Immunofunctional assay of human growth hormone (hGH) in serum: A possible consensus for quantitative hGH measurement

Confirmation of the diagnosis of GH deficiency in adults and children involves provocative testing for human (h) GH. Different commercially available immunoassays yield largely discrepant results in the measurement of GH levels in human serum. These discrepancies result in doubtful relevance of cut-off levels proposed for GH provocative testing. We have developed an immunofunctional assay method that allows quantitation of only those GH forms in circulation that possess both binding sites of the hormone for its receptor and thus can initiate a biological signal in target cells. An anti-hGH monoclonal antibody recognizing binding site 2 of hGH is immobilized and used to capture hGH from the serum sample. Biotin-labeled recombinant GH-binding protein in a second incubation step forms a complex with those hGH molecular isoforms that have both binding sites for the receptor. The signal is detected after a short third incubation step with labeled streptavidin. The assay is sensitive (detection range, 0.1-100 micrograms/L) and has average inter- and intraassay precisions of 10.3% and 7.3% respectively. Endogenous GH-binding protein does not interfere with the hGH result; placental lactogen slows no detectable cross-reaction in this immunofunctional assay. The degree of immunofunctionally active hGH forms in serum samples, calculated by comparison of immunofunctional assay and RIA results, varied between 52-93%. We propose this immunofunctional assay for GH measurement as a new reference method for hGH quantitation in serum. The immunofunction assay translates only hGH forms into an assay signal that are capable of dimerizing GH receptors and, thus, of initiating a biological effect in target cells

Effect of Growth Hormone (hGH) Replacement Therapy on Physical Work Capacity and Cardiac and Pulmonary Function in Patients with hGH Deficiency Acquired in Adulthood.

The effects of 6 months of replacement therapy with recombinant human GH (hGH) on physical work capacity and cardiac structure and function were investigated in 20 patients with hGH deficiency of adult onset in a double blind, placebo-controlled trial. The GH dose of 12.5 micrograms/kg BW was self-administered daily sc. Oxygen consumption (VO2), CO2 production, and ventilatory volumes were measured during exercise on a bicycle spiroergometer. M-Mode echocardiography was performed using standard techniques. The VO2 max data, expressed per kg BW (mL/min.kg BW) showed a significant increase from 23.2 +/- 2.4 to 30.0 +/- 2.3 (P < 0.01) in the hGH-treated group, whereas the VO2 max data, expressed per lean body mass (milliliters per min/kg lean body mass) did not change significantly in either group. Maximal O2 pulse (milliliters per beat) increased significantly from 15.2 +/- 5.6 to 19.6 +/- 3.3 mL/beat (P < 0.01), but remained constant in the placebo group. The maximal power output (watts +/- SE) increased significantly (P < 0.01) from 192.5 +/- 13.5 to 227.5 +/- 11.5 in the hGH-treated group, but remained constant in the placebo group. Cardiac structure (left ventricular posterior wall, interventricular septum thickness, left ventricular mass, left ventricular end-systolic dimension, and left ventricular end-diastolic dimension) as well as echocardiographically assessed cardiac function did not change significantly after 6 months of treatment in either group. We conclude that hGH replacement in hGH-deficient adults improves oxygen uptake and exercise capacity. These improvements in pulmonary parameters might be due to an increase in respiratory muscle strength and partly to the changes in muscle volume per se observed during hGH replacement therapy. Furthermore, an increased cardiac output might contribute to the improvement in exercise performance during hGH treatment. According to our data, hGH replacement therapy leads to an improvement of exercise capacity and maximal oxygen uptake, but has no significant effect on cardiac structure

Growth Hormone (GH)-Releasing Peptide Stimulation of GH Release from Human Somatotroph Adenoma Cells: Interaction with GH-Releasing Hormone, Thyrotropin- Releasing Hormone, and Octreotide.

