GRF Injection Research Articles

Diagnosis of growth hormone deficiency with the growth hormone releasing factor GRF 1-40. The potentials and limits

Eleven patients with constitutionally delayed development, seven patients with idiopathic growth hormone deficiency and ten patients with craniopharyngeoma received growth hormone releasing factor GRF 1-40 in a dose of 1 micrograms/kg body weight as a bolus injection. In the patients with constitutionally delayed development, a mean maximum HGH value of 86.7 microU/ml was measured 30 minutes after GRF injection. Considerably higher HGH stimulation values were thus obtained after GRF than with conventional stimulation methods. In the patients with idiopathic growth hormone deficiency or craniopharyngeoma, there was mostly only a slight rise of the growth hormone level with a delayed onset. Merely two patients with craniopharyngeoma could be adequately stimulated in accordance with conventional criteria (HGH greater than 12 microU/ml), although their values also remained markedly below those of the patients with constitutionally delayed development. Both patients had somatomedin C values which were below that normal for their age; together with the delayed rise in HGH after GRF, this allowed diagnosis of growth hormone deficiency.

Standardization and validation of a simple, sensitive, second antibody format enzyme immunoassay for growth hormone determination in mithun (Bos frontalis) plasma

To facilitate research into the action of growth hormone (GH) in mithun (Bos frontalis), we standardized and validated a simple and highly sensitive enzyme immunoassay (EIA) for GH determination in mithun blood plasma on microtiter plates using biotin-streptavidin amplification system and the second antibody coating technique. Biotin was coupled to GH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 25 µl mithun plasma. The GH standards ranging from 0.25 ng/well/25 µl to 128 ng/well/25 µl were prepared in charcoal-treated plasma collected from an aged (>10 years) senile mithun. The sensitivity of EIA procedure was 1.0 ng/ml plasma; the 50% relative binding sensitivity was seen at 36 ng/ml plasma. Plasma volumes for the EIA, namely 12.5 and 25 µl, did not influence the shape of standard curve even though a drop in the optical density (OD)450 observed with higher plasma volumes was due to higher inherent GH content in mithun plasma collected from an aged (>10 years) senile mithun. For the biological validation of assay, two mithuns were administered with synthetic bovine GH-releasing factor (GRF; 10 µg/100 kg body weight; intravenous) and another two were administered sterile normal saline (controls). Jugular blood samples were collected at −60, −45, −30, −15, −10, −5, 0, 5, 10, 15, 30 min and thereafter at 15-min intervals beginning 1 hour before GRF injection up to 8-hr post treatment, and samples were assayed for plasma GH. In two animals, a peak of GH was recorded at 15 min of GRF administration, which confirms the biological validation of the EIA. Plasma GH estimated in blood samples collected for 6 consecutive weeks from two different age groups of mithun (Group I, age 0–3 months; Group II, age 3–4 yr) showed higher (P < 0.0001) mean plasma GH in younger than in adult mithuns and consequently higher growth rates in the younger group. A parallelism test conducted between a buffer standard curve of bovine GH and GH measured from serial dilution of mithun plasma containing high concentration of endogenous GH showed good parallelism with a standard curve. In conclusion, the EIA developed for GH determination in mithun blood plasma is sufficiently reliable, economic, and sensitive enough to estimate mithun GH in all physiologic variations. Zoo Biol 0:1–11, 2005. © 2005 Wiley-Liss, Inc.

Development and Validation of a Simple Sensitive Enzyme Immunoassay (EIA) for GH Determination in Buffalo Plasma

A simple and highly sensitive enzymeimmunoassay (EIA) for GH determination in buffalo plasma on microtitreplates using biotin-streptavidin amplification system and the second antibody coating was developed. Biotin was coupled to GH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 100 µL buffalo plasma. The GH standards ranging from 0.05 ng/well/100 µL to 12.8 ng/well/100 µL were prepared in hormone free plasma collected from an aged (>15 years) senile buffalo. The sensitivity of the EIA procedure was 50 pg/well GH, which corresponded to 0.50 ng/mL plasma; the 50% relative binding sensitivity was seen at 800 pg/well/100 µL. Plasma volumes for the EIA, viz., 25, 50, and 100 µL did not influence the shape of standard curve, even though a slight drop in the OD450 was seen with higher plasma volumes. For the biological validation of the assay, 12 Murrah buffalo calves were used. Six of these were administered synthetic bovine growth hormone-releasing factor (10 µg/100 kg body weight, i.v., and the remaining six animals were administered sterile normal saline and kept as controls. Jugular blood samples were collected at −60, −45, −30, −15, −10, −5, 5, 10, 15, and 30 min and, thereafter, at an interval of 15 min using an indwelling jugular catheter, beginning 1h prior to GRF injection up to 8 h post treatment. In all animals, a peak of GH was recorded within 5 to 20 min of GRF administration, which confirms the biological validation of the EIA. To confirm homogeneity of buffalo GH with bovine GH, a parallelism test was conducted between the buffer standard curve of bovine GH and GH measured from serial dilution of buffalo plasma containing a high level of endogenous growth hormone.

