The role of serial sampling in the diagnosis of growth hormone deficiency.

We analyzed 12-hour serial sampling of growth hormone (GH) levels in two cohorts of short children: 96 children referred to a university endocrine clinic or studied on a research protocol and 825 children in the National Cooperative Growth Study of children treated with exogenous GH. The mean 12-hour GH levels correlated with growth velocity in 60 children with normal height and growth velocity in the university study, and this correlation was stronger in the boys. The testosterone levels also correlated with growth velocity and mean 12-hour GH levels in the boys. The mean 12-hour GH levels were lower in a group of 36 children with idiopathic short stature than in the control subjects, as were the peak GH levels within 1 hour after the onset of sleep and the insulin-like growth factor I levels. In the National Cooperative Growth Study cohort, pooled 12-hour GH levels were lower in the group with idiopathic GH deficiency (n = 300) than in the group with idiopathic short stature (n = 525), but the difference was not significant. The duration of GH treatment was the most significant predictor of change in the height SD score in both groups. Indices of spontaneous secretion of GH were not predictive of the response to GH treatment, nor were the results of provocative GH testing, the responses to GH treatment being similar in both groups over time. We conclude that the results of GH testing must be interpreted for each patient and that several testing modalities may be helpful in finding GH insufficiency that originates at various levels of the somatotropic axis.

Demonstration of a leptin binding factor in human serum.

Serum leptin levels are elevated in subjects with exogenous obesity, indicating that obesity is associated with leptin resistance. Since in man no abnormalities have yet been found in either the genes for leptin or its receptor, the mechanism of leptin resistance in obesity remains unknown. To determine if resistance might be related to leptin binding by a serum component, we assessed the carrier status of leptin in serum. The presence of a specific leptin binding factor in human serum has been established by (1) demonstrating [125I]-leptin binding to a serum component that is saturable and specifically displaceable only by unlabeled leptin and not by human growth hormone, pork insulin, insulin-like growth factors I and II, luteinizing or follicle stimulating hormones, transforming growth factor-beta 1, interleukin-6, or leukemia inhibiting factor; (2) fractionating the leptin bound serum complex and the serum leptin binding component on a molecular sieving column revealing a mass of approximately 450 kDa; and (3) identifying an inverse correlation between the concentration of serum leptin and the quantity of the leptin binding component. It is suggested that binding of leptin by this serum component may influence the physiologic response to leptin.

Autoimmune hyperthyroidism in prepubertal children and adolescents: comparison of clinical and biochemical features at diagnosis and responses to medical therapy.

We explored our clinical impression that young children with autoimmune hyperthyroidism are more thyrotoxic at presentation and require a longer course of medical therapy than do adolescents to achieve remission. A retrospective chart review of clinical and biochemical data at presentation and response to therapy in 32 prepubertal (PREPUB) and 68 pubertal (PUB) children and adolescents with autoimmune hyperthyroidism was undertaken. Initial therapy included prophylthiouracil or methimazole in all but 11 patients who chose radioactive iodine (131I); 30 additional patients ultimately chose 131I or surgery after an initial period of medical therapy. In PREPUB children there were significantly longer duration of symptoms (7.8+/-7.7 months) and higher serum concentrations of triiodothyronine (T3) 708+/-330 ng/dL) at presentation than in the PUB group (4.7+/-3.4 months; p < .05) (537+/-197 ng/dL; p < .01). Duration of symptoms correlated negatively with chronologic age (r = -0.24; p < .02) but not with T3 or thyroxine (T4) levels (p = .1). PUB children had significantly higher titers of thyroid microsomal antibodies (positive dilution factor 1:6022+/-14572) than did PREPUB children (1:592+/-1226; p < .05). There was a higher familial incidence of thyroid disease in boys (80%) than in girls (64%) (p < .02). The duration of medical therapy was significantly longer (3.5+/-2.9 years) in PREPUB children compared to the PUB group (2.2+/-1.8 years) (p < .05). Only 17% of PREPUB treated 5.9+/-2.8 years compared with 30% of PUB treated 2.8+/-1.1 years achieved a 1-year remission after stopping antithyroid medication (percentage between groups, p < .01; years of treatment, p < .05). The median time to remission after medical therapy was 8 years in PREPUB and 4 years in PUB (p < .02). PREPUB children continued to remit after prolonged medical therapy (>6 years) whereas PUB patients did not. Total treatment length correlated negatively with chronological age (r = -0.26; p < .05) and positively with T4 and T3 concentrations at diagnosis (r = 0.31; p < .01). The diagnosis of hyperthyroidism is delayed in prepubertal children compared to adolescents. This delay may contribute to the higher T3 levels observed in this group at presentation. Prepubertal children also appear to require longer medical therapy to achieve a lower rate of remission, but do continue to remit after prolonged treatment. These differences in response to therapy should be considered when discussing therapeutic options with the family.

