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 pH is a convenient method to remove the insulin-like growth factor binding proteins (IGFBPs); however, an incomplete removal of IGFBPs can occur and this can potentially interfere with the radioimmunoassay of insulin-like growth factors (IGFs). This study compared the removal of IGFBPs 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) treatments by western ligand blotting and slot-blotting analysis. In adult rat serum all three extraction methods removed nearly 75% of total IGFBPs present. For the neonatal serum, AE and FAA were very inefficient in eliminating the IGFBPs, while ACE was somewhat better, as it removed nearly 30% of IGFBPs. Ligand blots of extracted samples showed that IGFBPs of lower size range, 24 to 32 kDa (IGFBP-4, IGFBPs-1 and -2), were resistant to solvent extraction. Acid-gel filtration, in contrast, eliminated > 95% of IGF-binding components in both sera. Determination of IGF-I concentrations in samples after gel filtration and extraction methods revealed lower IGF-I values in neonatal serum in acid extracted samples. These data caution against using solvent extractions for IGFBP removal in fetal/neonatal serum.
Srivenugopal
K
K
Endocrine-Metabolic Division, Veterans Affairs Medical Center, North Chicago, Illinois.
Singh
S P
SP
Yuan
X H
XH
Ehmann
S
S
Snyder
A K
AK
D000375:Aging / Q000097:blood; D000818:Animals; D000831:Animals, Newborn; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002850:Chromatography, Gel / Q000379:methods; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012997:Solvents
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D012997:Solvents; D007334:Insulin-Like Growth Factor I