Correlative study of radioreceptor assay and radioimmunoassay of serum growth hormone in children. II: Primary, genetic and intra-uterine growth retardation

Growth hormone (GH) was measured by radioreceptorassay (RRA) and radioimmunoassay (RIA) in the sera of 24 children with idiopathic primary growth retardation (PGR), 15 with genetic short stature (GSS) and 11 with intra-uterine growth retardation (IUGR), and compared to results obtained in normal children. The average RRA/RIA ratio was close to normal in PGR (1.02 +/- 0.05) and in IUGR (1.06 +/- 0.07), and slightly though not significantly lower in GSS (0.86 +/- 0.06). Some variability in RRA/RIA ratio was found in individual patients of each group, and some sera gave a non-parallel displacement of the tracer when compared to the standard curve. But no genetic difference of RRA-assayable GH was found between the three groups studied and normal children.     

Characterization of dimeric forms of human pituitary growth hormone by bioassay, radioreceptor assay, and radioimmunoassay

Seven highly purified dimeric forms of human pituitary growth hormone, composed of the monomeric forms 20 K hGH, 22 K hGH and 24 K hGH linked together by noncovalent or covalent bonds, have been characterized by an in vitro bioassay (the Nb2 assay), a radioreceptor assay and a radioimmunoassay. Considerable differences in the ability to displace labelled recombinant hGH were observed in the radioreceptor assay. The seven dimeric forms varied over a range between 22 K hGH (most effective) and 20 K hGH. The three covalently-linked dimeric forms had nearly identical affinity constants. The mitogenic action of all but one of the hGH dimers in the Nb2 assay was in the same mutual order as the receptor binding activity in the radioreceptor assay. In the RIA, all dose-response curves were parallel except for those obtained with 20 K hGH and with the dimeric form (20 K-20 K)hGH. In this assay, dimeric variants of the constituents 22 K hGH and 24 K hGH were approximately twice as active as 22 K hGH on a molar basis, suggesting about the same affinity between the antibodies and each of the monomeric forms. Determination of the amino acid compositions of the dimeric forms provided support for their content of monomeric constituents as established earlier by electrophoretic analysis.     

Serum growth hormone levels measured by radioimmunoassay and radioreceptor assay: a useful diagnostic tool in children with growth disorders?

Serum GH levels were measured by RIA and RRA in 133 subjects (19 healthy controls and 114 patients with various growth disturbances, aged 2.3-24.8 yr). Serum samples obtained from 147 stimulation tests representing a total of 1065 samples were analyzed by both methods, and the results compared. The data are expressed in absolute values and in RRA/RIA ratios. The area under the curve after a stimulation test (area GH) was calculated by planimetry. RIA was performed by the classical double antibody method using a polyclonal anti-serum. For the RRA, human cultured lymphocytes (IM-9 cells) were used, and 125I-labeled human GH was purified by high performance liquid chromatography. The same human GH standard was used in both assay systems. In control subjects a significant (P less than 0.0001) positive correlation was found at all ages between GH levels measured by RIA and RRA (r = 0.69 after insulin and r = 0.77 after glucagon). The RRA/RIA ratio (mean +/- SEM) for the peak GH level was 0.88 +/- 0.05, and the area under the GH curve was 0.85 +/- 0.05. The peak mean RRA/RIA ratios were significantly lower (P less than 0.05 and P = 0.03, respectively). No relationship was found with the absolute value of either peak or area GH. In patients with growth delay and Turner's syndrome, lower GH levels were found than in control subjects in both assay systems. The RRA/RIA ratios were also lower. In the other patients with some growth disorder, normal GH levels and ratios were found. In patients with renal failure, high levels of RIA-GH and RRA-GH were found, with a normal RRA/RIA ratio. In patients with documented pituitary GH deficiency, GH-releasing factor administration resulted in an increase in GH levels that was identical in both assays. The RRA/RIA ratio remained constant throughout the test. No correlation was found between the ratio and the absolute value of either RIA-GH or RRA-GH regardless of the stimulation test used. It is concluded that the presence of an abnormal GH molecule is extremely rare in patients with short stature. Thus, the presence of a bioinactive hormone is not a common cause of growth failure. During provocative testing some changes in the ratio may occur that do not appear after GH-releasing factor, further illustrating the different mechanisms involved in GH secretion.     

A radioreceptor assay for purified teleost growth hormone.