Serum somatotropin concentrations in Holstein heifers administered growth hormone-releasing factor and somatostatin

AbstractDose-response experiments were designed to determine the dosage of somatostatin (SRIF) necessary to alter serum growth hormone (GH) response to administration of GH-releasing factor (1·44; GRF) in cattle. The objective of this experimentation was to develop a GRF/SRIF challenge model that might be useful in early identification of animals which are genetically superior for milk production traits. In experiment 1, calves received either 0, 5 or 10 /JLg GRF per 100 kg body weight (BW) or GRF 1·29 at 1 ug per 100 kg BW. Both the 5 and 10 ug GRF per 100 kg BW dosages increased GH concentrations (P &lt; 0·05). In experiment 2, animals received 3 ug GRF per 100 kg BW concurrent with administration of 0, 0·5, 1 or 2 ug SRIF per 100 kg BW at 0 min, with second dosage of GRF at +120 min. Administration of SRIF in those dosages did not alter GH response to GRF. In experiment 3, animals received 5 ug SRIF per 100 kg BW at -2, -1, 0 or +1 min relative to 3 ug GRF per 100 kg BW, with a second GRF injection at +120 min. No differences (P &lt; 0·05) were found in response due to timing of SRIF administration. In experiment 4, animals were administered 0, 5, 10 or 20 Xg SRIF per 100 kg BW concurrent with 3 ug GRF per 100 kg BW. SRIF delayed (P &lt; 0·05) the occurrence of maximum GH concentrations but did not affect the maximum concentration nor the area under the GH response curve (AUC) following either the first or second GRF injection. Utilizing data from experiment 4, individual animal response to GRF was reasonably consistent, as repeatabilities of AUCs for 1 h following first and second GRF injections were 0·80 and 0·65, respectively. Furthermore, sufficient among-animal variation existed so that animals could be distinguished from each other. Thus we conclude the method described herein might be useful in identifying superior dairy calves but accuracy would potentially be enhanced by collecting blood samples more frequently during the 20 min immediately after GRF injection.

No desensitization of the growth hormone (GH) response between GH-releasing peptide-2 and GH-releasing factor in calves.

We investigated the desensitization caused by growth hormone (GH)-releasing peptide-2 (D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2; GHRP-2) in vivo using calves. The GHRP-2 (12.5 micrograms.kgBW-1.h-1) or GH-releasing factor (GRF; .125 microgram.kgBW-1.h-1) were infused for 180 min into four calves, each followed by either GHRP-2 (12.5 micrograms/kg BW) or GRF (.125 microgram/kg BW) injection after 60 min. All treatments were given to all of the calves. The continuous infusion of GHRP-2 did not attenuate the GH secretion caused by a GRF bolus, and vice versa. Continuous exposure to GHRP-2 attenuated the GH secretion caused by a GHRP-2 bolus. In an additional experiment, two repetitive injections of GHRP-2 (12.5 micrograms/kg BW) at 1-, 2-, 3-, or 4-h intervals were administered. Attenuation of the GH response caused by GHRP-2 was maintained until 4 h. These results suggest that GHRP-2 is not cross-desensitized with GRF and that the mechanisms by which they cause GH release may be different in calves.

PRETREATMENT WITH SMS 201–995 POTENTIATES GRF RESPONSE IN SHORT CHILDREN

We tested the hypothesis that prior inhibition of spontaneous GH secretion by administration of the somatostatin analog SMS 201-995 (SMS) will enhance the GH response to a subsequent GRF challenge. Two controlled protocols were employed in 37 short children [M=31, F=6, aged 11.8 ± 1.6 yr (M ± SEM), height SDS −2.25 ± 0.55]. Study I determined an optimal regimen: SMS (0.8-2.2 ug/kg, sc) was randomly administered or not at 08:00 h and a GRF bolus (50 ug, iv) was given 4 h later. Study 2 used standard doses of SMS (1 ug/kg, sc) and GRF (1 ug/kg, iv), and a 1 h delay of the GRF injection. GH levels were measured q 20 min until 2 h following the GRF and expressed as the GH area under the curve during the 4-5 h SMS-GRF interval (AUC 1, ug/L/h) and as GH response to GRF for 2 h following GRF (AUC 2, ug/L/h; GH peak, ug/L).In study 1, SMS significantly suppressed spontaneous GH secretion and GH response to GRF, compared to control. In study 2, GH secretion was still suppressed during the 5-h SMS-GRF interval, but both the GH area and peak GH response to GRF were significantly increased by pretreatment with SMS. Thus, a “priming” SMS dose of 1 ug/kg significantly augmented the GH response to GRF given 5 h later, enhancing the diagnostic value of the 'standard” GRF test.

