Development of a simple valid method for the complete removal of insulin-like growth factor (IGF)-binding proteins from IGFs in human serum and other biological fluids: comparison with acid- @entity1068 treatment and XXXX Sep-Pak separation.

@entity1 @entity2301 @entity1219 @entity442 @entity5100 @entity6173 @entity2975 @entity1068 @entity1543
Insulin-like growth factor (IGF)s circulate in plasma as large mol wt proteins bound to specific proteins, termed IGF-binding proteins (IGFBPs). 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- @entity1068 and @entity6173 Sep-Pak extraction methods, the two most widely used procedures for separation of IGFs from IGFBPs in human serum samples, with the established gold standard (Sephadex G-75 acid gel filtration) revealed that a significant amount of IGFBP activity survived the acid- @entity1068 extraction and @entity6173 Sep-Pak separation techniques. We, therefore, have developed a simple novel method comprising a combination of two techniques, involving separation based on size and separation based on centrifugation. In this method, serum samples were acidified and applied to Bio-Spin columns containing BSA-pretreated Bio-Gel @entity2975 -10 (P-10). Upon centrifugation, IGFBPs eluted in the void volume. IGFs were then eluted with 1 mol/L @entity1543 containing 0.1 mol/L @entity1219 upon subsequent centrifugation. The efficacy of Bio-Spin P-10 separation for the complete removal of IGFBPs was determined by Western ligand blot analysis and determination of @entity2301 levels by RIA in the extracted serum samples. The recovery of exogenously added @entity442 to the serum samples was greater than 90%. Comparison of @entity442 and @entity5100 values determined in 12 human serum samples after Bio-Spin P-10 separation with those obtained after separation with the established gold standard method (Sephadex G-75) revealed a correlation greater than 0.9. In contrast to the established gold standard method, which is tedious and time consuming, Bio-Spin P-10 separation offers the advantage 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 the @entity442 and @entity5100 levels in 14 normal and 15 age-matched postmenopausal osteoporotic @entity1 revealed that 1) both @entity442 and @entity5100 levels were reduced by 30% (P < 0.01) and 20% (P < 0.05), respectively, in osteoporotics; and 2) both @entity442 and @entity5100 levels correlated with bone mineral density in the pooled data from normal and osteoporotic populations even when age was held constant (P < 0.05).