Highly purified ¹²⁵I-labelled tilapia (Sarotherodon mossambicus) growth hormone (GH) binds to membrane fractions prepared from tilapia liver. Specific (displaceable) binding occurred with the 600, 15,000, and 90,000g fractions with the greatest binding observed with the 90,000g microsomal membrane fraction. Binding was dependent on time and membrane concentration. Specificity studies showed that up to 60% of the ¹²⁵I-labelled tilapia GH bound to the liver microsomal membrane fraction could be displaced by 500 ng of unlabelled hormone. Scatchard analysis of the binding of ¹²⁵I-labelled tilapia GH revealed a single class of binding site with a binding capacity of 125 ± 7.7 fmol/mg protein and a binding affinity of 1.5 × 10¹⁰ ± 0.4 × 10¹⁰ (SEM) l/mol. GH preparations from several vertebrate classes and tilapia prolactin and ovine prolactin at high concentrations displayed competition for the tilapia GH-binding site.¹²⁵I-Labelled tilapia GH demonstrated specific binding to liver microsomal membrane fractions of the teleosts Gillichthys mirabilis, Salmo gairdneri, and Oncorhynchus tschawytscha but not to those of an amphibian Necturus maculosus or a bird Coturnix japonica. Slight, but significant, specific binding was observed with the microsomal membrane fraction of tilapia kidney and gill and of rat liver. These data suggest that this tilapia GH radioreceptor assay may have an application in the detection of teleost GH-like activity.     

Evaluation of the bovine testicular radioreceptor assay for pituitary follicle-stimulating hormone.

A modified bovine testicular receptor was used to evaluate highly purified follicle-stimulating hormone (FSH) from a number of species. The particulate receptor obtained from adult bovine testes could be stored frozen or lyophilized for long periods without appreciable decrease in the binding of the ligand facility or in loss of specificity. The bovine testis receptor binds twice as much 125I-labelled ovine FSH as 125I-labelled human hormone. When FSH from different species was compared against NIH-FSH-S10, using various FSH ligands, the ovine hormone was clearly the most active, although many had comparable in-vivo biological potencies. The results suggest that there is probably some species specificity in the hormone-receptor interactions. As the ovine hormone is structurally closer to the bovine, from which the receptor was derived, it appears to have the highest activity in vitro. Marked differences in the biological activities of the different preparations between the human chorionic gonadotrophin-augmentation test and the in-vitro assays have been observed. In the in-vitro assays, all preparations, with the exception of the porcine hormone preparation, were less active and the ratio of bioassay/radioreceptor assay varied widely. In the radioreceptor assays, all FSH preparations except pregnant mare serum gonadotrophin (PMSG) showed parallel inhibition curves. The three different PMSG preparations examined gave inhibition lines that were parallel to each other.     

Direct pituitary effects of testosterone and luteinizing hormone-releasing hormone upon follicle-stimulating hormone: analysis by radioimmuno- and radioreceptor assay

The present studies examine the effects of testosterone (T) and LHRH, alone or in combination, on the amount of FSH secreted by pituitary cells in culture. FSH was quantified by RIA and radioreceptor assay (RRA). Half of the cultures were exposed to T for 3 days. The remainder served as controls. Each of these two groups was divided in half and exposed to medium only or LHRH (10(-8) M) for 4 h. Medium was collected from all cultures after 3 days +/- T (medium 1) and after 4 h +/- LHRH (medium 2). After medium 2 collection, cell homogenates were prepared. In a second study, T-treated cell cultures also received 0, 0.5, or 5.0 micrograms/dish tunicamycin for the last 16 h of the 3-day incubation. During the 3 days of culture, the T-treated group secreted greater amounts of immunoactive FSH than controls. However, LHRH-induced FSH release measured by RIA was blunted as a result of T exposure compared with untreated controls. T treatment elevated intracellular immuno-FSH stores. Each sample was quantitated for FSH activity by RRA, and the FSH RRA/RIA was calculated. T and/or LHRH treatment, while eliciting FSH hypersecretion, caused a reduction in the RRA/RIA of secreted FSH. Changes in the RRA/RIA are thought to occur as a result of alterations in the glycosylation of FSH. To test this hypothesis, T-treated cells were exposed to tunicamycin, a drug that reduces the rate of glycosylation of secreted proteins. Exposure of cells to this drug prevented the reduction in the RRA/RIA of secreted FSH caused by T and/or LHRH. FSH secreted from control, T-treated, or T-treated plus tunicamycin-exposed cells was examined by isoelectric focusing. T-Treated cells released a greater proportion of FSH forms with lower isoelectric points (indicative of a greater degree of glycosylation) compared with controls. Tunicamycin exposure reversed T's effect upon the isoelectric profile. These studies demonstrate a direct pituitary action of LHRH and T upon the type of FSH released. During times of hormonally induced increases in the rate of FSH secretion, the pituitary releases FSH forms that are more heavily glycosylated, exhibit a lower isoelectric point, and show a reduced RRA/RIA. FSH secreted after T treatment would be expected to have an increased plasma half-life due to the protective effects of the sugar residues. Thus, the existing hormonal milieu exerts a multidimensional effect upon FSH released by pituitary cells in culture that cannot be appreciated by RIA assessment alone.     