Facilitation of the growth promoting effect of growth hormone (GH) by an antibody to methionyl-GH.

Despite the development of antibodies to methionyl growth hormone in a child with hypopituitarism, the patient grew at a rapid rate on low doses of somatotropin. Serum immunoglobulins from this patient stimulated the growth of Nb2 lymphoma cells in vitro in samples obtained within 48 hours after the last dose of growth hormone, while samples obtained several weeks after an injection of methionyl growth hormone did not. Immunoglobulins from normal subjects or from hyposomatotropic patients being treated with methionyl growth hormone who had not developed antibodies did not stimulate Nb2 lymphoma cell growth. We suggest that the antibodies to methionyl growth hormone in this child served as a reservoir for exogenous growth hormone or facilitated the interaction of growth hormone with the prolactin receptor on the Nb2 lymphoma cell.

Neurotransmitter control of growth hormone secretion in children after cranial radiation therapy.

Cranial radiation for childhood cancer can cause growth hormone deficiency (GHD), usually due to hypothalamic rather than pituitary dysfunction. To investigate whether this hypothalamic dysfunction is secondary to altered neurotransmitter input from other brain centers, we used neurotransmitter-excitatory substances to study the GH secretory response in 17 children who had received 12 to 60 Grey (Gy) to the cranium and 40 short children with normal endocrine function. As expected, the irradiated children had decreased mean GH secretion in response to insulin-induced hypoglycemia and arginine infusion, and decreased mean 24 hour GH concentrations, compared to the control group. In contrast, the two groups had similar GH secretory responses to GHRH stimulation and somatostatin suppression. Assessment of neurotransmitter pathways in the irradiated children revealed significantly lower mean peak GH concentrations in response to 5 of the 6 substances tested compared to control children: alpha-adrenergic stimulation (clonidine), beta-adrenergic blockade (propranolol), cholinergic stimulation, dopaminergic stimulation (L-dopa), and GABA-ergic stimulation (valproic acid). Results of serotonergic stimulation (L-tryptophan) were not statistically significant. Eleven patients who had abnormal GH secretion underwent 4 or more tests with neurotransmitter-stimulatory agents; 3 patients had peak GH concentrations of < 2.5 micrograms/l to all tests, whereas 4 patients had a peak GH concentration of > or = 7 micrograms/l to one or more tests but < 5 micrograms/l to one or more other tests. These observations suggest that radiation damage may sometimes spare growth hormone-releasing hormone (GHRH) and somatostatin secretion while affecting neurotransmitter pathways. We postulate that the hierarchy of sensitivity to radiation damage may be hypothalamic and extra-hypothalamic neurotransmitters > hypothalamic GHRH and/or somatostatin secretion > pituitary GH secretion.

High density lipoprotein3 binding and biological action: high affinity binding is not necessary for stimulation of placental lactogen release from trophoblast cells.