The synthetic hexapeptide GH-releasing peptide (GHRP; His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) specifically stimulates GH secretion in humans in vivo and in animals in vitro and in vivo via a still unknown receptor and mechanism. To determine the effect of GHRP on human somatotroph cells in vitro, we stimulated cell cultures derived from 12 different human somatotroph adenomas with GHRP alone and in combination with GH-releasing hormone (GHRH), TRH, and the somatostatin analog octreotide. GH secretion of all 12 adenoma cultures could be stimulated with GHRP, whereas GHRH was active only in 6 adenoma cultures. In GHRH-responsive cell cultures, simultaneous application of GHRH and GHRP had an additive effect on GH secretion. TRH stimulated GH release in 4 of 7 adenoma cultures; in TRH-responsive cell cultures there was also an additive effect of GHRP and TRH on GH secretion. In 5 of 9 adenoma cultures investigated, octreotide inhibited basal GH secretion. In these cell cultures, GHRP-induced GH release was suppressed by octreotide. In 5 of 5 cases, the protein kinase-C inhibitor phloretin partly inhibited GHRP-stimulated GH release, but not basal GH secretion. In summary, GH secretion was stimulated by GHRP in all somatotroph adenomas investigated, indicating that its unknown receptor and signaling pathway are expressed more consistently in somatotroph adenoma cells than those for GHRH, TRH, and somatostatin. Our data give further evidence that GHRP-stimulated GH secretion is mediated by a receptor different from that for GHRH or TRH, respectively, and that protein kinase-C is involved in the signal transduction pathway. Because human somatotroph adenoma cell cultures respond differently to various neuropeptides (GHRH, TRH, somatostatin, and others), they provide a model for further investigation of the mechanism of action of GHRP-induced GH secretion

Cortisol and 17-hydroxyprogesterone levels in saliva of healthy neonates - Normative data and relation to body mass index, arterial cord blood pH and time of sampling after birth

The measurement of cortisol and 17-hydroxyprogesterone (17-OHP) in saliva has become a reliable tool for both the scientist and the clinician for studying adrenal cortical function in the adult and the older child. We have now established in parallel normative data for salivary cortisol and 17-OHP levels in healthy neonates. We have asked whether or not there is a circadian rhythm of cortisol and 17-OHP saliva levels in neonates. Furthermore, we have asked whether salivary hormone levels correlated with auxologic and clinical data and time of sampling. Cortisol and 17-OHP levels in saliva samples from 119 healthy neonates (55 girls, 64 boys) were measured using in-house time-resolved fluorescent immunoassays. Saliva samples were obtained using a saliva collecting tube three times a day on the first or second day of life. Gender and gestational age did not influence salivary cortisol and 17-OHP levels. No significant circadian rhythm of salivary hormone levels was detected in this group of newborns. However, body mass index, arterial cord blood pH and time of saliva sampling significantly influenced salivary hormone levels. In conclusion, measurement of cortisol and 17-OHP in saliva is feasible in healthy neonates. The existence of normative data forms the basis for future studies on pathophysiologic states in the newborn period. Copyright (C) 2000 S. Karger AG, Basel

Regulation of growth-hormone-receptor gene expression by growth hormone and pegvisomant in human mesangial cells

Regulation of growth-hormone-receptor gene expression by growth hormone and pegvisomant in human mesangial cells.BackgroundMice transgenic for growth hormone develop mesangial proliferation, glomerular hypertrophy, and progressive glomerular sclerosis suggesting that the growth hormone–insulin-like growth factor I (IGF-I) pathway plays an important role. Therefore, we studied the impact of variable concentrations of 22 kD, 20 kD growth hormone, as well as of the growth hormone receptor antagonist pegvisomant (B2036-PEG), on both the growth hormone receptor (GHR/GHBP) gene expression and growth hormone binding protein (GHBP) formation in a human glomerular mesangial cell line. Further, the impact on collagen, IGF-I and IGF binding protein-1 (IGFBP-1) formation was studied.MethodsIn order to assess transcription, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used.ResultsPhysiologic doses of 22 kD or 20 kD growth hormone caused a dose-dependent and significant (P < 0.01) up-regulation of GHR/GHBP gene transcription, whereas supraphysiologic doses (50 and 500ng/mL) resulted in down-regulation (P < 0.001). Whenever pegvisomant was used, there was no increase in GHR/GHBP expression. These data were confirmed using run-on experiments. Further, the assessment of GHBP presented a constant, dose-dependent increase, which was completely abolished in the experiments where pegvisomant was used.ConclusionWe present data showing that growth hormone has a direct impact on GHR/GHPB gene transcription and that pegvisomant is a potent growth hormone receptor antagonist in human mesangial cells. In addition, although the GHR/GHBP gene transcription is down-regulated by supraphysiologic growth hormone concentrations, this effect was not found when GHBP levels were measured. This finding may reflect a self-inhibitory effect of growth hormone on the level of GHR/GHBP gene transcription, which does not involve the regulation of the shedding of GHBP and may, therefore, be of physiologic interest

