar GH tumor who had been referred to the neuroendocrine department of the HGF, Brazil. The patients underwent a transsphenoidal endoscopic adenomectomy for acromegaly between 2000 and 2005. Their ages were between 20 and 67 years (mean, 44 years) at the moment of surgery. No cavernous sinus invasion was present. Follow-up was a median of 2 years (range, 12 months-6 years).

Results

All 33 patients had intrasellar adenoma, 84.84% of patients achieved remission by surgery. One patient was operated twice and reached hormonal normalization. Five patients still had the disease and refused a second surgery. A treatment with octreotide was started for these 5 patients and resulted in an adequate control of GH and IGF-1 levels. No patients had radiotherapy.

Conclusion

Our patients, with intrasellar GH tumor, operated by a pituitary specialist neurosurgeon had remission rates approaching those obtained by most specialized neurosurgical centers worldwide. For equal results, our study shows that the surgical treatment is the best issue for the patient and for the NHS.     

Aging and the growth hormone/insulin like growth factor-I axis

Growth hormone release and IGF-I synthesis decrease with increasing age. The regulation of the GH/IGF-I system is dependant on the integrity of the hypothalamus, pituitary and liver. During aging there are several changes which contribute to the decline in GH/IGF-I including changes in signal to the somatotrophs from growth hormone releasing hormone, somatostatin and other factors such as body composition, exercise, diet and sleep. All of these factors are discussed in detail within this review. The phenotypic similarities between aging and adult growth hormone deficiency syndrome combined with this decrease in GH/IGF-I with aging have prompted the question whether aging is a GH deficient state. The advent of recombinant growth hormone has led to a number of studies treating elderly patients with GH alone or in combination with sex steroids or exercise. The results of these studies would not back up the use of GH in elderly non-hypopituitary patients as they did not show efficacy, showed high rates of adverse events and there is also some evidence associating GH/IGF-I and risk of neoplasia. If GH therapy is to be used in this cohort of patients further long term efficacy and safety studies are required.     

Acromegaly caused by growth hormone-releasing hormone-producing tumors: long-term observational studies in three patients

We report on three newly diagnosed patients with extracranial ectopic GHRH-associated acromegaly with long-term follow-up after surgery of the primary tumor. One patient with a pancreatic tumor and two parathyroid adenomas was the index case of a large kindred of MEN-I syndrome. The other two patients had a large bronchial carcinoid. The first patient is still in remission now almost 22 years after surgery. In the two other patients GHRH did not normalize completely after surgery and they are now treated with slow-release octreotide. IGF-I normalized in all patients. During medical treatment basal GH secretion remained (slightly) elevated and secretory regularity was decreased in 24 h blood sampling studies. We did not observe development of tachyphylaxis towards the drug or radiological evidence of (growing) metastases. We propose life-long suppressive therapy with somatostatin analogs in cases with persisting elevated serum GHRH concentrations after removal of the primary tumor. Independent parameters of residual disease are elevated basal (nonpulsatile) GH secretion and decreased GH secretory regularity.