Margaret C. Stuart

Circulating insulin-like growth factor I levels in newborn premature and full-term infants followed longitudinally.

Longitudinal circulating levels of insulin-like growth factor I (IGF-I) were measured by radioimmunoassay after acid/ethanol extraction of serum or plasma in 44 appropriate-for-gestational age (AGA) premature infants, 7 small-for-gestational age (SGA) premature infants and 9 AGA full-term infants. The subjects were divided into cohorts with gestational age at birth 26-29 weeks, 30-33 weeks, 34-37 weeks and 38-42 weeks (full-term). The premature infants in this study exhibited diminished growth as compared with normal intrauterine growth. In all but the earliest premature infant cohort there was an immediate fall from the mean fetal IGF-I level, as reflected by the cord value, to a basal postnatal circulating level of IGF-I. The basal level of circulating IGF-I in premature infants was related only to gestational age. It increased slowly from 25 weeks gestation until four weeks after full-term equivalent and was independent of time of birth. Full-term infants were distinguished from early premature infants by the occurrence of a prominent postnatal surge in circulating IGF-I levels that was characterised by a significant (P less than 0.02) increase between day 1 and days 10-15. The SGA and AGA infants in the 34-37 week cohort showed similar profiles of circulating IGF-I with no significant difference in cord values between the two groups.

Fetal Bin Growth: Selective Action by Growth Hormone

Growth hormone was administered to pregnant rats maintained under dietary control, and fetal and placental growth and nutrition were examined. Growth hormone had a selective action on brain growth that could not be attributed to nutrient mobilization but suggested a trophic factor which is unique to the brain.

Effect of biosynthetic human growth hormone on insulin action in individual tissues of the rat in vivo

Excessive endogenous production or exogenous administration of human growth hormone (hGH) causes insulin resistance at both the hepatic and extrahepatic levels. However, which extrahepatic tissues are involved have not been defined. We have examined the diabetogenic action of authentic biosynthetic hGH on whole body glucose disposal, hepatic glucose output, and glucose metabolism in individual peripheral tissues. The use of a highly purified preparation of the hormone allowed us to examine the isolated effects of 22K hGH. The euglycemic hyperinsulinemic (approximately 100 mU/L) clamp plus 3H-2-deoxyglucose technique was used to quantitate the effects of hGH on insulin action in vivo. Administration of biosynthetic hGH at a dose of 10 IU/kg/24 h for 48 hours in male Wistar rats (approximately 340 g) produced a highly significant decrease in the steady state clamp glucose infusion rate (GIR) when compared with controls (8.1 +/- 0.6 v 18.7 +/- 0.7 mg/kg/min, P less than .001), reduced insulin-mediated suppression of hepatic glucose output (Ra) (3.9 +/- 0.6 v 0.7 +/- 0.3 mg/kg/min, P less than .05) and a decreased clamp glucose disposal rate (Rd) (12.0 +/- 0.4 v 18.10 +/- 1.1 mg/kg/min, P less than .001). There was a significant decrease in insulin-mediated glucose uptake as indicated by tissue accumulation of [3H]-2-deoxyglucose phosphorylation in diaphragm and hindlimb muscles. Insulin action was more substantially reduced in muscles (approximately 50%) than in adipose tissues (approximately 20%). These studies confirm that the diabetogenic action of hGH in the rat is due to a combination of inhibition of insulin suppression of hepatic glucose output and inhibition of the uptake and subsequent utilization of glucose in skeletal muscles.

Biogenic Amine Control of Growth Hormone Secretion in the Fetal and Neonatal Rat

The development of the hypothalamic-pituitary axis for growth hormone (GH) secretion has been studied in the rat fetus and in the neonate 4, 24, 48 and 72 h after birth. Injections of the serotonin blocker cyproheptadine (Cypro) and a catecholamine, dopamine (DA), each led to reductions in the level of serum GH in 21 to 22 day fetuses and in neonates up to 3 days after birth. The O-methylated derivative of dopamine, dimethoxyphenylethylamine (DMPEA), did not alter serum GH levels from those seen in saline-treated control animals. These results indicate that biogenic amines exert control over GH secretion in the fetus, close to term, and in early neonatal period. They suggest that this control is similar to that seen in the adult rat and in man and that such control may operate through serotonin receptors.

Specificity of two-site immunoassays.

When cross-reaction in two-site immunoassays was investigated both theoretically and experimentally it was found that such systems do not always result in enhanced specificity. Computer simulation studies indicated that substances which display negligible cross-reaction in a radioimmunoassay could produce an assay response identical to that of the analyte in a two-site immunoassay using excess antibody. Cross-reactivity in two differing two-site immunoassays was compared to that obtained in radioimmunoassays using the same monoclonal antibodies for human chorionic gonadotrophin. In addition to the effects of excess antibody, cross-reactivity was observed in one of the two-site immunoassays which could not have been predicted from the specificity of the antibodies or cross-reactivity in the radioimmunoassays. The unexpected cross-reaction of the beta subunit of human chorionic gonadotrophin in the assay resulted from an apparent alteration in the specificity of one of the antibodies following binding of the beta subunit to the second antibody. These studies emphasise the complexity of binding reactions in two-site immunoassays.

The production of high affinity monoclonal antibodies to human growth hormone

The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4 X 10(10) l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay.

Biological Activities of Recombinant Human Follicle Stimulating Hormone

Follicle stimulating hormone (FSH), a member of the glycoprotein hormone family produced in the anterior pituitary, plays a major role in the maturation of ovarian follicles. FSH is a 32-kD heterodimer consisting of 2 noncovalently linked subunits, α and β. Each subunit contains 2 asparagine-linked carbohydrate complexes that are required for full biological activity of the hormone (e.g., circulatory half-life, signal transduction, etc.). Variation in glycosylation patterns at these sites results in multiple hormone isoforms that possess differing biological activities.

The Evaluation of Imprecision in Collaborative Immunoassay Quality-Assessment Programmes

The calculation of within-laboratory imprecision in quality-assessment (QA) programmes normally involves combining data from different analyte concentrations to calculate an average standard deviation (SD) or coefficient of variation. However, for immunoassay neither of these parameters is concentration independent. This paper describes a method of calculating within-laboratory imprecision in QA programmes by assuming a linear relationship between SD and analyte concentration. This method is used in programmes conducted by the Australian Joint Working Party for Quality Control in Immunoassay to calculate imprecision at the limits of the reference range. Results from these programmes show that this method better represents the differences in imprecision between analytes, methods and laboratories than the calculation of a single imprecision parameter. The method is trivial for a computer and its robustness has been validated by Monte Carlo simulation. It is suggested that major differences in laboratory performance between different QA programmes may be due to inappropriate calculation of single imprecision parameters.