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P

Selenoprotein P (SELENOP) is a liver-derived transporter of selenium (Se) in blood, and a meaningful biomarker of Se status. Se is an essential trace element for the biosynthesis of enzymatically-active selenoproteins, protecting the organism from oxidative damage. The usage of uncalibrated assays hinders the comparability of SELENOP concentrations and their pathophysiological interpretation across different clinical studies. On this account, we established a new sandwich SELENOP-ELISA and calibrated against a standard reference material (SRM1950). The ELISA displays a wide working range (11.6–538.4 µg/L), high accuracy (2.9%) and good precision (9.3%). To verify whether SELENOP correlates to total Se and to SELENOP-bound Se, serum samples from healthy subjects and age-selected participants from the Berlin Aging Study II were analyzed by SELENOP-ELISA and Se quantification. SELENOP was affinity-purified and its Se content was determined from a subset of samples. There was a high correlation of total Se and SELENOP concentrations in young and elderly men, and in elderly women, but not in young women, indicating a specific sexual dimorphism in these biomarkers of Se status in young subjects. The Se content of isolated SELENOP was independent of sex and age (mean±SD: 5.4±0.5). By using this calibrated SELENOP-ELISA, prior reports on pathological SELENOP concentrations in diabetes and obesity are challenged as the reported values are outside reasonable limits. Biomarkers of Se status in clinical research need to be measured by validated assays in order to avoid erroneous data and incorrect interpretations, especially when analyzing young women. The Se content of circulating SELENOP differs between individuals and may provide some important diagnostic information on Se metabolism and status

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P

Selenoprotein P (SELENOP) is a liver-derived transporter of selenium (Se) in blood, and a meaningful biomarker of Se status. Se is an essential trace element for the biosynthesis of enzymatically-active selenoproteins, protecting the organism from oxidative damage. The usage of uncalibrated assays hinders the comparability of SELENOP concentrations and their pathophysiological interpretation across different clinical studies. On this account, we established a new sandwich SELENOP-ELISA and calibrated against a standard reference material (SRM1950). The ELISA displays a wide working range (11.6–538.4 µg/L), high accuracy (2.9%) and good precision (9.3%). To verify whether SELENOP correlates to total Se and to SELENOP-bound Se, serum samples from healthy subjects and age-selected participants from the Berlin Aging Study II were analyzed by SELENOP-ELISA and Se quantification. SELENOP was affinity-purified and its Se content was determined from a subset of samples. There was a high correlation of total Se and SELENOP concentrations in young and elderly men, and in elderly women, but not in young women, indicating a specific sexual dimorphism in these biomarkers of Se status in young subjects. The Se content of isolated SELENOP was independent of sex and age (mean±SD: 5.4±0.5). By using this calibrated SELENOP-ELISA, prior reports on pathological SELENOP concentrations in diabetes and obesity are challenged as the reported values are outside reasonable limits. Biomarkers of Se status in clinical research need to be measured by validated assays in order to avoid erroneous data and incorrect interpretations, especially when analyzing young women. The Se content of circulating SELENOP differs between individuals and may provide some important diagnostic information on Se metabolism and status

Consensus on criteria for acromegaly diagnosis and remission

Purpose: The 14th Acromegaly Consensus Conference was convened to consider biochemical criteria for acromegaly diagnosis and evaluation of therapeutic efficacy. Methods: Fifty-six acromegaly experts from 16 countries reviewed and discussed current evidence focused on biochemical assays; criteria for diagnosis and the role of imaging, pathology, and clinical assessments; consequences of diagnostic delay; criteria for remission and recommendations for follow up; and the value of assessment and monitoring in defining disease progression, selecting appropriate treatments, and maximizing patient outcomes. Results: In a patient with typical acromegaly features, insulin-like growth factor (IGF)-I &gt; 1.3 times the upper limit of normal for age confirms the diagnosis. Random growth hormone (GH) measured after overnight fasting may be useful for informing prognosis, but is not required for diagnosis. For patients with equivocal results, IGF-I measurements using the same validated assay can be repeated, and oral glucose tolerance testing might also be useful. Although biochemical remission is the primary assessment of treatment outcome, biochemical findings should be interpreted within the clinical context of acromegaly. Follow up assessments should consider biochemical evaluation of treatment effectiveness, imaging studies evaluating residual/recurrent adenoma mass, and clinical signs and symptoms of acromegaly, its complications, and comorbidities. Referral to a multidisciplinary pituitary center should be considered for patients with equivocal biochemical, pathology, or imaging findings at diagnosis, and for patients insufficiently responsive to standard treatment approaches. Conclusion: Consensus recommendations highlight new understandings of disordered GH and IGF-I in patients with acromegaly and the importance of expert management for this rare disease.</p