Pregnant mare serum gonadotrophin: ratio of follicle-stimulating hormone and luteinizing hormone activities measured by radioreceptor assay.

Specific radioreceptor assays for FSH and LH, which employ tissue receptors from rat testis and highly purified human FSH (LER 1575-C) and LH (Hartree IRC-2, 24/6/69) as standards, have been developed to determine the FSH-like and LH-like activities in pregnant mare serum gonadotropin (PMSG). Measurements of FSH and LH concentrations in the serum of six pregnant Pony mares showed that the ratio of these two activities did not vary significantly between mares and remained constant between days 40 and 80 of gestation with a value of 1-45 +/- 0-04 (S.E.M.). The FSH:LH ratio of PMSG produced by cultured equine trophoblast cells was found to be 0-72 +/- 0-03 (S.E.M.) and that of partially purified serum extracts of PMSG 1-08 (range 0-87-1-30).     

Heterogeneity of human growth hormone and prolactin secreted in vitro: Immunoassay and radioreceptor assay correlations.

Gel filtration of sera and of in vitro pituitary incubation media from a normal subject and from subjects with pituitary tumors or physiologically elevated growth hormone (hGH) and/or prolactin (hPRL) levels, has been performed. Heterogeneous immunoreactive forms of both hormones with comparable elution profiles in sera and in in vitro incubation media were identified. In each instance the most retarded component ('little hPRL' and 'little hGH') migrated identically with the respective radioiodinated pituitary standard and constituted the major component identified. Several components of intermediate mobility were identified and characterized as 'big hPRL', 'big hGH,' and 'big big hGH'. In addition, a void volume immunoreactive peak was often identified. No interconversion was demonstrated on refiltration of prolactin tumor sera or normal pituitary incubation media. Radioreceptor activity utilizing pregnant rabbit liver membranes for hGH and rabbit mammary membranes for hPRL indicates comparable immunologic and receptor activity in all forms identified following gel filtration of prolactin tumor sera (for hPRL) and normal pituitary incubation media (for hGH). Only for the larger species of HGH, identified by gel filtration of clinical grade hGH, was diminished receptor activity demonstrated. These data suggest that the human pituitary synthesizes and secretes protein hormones of different molecular size according to gel filtration elution volume profiles but with comparable receptor and immunologic assay reactivity. These results support but do not establish the proposal that pituitary hormones are also secreted as prohormones.     

Measurement of developmental changes in plasma insulin-like growth factor-I levels of broiler chickens by radioreceptor assay and radioimmunoassay

The main purpose of this study was to examine the relationship between insulin-like growth factor-I (IGF-I) and growth hormone (GH) during embryonic and posthatching development of broiler chickens. Two heterologous assays were validated for measurement of IGF-I in chicken and turkey plasma. A radioreceptor assay (RRA), utilizing microsomal membranes prepared from human placenta, was modified and validated for measurement of IGF peptide (mainly IGF-I). A double-antibody radioimmunoassay (RIA) was validated for measurement of immunoreactive IGF-I levels in chicken and turkey plasma. In both assay systems, recombinant-derived human IGF-I was used for standards and trace hormone. Hypophysectomy in turkey poults reduced plasma levels of IGF (RRA) by 35% and IGF-I (RIA) by 59% as compared to intact control turkeys. In Experiment 1, 14 chicken embryos were bled at 15, 17, 19, and 21 days of incubation and at 1 week of age to determine plasma levels of IGF-I and GH. Plasma IGF levels (RRA) remained constant during late incubation, but increased significantly (P less than 0.05) at 1 week of age. Plasma IGF-I levels (RIA) declined 2 days before hatching; however, plasma levels of IGF-I were sharply elevated (P less than 0.05) at 1 week of age. Plasma GH concentrations were low in embryos and were greatly elevated (P less than 0.05) at hatching (21 days of incubation) and at 1 week of age. In Experiment 2, 12 different broiler cockerels were weighed and then bled by cardiac puncture each week from hatching (1 day of age) to 7 weeks of age. The plasma profiles of IGF, IGF-I, GH, triiodothyronine (T3), and thyroxine (T4) were each compared to relative growth rate by analysis of covariance. Plasma IGF and IGF-I levels increased progressively from 0 to 3 weeks of age and were maintained in a plateau from 3 to 7 weeks of age. Plasma GH levels reached a peak at 4 weeks of age, but declined sharply thereafter, while IGF and IGF-I levels remained elevated. Plasma T3 concentrations were progressively increased and reached peak concentrations at 3 weeks of age, while plasma T4 levels increased only at 6 and 7 weeks of age. There was a high correlation (P less than 0.01) between relative growth rate and age-related changes in plasma levels of IGF (r = 0.96), IGF-I (r = 0.97), and T3 (r = 0.94); however, there was no correlation between relative growth rate and changes in plasma GH or T4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay

Goldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein. This fusion protein was purified, refolded, and (125)I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the (125)I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca(2+) ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca(2+) influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish.     

