COLLECTION OF BLOOD IN HEPARINIZED TUBES DOES NOT ALTER THE MOLECULAR-DISTRIBUTION OR FORMS OF IGFBP-3 AND IGF

The major serum carrier of the insulin-like growth factors (IGFs) is I GF-binding protein-3 (IGFBP-3) that exists in the circulation associat ed with ICF and an acid labile subunit to form a ternary (158-kDa) com plex. It has been reported that heparin disrupts the IGF carrying capa city of the ternary complex and is a potent inhibitor of ternary compl ex reformation (Clemmons et al., 1983; Baxter, 1990). Thus, the aim of this study was to determine if, in a clinical setting where blood may be collected in both nonheparinized and heparinized tubes, heparin al ters the molecular distribution or immunoreactive measurement of IGFBP -3 and IGF-I. Two different collection modalities were examined: proto col 1, blood was drawn and immediately centrifuged and aliquotted; and protocol 2, blood was drawn, left at room temperature for 2 h and the n at 4 degrees C overnight prior to centrifugation. Samples were drawn from a normal adult and from a growth hormone-deficient (GHD) child a nd subjected to neutral size-exclusion chromatography to separate the ternary 158-kDa complex from the binary IGFBP-3-IGF (approx 50 kDa) co mplex. Fractions were then subjected to Western ligand blot (WLB), wes tern immunoblot (WIB), and measurement of IGFBP-3 by immunoradiometric assay (IRMA), while the IGF distribution was measured by radioimmunoa ssay (RIA) following acidic size-exclusion chromatography. In both ser um and plasma of a normal adult, WLB detected a 45-40-kDa IGFBP-3 doub let eluting primarily within the 158-kDa IGFBP region (i.e., ternary c omplex). Similarly, assessment of immunoreactive IGFBP-3 by WIB showed a 45-40-kDa IGFBP-3 doublet, as well as a 29 kDa immunoreactive form primarily eluting in the 158-kDa IGFBP region of the chromatograph. Me asurement of IGFBP-3 by IRMA confirmed these findings. No difference b etween serum and plasma was detected in either collection protocol. RI A of IGF-I revealed that the ternary complex carried the majority of t he circulating IGF-I and that there was no difference between serum an d plasma. Assessment of serum and plasma of a GHD child showed reduced serum concentrations of IGFBP-3 but no difference in the IGFBP profil es between serum and plasma. These data demonstrate that the collectio n of blood in heparinized tubes does not alter the molecular distribut ion or forms of IGFBP-3 and IGF-I.