DEVELOPMENT OF A SIMPLE VALID METHOD FOR THE COMPLETE REMOVAL OF INSULIN-LIKE GROWTH-FACTOR (IGF)-BINDING PROTEINS FROM IGFS IN HUMAN SERUMAND OTHER BIOLOGICAL-FLUIDS - COMPARISON WITH ACID-ETHANOL TREATMENT AND C-18 SEP-PAK SEPARATION

Insulin-like growth factor (IGF)s circulate in plasma as large mol wt proteins bound to specific proteins, termed IGF-binding proteins (IGFB Ps). As IGFBPs have been shown to produce artifacts in IGF radioligand assays, various extraction procedures have been proposed to eliminate IGFBPs from biological samples before radioligand assays. Comparison of acid-ethanol and C-18 Sep-Pak extraction methods, the two most wide ly used procedures for separation of IGFs from IGFBPs in human serum s amples, with the established gold standard (Sephadex G-75 acid gel fil tration) revealed that a significant amount of IGFBP activity survived the acid-ethanol extraction and C-18 Sep-Pak separation techniques. W e, therefore, have developed a simple novel method comprising a combin ation of two techniques, involving separation based on size and separa tion based on centrifugation. In this method, serum samples were acidi fied and applied to Bio-Spin columns containing BSA-pretreated Bio-Gel Polyacrylamide-10 (P-10). Upon centrifugation, IGFBPs eluted in the v oid volume. IGFs were then eluted with 1 mol/L acetic acid containing 0.1 mol/L NaCl upon subsequent centrifugation. The efficacy of Bio-Spi n P-10 separation for the complete removal of IGFBPs was determined by Western ligand blot analysis and determination of IGFBP-3 levels by R IA in the extracted serum samples. The recovery of exogenously added I GF-I to the serum samples was greater than 90%. Comparison of IGF-I an d IGF-II values determined in 12 human serum samples after Bio-Spin P- 10 separation with those obtained after separation with the establishe d gold standard method (Sephadex G-75) revealed a correlation greater than 0.9. In contrast to the established gold standard method, which i s tedious and time consuming, Bio-Spin P-10 separation offers the adva ntage of speed, such that 50 or more samples can be processed in less than 4-6 h. Application of Bio-Gel P-10 gel filtration to determine th e IGF-I and IGF-II levels in 14 normal and 15 age-matched postmenopaus al osteoporotic women revealed that 1) both IGF-I and IGF-II levels we re reduced by 30% (P < 0.01) and 20% (P < 0.05), respectively, in oste oporotics; and 21 both IGF-I and IGF-II levels correlated with bone mi neral density in the pooled data from normal and osteoporotic populati ons even when age was held constant (P < 0.05).