GH Secretion in children. Methodological aspects of determining immunoreactive and bioactive isoforms

Abstract: A child's hormone-dependent growth is dependent on multiple factors, although linear growth is closely associated with levels of growth hormone (GH) secretion. As GH in serum exists in several isoforms, some of which may not be identified by immunoassay, possible alterations in the secretion of these different isoforms in childhood may influence growth and/or metabolism. Changes in GH sensitivity may also affect growth. These changes may be primary, i.e. caused by defects in the GH receptor (GHR) or defects further along the GH ? insulin-like growth factor 1 (IGF-I) axis, or secondary, resulting from a variety of illnesses or malnutrition affecting various steps in the pathway from GH binding to IGF-I action.The general aim of the work reported in this thesis was to improve the diagnostic tools available to evaluate GH secretion in children.This work attempted to characterise immunoreactive serum GH measurements. In addition, the proportion of non-22 kDa GH isoforms in children of short stature and in children of normal stature was evaluated. This indicated that GH measurements should indeed be standardised, and assays cross-reactivity patterns explored to provide well-defined methods that can be used for screening children. It was found that the ratio of the total amount of GH compared with the amount of the 22 kDa isoform differed significantly between children of short stature and children of normal stature. The use of ratios on an individual level might serve as a valid screening method for identifying children for further studies. However, it is important to stress that immunoassays detect GH-like molecules in biological samples that interact with the antibodies used in the particular assay rather than measuring the biological activity of GH.The second part of this work focuses on the possibility of developing a bioassay that involves the concept of ligand-induced receptor dimerization, which is necessary for GH receptor activation. As a first step in the development of a cell-free assay we have constructed a soluble hybrid receptor consisting of the extracellular domain of the GH receptor linked to the intracellular domain of epidermal growth factor (EGF), containing a tyrosine kinase domain. A fusion protein has been constructed and expressed in a baculovirus system. The protein had GH-binding capacity as well as tyrosine kinase activity, indicating that both the GHR and the EGF receptor (EGFR) domains were functional when expressed as a chimeric protein. Further development of an assay that measures biologically active GH and is simple enough to use in a large-scale clinical setting would be a considerable improvement on present assay methods. Finally, a model was constructed that converted the IGF-I level of a child into an SD score. The objective was to produce reference values to be used in clinical practice in a model that would explain the variation in IGF-I levels throughout a child's growth phases without including too many variables. The study clearly showed that correct interpretation of IGF-I measurements is highly dependent on the relationship between age, gender and puberty.