Effect of human growth hormone-releasing factor on bone and mineral metabolism in growing pigs.

Little information is available on the effects of growth hormone (GH) and growth hormone-releasing factor (GRF and GHRH) treatment on bone metabolism in pigs. Thus, tibial bending moments and ash contents were studied in 12, 6-wk-old pigs weighing 13 +/- .2 kg. Six pigs (GRF group) were injected s.c. twice daily with 75 micrograms GRF (hGRF [1-29] NH2)/kg BW for 52 d and six remained untreated (control group, C). Average daily gain was slightly (5%; P less than .10) increased in treated pigs. At slaughter, plasma measurements related to calcium homeostasis, such as concentrations of Ca, inorganic P, and vitamin D metabolites (25-OH and 1,25-(OH)2 vitamin D3), were not changed by GRF injection. At slaughter, plasma GH levels were 3.3 times greater in treated (11.3 +/- 3 ng/ml) than in untreated pigs (3.4 +/- .5 ng/ml, P less than .02), whereas those of insulin-like growth factor I were increased by approximately 38%. No difference was observed between the two groups at slaughter in tibial weight, density, bending moment, ash relative to bone volume (29 +/- 1 vs 30 +/- 2 g/100 cm3, GRF vs C), total ash content, or ash relative to dry matter in cortical or medullary bone. Our GRF treatment did not affect bone and mineral metabolism in young, growing pigs.

Effect of triglycerides on growth hormone (GH)-releasing factor-mediated GH secretion in newborn calves

The effect of triglycerides (Tg) on GRF-mediated GH secretion was examined in 2 groups of twelve ten-day old male calves. Twelve calves were intravenously infused with a lipid-heparin solution (5 mg Tg and 0.3 IU heparin/kg body wt/min for 90 min). The twelve control calves received in the same way, the same volume of saline. Thirty minutes after the start of infusion, GRF 1–29 (human amide, 0.16 μg/kg body wt) was intravenously injected in six animals of each group. Mean plasma GH levels reached peak concentrations in the 2 groups 5 min after GRF injection. However the area under the GH response curve, when lipid-heparin was given, was significantly diminished compared to the response when saline was given. In the same time, lipid-heparin treatment increased plasma SRIF concentration. These data suggest that an increase in plasma Tg concentration, induced by lipid-heparin infusion, inhibits GRF-mediated GH secretion, possibly through stimulation of SRIF secretion.

Expression of human or bovine growth hormone gene with a mouse metallothionein-1 promoter in transgenic swine alters the secretion of porcine growth hormone and insulin-like growth factor-I.

Endocrine profiles were examined in swine that had integrated and expressed a fusion gene consisting of mouse metallothionein-1 (MT) promoter fused to either a human (h) or bovine (b) GH structural gene. Eleven of 18 pigs that had integrated MT-hGH and eight of nine pigs that had integrated MT-bGH expressed the genes. The level of expression varied widely among pigs (14-4551 micrograms/l for MT-hGH and 23-1578 micrograms/l for MT-bGH). The level of expression varied over time within each pig with no general pattern. Concentrations of porcine GH (pGH) were lower in MT-hGH pigs that expressed the gene than in non-expressors or in littermate controls. Insulin-like growth factor-I (IGF-I) concentrations increased with age in all pigs and were raised threefold in pigs expressing either the MT-hGH or MT-bGH genes. Measurement of the foreign GH in samples taken at 15-min intervals failed to reveal any short-term fluctuations in concentration. Administration of hGH releasing factor (GRF) to pigs expressing MT-bGH resulted in attenuated release of pGH compared with that of contemporary controls. Concentrations of bGH did not change after GRF injection. Human and bovine GH expressed in transgenic pigs appear to be biologically active in that they induce IGF-I and suppress endogenous pGH secretion. The failure to find short-term fluctuations and the lack of response to GRF injections are consistent with a non-pituitary and non-GRF regulatable site of production.