Homologous IM-9 lymphocyte radioreceptor and receptor modulation assays for human serum growth hormone

Radioreceptor assays for human GH (hGH) have been developed using the IM-9 cultured human lymphoid cell receptor. Varying degrees of nonspecific interference with [125I] hGH binding to these cells occurs in the presence of serum. We have modified the traditional IM-9 competitive binding assay for hGH and abolished the nonspecific serum effects. The modified competitive assay is sensitive to as little as 2 ng/ml hGH, and it has been validated by the quantitative recovery of purified pituitary hGH in hypopituitary serum. Sera from stimulated normals, acromegalics, and patients with severe growth retardation were assayed. The RIA to radioreceptor assay ratios for these groups were 0.98 +/- 0.10, 0.97 +/- 0.18, and 2.43 +/- 0.54, respectively. The assay has potential usefulness in screening and predicting growth-retarded patients most likely to respond to exogenous hGH therapy. In addition, a sensitivity receptor modulation assay, which uses the ability of hGH to regulate its homologous IM-9 receptors, is described. This is 8- to 10-fold more sensitive than the nonregulatory assays and has been applied to the measurement of hGH in sera from unstimulated normals and acromegalics. The ratios of RIA to receptor modulation assay for the two groups were 1.17 +/-0.68 ad 1.07 +/- 0.26, respectively. These sensitive receptor assays offer a powerful technique for the measurement of biologically active and inactive GH peptide in serum, and may be particularly useful in the evaluation of statural growth disorders.     

Regulation of receptor by homologous hormone enhances sensitivity and broadens scope of radioreceptor assay for human growth hormone.

In standard competitive binding assays (including radioreceptor assays) unlabeled ligand (hormone) competes with labeled ligand (hormone) for binding to a fixed number of binding (receptor) sites. Detection of the unlabeled ligand occurs when the occupancy of binding sites by the unlabeled ligand is sufficient to reduce the binding of labeled ligand. A common feature of the hormone-receptor interaction is the ability of the hormone to regulate the affinity and/or the concentration of its homologous receptor. In the present study, by exploiting the ability of human GH to regulate by negative feedback the concentration of its own receptors, we have enhanced the sensitivity of the human GH radioreceptor assay 5-fold. The ability of hormone to regulate receptor concentration and affinity affords wide opportunities to broaden the scope as well as to enhance the sensitivity of radioreceptor assays.     

The assay of urinary growth hormone in normal and acromegalic adults

OBJECTIVES: To establish a normal range for urinary growth hormone in adults and to investigate the urinary growth hormone levels in patients with acromegaly, comparing these with the serum growth hormone results of a glucose tolerance test. We also studied the molecular identity of the growth hormone recognized by our assay method. DESIGN: Overnight urine samples and, in some cases, timed urine samples taken during the day were obtained from healthy volunteers and acromegalic patients. A standard glucose tolerance test with serum growth hormone measurements was performed on the acromegalic patients. PATIENTS: One hundred and thirty-five normal adults and 33 acromegalic patients were studied. MEASUREMENTS: Urinary growth hormone was measured using a sensitive and precise assay developed previously. RESULTS: In healthy volunteers overnight urinary growth hormone values fell gradually with increasing age, but there was no significant difference between men and women in any decade or between smokers and non-smokers. Sexual intercourse had no detectable effect on the values, but there was a large increase following strenuous exercise. Studies of the diurnal patterns in normal and abnormal adults suggested that it might be possible to diagnose acromegaly on a random urine sample. Gel filtration studies on a urine sample from an acromegalic patient showed a single peak of molecular weight 22,000. Using overnight collections there was clear discrimination between the values given by the normal adults and the acromegalic patients and an excellent correlation between urinary growth hormone levels in acromegalic patients and the mean serum growth hormone in a glucose tolerance test. CONCLUSIONS: In contrast to some other groups we conclude that urinary growth hormone provides a useful, non-invasive screening test for acromegaly, but this conclusion depends crucially on the assay being sensitive and precise at low values.