COMPARISON OF EXTRACTION METHODS FOR INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS PRIOR TO MEASUREMENT OF INSULIN-LIKE GROWTH-FACTOR-I IN UNDERNOURISHED NEONATAL AND ADULT-RAT SERUM

Insulin-like growth factors (IGFs) are considered to be endocrine regulators during development, and different species have been used for the study of these factors during te perinatal period. The neonatal rat is a very useful model widely utilized to study endocrine alterations throughout the developmental period; few references in the literature present the neonatal rat as a model for the study of IGFs however. This study was undertaken to compare two extraction methods, acid-ethanol cryoprecipitation (A-EC) and formic acid-acetone (FA), for the removal of IGF-binding proteins (IGFBPs) from neonatal and adult rat serum in ted (control) and undernourished populations prior to measurement of IGF-I by radioimmunoassay (RIA). The IGF-I values obtained by PIA following A-EC or FA extraction were compared with those obtained following gel filtration (GF), which is considered to be the reference method. Western ligand blotting was used to determine IGFBPs in unextracted serum and after AEC or FA extraction of serum from rats of 10 and 20 days of age and adult rats in both populations. Although serum IGF-I levels after A-EC or FA in adult control rats were comparable with those obtained following GF, a significant correlation was found only after AEC extraction both in fed and undernourished adult rats. During the neonatal period, at 10 and 20 days, serum IGF-I levels after AEC or FA extraction were very different from those obtained after GF, especially in undernourished populations, and the correlation was very poor at 10 and 20 days of age in both populations studied. The IGFBP radioligand blots showed a different rate of change in control and undernourished rats during the period studied. Changes in IGFBP-3 and the band at 30 kDa are reported in both control and undernourished animals. The different proportions of the three major IGFBPs remaining after A-EC or FA extraction were also shown. We conclude that AEC extraction, originally validated for use in human serum, is also satisfactory for use in adult but not in neonatal rat serum. However, FA extraction was not a useful method for the rat serum. In neonatal rat serum, the only reliable extraction method was GF.