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-Ethanol Treatment and C,, Sep-Pak Separation*

Insulin-like growth factor (1GFJ.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-ethanol and C,, 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-ethanol extraction and C,, SepPak 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 Polyacrylamide-10 (P10). Upon centrifugation, IGFBPs eluted in the void volume. IGFs were then eluted with 1 mol/L acetic acid containing 0.1 mol/L NaCl 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 IGFBP-3 levels by RIA in the extracted serum samples. The recovery of exogenously added IGF-I to the serum samples was greater than 90% Comparison of IGF-I and IGF-II 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, BioSpin 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 IGF-I and IGF-II levels in 14 normal and 15 age-matched postmenopausal osteoporotic women revealed that 1) both IGFI and IGF-II levels were reduced by 30% (P i 0.01) and 20% (P < 0.051, respectively, in osteoporotics; and 2) both IGF-I and IGF-II levels correlated with bone mineral density in the pooled data from normal and osteoporotic populations even when age was held constant (P < 0.05). (J Clin Endocrinol Metab SO: 637-647, 1995) I NSULIN -like growth factor-I (IGF-I) and IGF-II, previously known as somatomedins, are structurally related to insulin and are the two most abundant polypeptide growth factors that circulate in human plasma. IGF-I and IGF-II are of recognized importance for regulating the proliferation and differentiation in a multitude of cell types, including cells derived from brain, thyroid, liver, muscle, kidney, blood, and bone. Many of these cell types have been shown to produce IGFs in culture, contain high affinity receptors for IGFs, and exhibit biological responses to exogenously added IGFs. Thus, IGFs have been proposed to play a significant role in local regulation, such that the IGFs produced by one cell type can act on the same cell type in an autocrine manner or on a neighboring cell type in a paracrine manner (l-5). IGFs circulate in plasma as large mol wt proteins bound to specific proteins, which are termed IGF-binding proteins Received June 8, 1994. Revision received October 10, 1994. Accepted October 25, 1994. Address all correspondence and requests for reprints to: Dr. Subburaman Mohan, Research Service, Pettig Veterans Administration Medical Center, 11201 Benton Street, Loma Linda, California 92357. * This work was supported by funds from the NIH (AR-31062), the V.A., and the Departments of Medicine and Pediatrics, Loma Linda University. (IGFBPs). Six different IGFBPs (designated IGFBP-1 through IGFBP-6) have been purified and cloned from a variety of human tissues thus far (6-8). Recent studies emphasize a number of important functions for IGFBPs, including 1) prevention of acute metabolic effects of free IGFs, specifically hypoglycemia; 2) increasing the half-life of the IGFs in the circulation; 3) regulation of the bioavailability of IGFs in the local areas; and 4) providing tissue specificity for the local actions of IGFs (6-8). The existence of these IGFBPs in serum and other biological fluids, such as conditioned medium, has complicated the application of specific radioligand assays (RIA and RRA) for measurement of IGFs, because IGFBPs can compete with the antibody or receptors for tracer binding (9-14). Previous studies have shown that IGF measurements made using methods in which IGFBP artifacts were not removed completely could lead to erroneous conclusions. For example, forskolin and dibutyryl CAMP treatment increased IGF-Iand IGF-II-like activities in the conditioned medium of TE85 human osteosarcoma cells; however, this increase in IGF radioligand activity was due to increased production of IGFBI’-4 and not IGF-I or IGF-II (15). Thus, for valid measurements of IGF concentrations, the dissociation of IGFs from by on April 14, 2008 jcem.endojournals.org Downloaded from 638 MOHAN AND BAYLINK JCE & M . 1995 Vol80. No 2 IGFBPs and complete elimination of IGFBPs from the sample must precede the measurement of IGFs with the radioligand assay. During the past few years, a number of strategies have been used to eliminate IGFBPs in human serum samples, including acid gel filtration, acid-ethanol extraction, acidethanol cryoprecipitation, C,, Sep-Pak separation, formic acid extraction, or Sephacryl extraction (ll-14,16-20). Many of these techniques are either inconvenient and time consuming or give incomplete separation of IGFBPs. Of the various procedures established for removal of IGFBPs, acidethanol extraction has been the most widely used because of its simplicity. However, the reliability of acid-ethanol treatment to prevent interference by IGFBPs in serum has been controversial, based on recent studies (14,211. In the present study, we, therefore, systematically examined the effectiveness of both acid-ethanol extraction and C,s Sep-Pak separation, the two most commonly used procedures, as means for the elimination of IGFBPs from human serum and compared the validity of these methods with the size-exclusion chromatography under acidic conditions, the established gold standard, for removal of IGFBPs in human serum before IGF radioligand assays. Finally, we describe the development and application of a simple valid method that can be used to completely separate IGFBP artifacts from IGFs in biological fluids. Materials and Methods