DIFFERENTIAL REMOVAL OF INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEINS IN RAT SERUM BY SOLVENT-EXTRACTION PROCEDURES

Solvent extraction of serum and other biological fluids at an acidic p H is a convenient method to remove the insulin-like growth factor bind ing proteins (IGFBPs); however, an incomplete removal of IGFBPs can oc cur and this can potentially interfere with the radioimmunoassay of in sulin-like growth factors (IGFs). This study compared the removal of I GFBPs from normal adult rat serum and 5-day old neonatal rat serum by acid-gel filtration, and three solvent extraction methods, i.e., acid- ethanol (AE), acid-cryo-ethanol (ACE) and formic acid-acetone (FAA) tr eatments by western ligand blotting and slot-blotting analysis. In adu lt rat serum all three extraction methods removed nearly 75% of total IGFBPs present. For the neonatal serum, AE and FAA were very inefficie nt in eliminating the IGFBPs, while ACE was somewhat better, as it rem oved nearly 30% of IGFBPs. Ligand blots of extracted samples showed th at IGFBPs of lower size range, 24 to 32 kDa (IGFBP-4, IGFBPs-1 and -2) , were resistant to solvent extraction. Acid-gel filtration, in contra st, eliminated >95% of IGF-binding components in both sera. Determinat ion of IGF-I concentrations in samples after gel filtration and extrac tion methods revealed lower IGF-I values in neonatal serum in acid ext racted samples. These data caution against using solvent extractions f or IGFBP removal in fetal/neonatal serum.