75 TRANSIENT GROWTH HORMONE DEFICIENCY DURING BROMOCRYPTINE TREATMENT FOR HYPERPROLCATINEMIA

Growth hormone (GH) deficiency has been demonstrated in several patients with prolactinoma. Very often it was attrituted to tumor expansion and found to be permanent after surgery. In a 14-year-old boy with galactorrhea, arrested puberty (testesvolume 4ml) and poor growth (3cm/year) elevated prolactin (PRL) levels (5800 mU/l) were found. MR imaging showed no abnormalities of the pituary gland. Nighttime sampling revealed persistent PRL elevation and low GH peaks (max. 12 mU/l). GH response to insuline induced hypoglycemia was also low (max. 10 mU/l) but GH increased normally after GRF injection (38 mU/l). Basal and stimulated LH and FSH values were prepubertal. 3 h. after the administration of 10 mg bromocryptine (B) PRL dropped to normal values and GH increased to 22 mU/l. After six months of treatment with B, height velocity doubled and normalization of GH secretion was observed. Basal LH, FSH and testosterone values remained prepubertal. In conclusion, GH deficiency in hyperprolactinemia can be restored during B treatment. The acute GH response to B may be predictive of the subsequent normalization of GH secretion during long term treatment. In Isolated hyperprolactinemia a central dopaminergie defect is probably responsible for this deficient GH secretion.

Characteristics of growth hormone response to the administration of growth hormone-releasing hormone (GRF) in the lamb.

Human growth hormone releasing hormone (GRF 1-44 or GRF 1-29) was administered to lambs at two different physiological stages (suckling: 5-6 week-old and weaned: 14-15 week-old) when growth hormone (GH) secretory patterns were different: suckling lambs exhibited flat basal GH profiles (5-10 ng/ml) while the weaned lambs had frequent spontaneous episodes of GH release (15-65 ng/ml). The iv injection of GRF evoked an immediate release of GH. In each case, plasma GH levels reached a maximum 1-4 min after the injection. The secretory spike was multiphasic and lasted 30-60 min. Administration of GRF (0.1 microgram/kg) in weaned lambs induced GH pulses with an amplitude comparable to that of endogenous peaks. The induction of a GH peak occurred even when a spontaneous peak immediately preceded the GRF injection. Also, spontaneous peaks were observed during the hour following a GRF-induced GH peak. In suckling lambs, GRF injected intravenously as a single bolus in a range of 0.01 to 0.5 microgram/kg (2 to 100 pmoles/kg) stimulated GH release in a dose-dependent manner. Chronic administration of GRF (0.75 nmole GRF 1-44 or GRF 1-29 per kg twice daily for 21 days) in newborn lambs increased significantly (p less than 0.001) the acute response to GRF during the course of the treatment. GH response to GRF 1-44 and GRF 1-29 was the same. These data show that lambs are highly responsive to GRF action during both suckling and weaning and suggest that there is no in vivo desensitization of the pituitary gland after acute or chronic GRF administration.

Growth hormone responses to growth hormone-releasing factor (1-29) in euthyroid, hypothyroid and hyperthyroid rats.

In order to investigate whether the impaired GH secretion associated with hypothyroidism and hyperthyroidism is due to a hypothalamic or a pituitary disorder, we have studied plasma GH responses to GH-releasing factor (1-29) (GRF) in euthyroid, hypothyroid and hyperthyroid rats. Hypothyroid rats showed a significant (P less than 0.001) reduction in GH responses to GRF (5 micrograms/kg) at 5 min (350 +/- 35 vs 1950 +/- 260 micrograms/l), 10 min (366 +/- 66 vs 2320 +/- 270 micrograms/l) and 15 min after GRF injection (395 +/- 72 vs 1420 +/- 183 micrograms/l; mean +/- S.E.M.) compared with euthyroid rats. Hyperthyroid rats showed a significant (P less than 0.05) decrease in GH responses to 5 micrograms GRF/kg after 30 min (200 +/- 14 vs 325 +/- 35 micrograms/l) but not at other time-points, or after the administration of 1 microgram GRF/kg. These data indicate that in hypothyroidism and perhaps hyperthyroidism there is an alteration in the responsiveness of the somatotroph to GRF administration.

Stimulation of growth hormone secretion with human growth hormone releasing factors (GRF1–44, GRF1–40, GRF1–29) in normal subjects

Peptides of different chain lengths (GRF1-44, GRF1-40, GRF1-29) were given as a 50 micrograms i.v. bolus to five normal volunteers. Blood was collected before and until 120 min after GRF injection. No serious side effects were recorded. All GRF peptides led to a clearcut and significant increase of GH levels compared to placebo controls with the maximum occurring 15-30 min after GRF injection. There was no significant difference in the maximal GH increase after the different GRF peptides. When GRF1-44 was administered to five normal subjects over 2 days in 6- and 18-h intervals, respectively, significant increase of GH levels were recorded after each injection compared to placebo controls. Again, there was no significant difference between the maximal GH rises during the different tests. These findings show that the short GRF1-29 peptide may be used for diagnosis and therapy. In addition, in contrast to continuous infusion and administration of GRF in short intervals, GRF application in 6-h intervals leads to adequate GH responses.