High density lipoprotein (HDL3) binds with high affinity to many types of cells, but controversy exists concerning the nature and biological significance of the binding. We have recently demonstrated that HDL and apoproteins (apo)-AI, -AII, and -CI stimulate a specific and dose-dependent increase in placental lactogen (hPL) release from human trophoblast cells. To examine the possible relationship between HDL3 binding and stimulation of hPL release, we have characterized the binding of [125I]HDL3 to an enriched fraction of hPL-producing trophoblast cells. Binding studies were performed on trophoblast cells isolated by isopycnic centrifugation of collagenase/hyaluronidase-dispersed placental tissue and apo-E free-HDL3 (density, 1.125-1.215 g/ml). Scatchard analysis of binding studies performed at 37 C for 2 h revealed two classes of binding sites: 1) high affinity binding sites with a Kd of 9.7 +/- 2.2 micrograms/ml (1.3 x 10(-7) M) and 9.8 +/- 3.2 x 10(5) binding sites/trophoblast cell, and 2) low affinity binding sites with a Kd of 172.8 +/- 64.8 micrograms/ml (2.3 x 10(-6) M) and an estimated 3.2 x 10(6) sites/cell. As has been found in hepatocytes and other cells, the number of HDL3-binding sites per trophoblast cell (but not the binding affinity) decreased at lower incubation temperatures. In addition, HDL3 binding to trophoblasts cells did not require calcium and was not affected by prior treatment of the cells with pronase or trypsin. HDL3-binding sites on trophoblast cells, however, were not specific for HDL3. Low density lipoprotein (density, 1.063-1.055 g/ml), which does not stimulate hPL release, was nearly as potent on a molar basis as HDL3 in binding to the high and low affinity binding sites on trophoblast cells. Furthermore, nitrated HDL3, which does not compete for high affinity binding to trophoblast cells, stimulated hPL release. Although the characteristics of HDL3 binding to trophoblast cells are similar to those of other cells, these results strongly suggest that the binding of HDL3 to high affinity binding sites is not essential for HDL-mediated hPL release.

Synthetic amphipathic peptides resembling apolipoproteins stimulate the release of human placental lactogen.

Previous studies from our laboratory demonstrated native high density lipoproteins and apolipoproteins AI, AII, and CI, stimulate the release of human placental lactogen (hPL) from trophoblast cells in culture. To examine the mechanisms by which these apolipoproteins stimulate hPL release, we have studied hPL secretion in response to several synthetic peptide analogs of the amphipathic helical structure of the apolipoproteins. The magnitude of the stimulation of hPL release in response to the analog peptides correlated with the ability to displace apolipoproteins from high density lipoprotein and with other measures of phospholipid binding affinity such as the increase in alpha-helicity and the size of complexes formed between the peptide and phospholipid. The correlation of stimulatory ability and lipid affinity suggests that the action of the apolipoproteins on hPL release may be mediated through an interaction with plasma membrane phospholipids.

High density lipoproteins stimulate placental lactogen release and adenosine 3',5'-monophosphate (cAMP) production in human trophoblast cells: evidence for cAMP as a second messenger in human placental lactogen release.

Recent studies from our laboratory indicate that high density lipoproteins (HDL) and apolipoproteins-AI, -AII, and -CI stimulate the release of human placental lactogen (hPL) from human placental explants and trophoblast cells. However, the intracellular mechanisms by which HDL stimulate hPL release are unknown. Since earlier investigations from our laboratory indicated that pharmacological agents that increase intracellular cAMP levels [such as (Bu)2cAMP, isobutylmethylxanthine, and cholera toxin] also stimulate hPL release, we have performed studies to determine whether the stimulation of hPL release by HDL may be mediated by cAMP. Exposure of an enriched fraction of enzymatically dispersed hPL-producing cells to HDL (10-1500 micrograms/ml) for 0.5 h resulted in a significant dose-dependent stimulation of both cAMP production and hPL release. The half-maximal effective HDL dose for cAMP production and hPL release was 60-100 micrograms/ml. When perifused trophoblast cells were exposed to HDL (150 micrograms/ml) for 25 min, the increase in cAMP production preceded the increase in hPL release. The increase in cAMP production occurred during the first 5 min of exposure to HDL and reached a maximum (12.1 +/- 1.6-fold greater than base-line levels) 10 min after exposure. The increase in hPL release occurred about 10 min after the initial increase in cAMP production, reaching a maximum (9.7 +/- 1.4-fold greater than base-line levels) 15 min after exposure to HDL. HDL (300 micrograms/ml) also stimulated a 2.7-fold (P less than 0.001) increase in adenylate cyclase activity in placental plasma membranes, a magnitude of stimulation comparable to that caused by epinephrine (1 mM). Apolipoprotein-AII stimulated cAMP formation and hPL release in trophoblast cells with a pattern identical to that of HDL. These results, the first to demonstrate an effect of HDL on adenylate cyclase activity and cAMP production, strongly suggest a role for cAMP as a second messenger in HDL-stimulated hPL release.