PLASMA-CLEARANCE OF CHICKEN AND HUMAN INSULIN-LIKE GROWTH FACTOR-I AND THEIR ASSOCIATION WITH CIRCULATING BINDING-PROTEINS IN CHICKENS

We have investigated the clearance of 125I-labelled chicken and recombinant human insulin-like growth factor-I (IGF-I) from the circulation of chickens as well as the role that IGF-binding proteins play in this process. Analysis of plasma samples by high-performance liquid chromatography (HPLC) neutral gel permeation on a TSK G3000SW column indicated that the i.v. injected radioactivity was rapidly partitioned between at least three pools. Most of the radioactivity occurred in a complex with binding protein(s), while smaller amounts of radioactivity chromatographed in the free IGF-I peak or appeared as low molecular weight degradation products. The labelled chicken and human IGF-I were rapidly cleared during the first 90 min. The calculated half-life for total labelled IGF-I during this period was 54 min for the chicken tracer and 33 min for the human tracer. The clearance was monitored for 10 h during which the human tracer continued to be cleared more rapidly than the chicken tracer. The proportion of radioactivity appearing as low molecular weight degradation products increased with time. Acid gel permeation and reverse-phase HPLC of the binding protein-associated radioactivity demonstrated that the labelled IGF-I bound was intact IGF-I. Sephadex G-200 gel permeation chromatography of chicken plasma samples at pH 7.4 showed that the binding protein complex labelled in vivo with chicken IGF-I tracer had a molecular mass of 55 kDa. Furthermore, the tracer associated with the binding protein coeluted with the major peak of endogenous IGF-I, suggesting that the tracer was bound to the physiologically relevant binding protein. Similar results were obtained with Superose 12 chromatography. Human plasma chromatographed as expected with much of the immunoreactivity and the labelled IGF-I eluting with a mass of 150 kDa on both Sephadex G-200 and Superose 12 columns. Ligand blotting of plasma binding proteins with 125I-labelled IGF-I after first subjecting the samples to polyacrylamide gel electrophoresis and transfer to nitrocellulose confirmed that the predominant binding protein in chicken plasma differed from the major forms in humana plasma.