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 IGF-binding protein-3 (IGFBP-3) that exists in the circulation associated with IGF and an acid labile subunit to form a ternary (158-kDa) complex. It has been reported that heparin disrupts the IGF carrying capacity of the ternary complex and is a potent inhibitor of ternary complex 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 alters the molecular distribution or immunoreactive measurement of IGFBP-3 and IGF-I. Two different collection modalities were examined; protocol 1, blood was drawn and immediately centrifuged and aliquotted; and protocol 2, blood was drawn, left at room temperature for 2 h and then at 4°C overnight prior to centrifugation. Samples were drawn from a normal adult and from a growth hormone-deficient (GHD) child and subjected to neutral size-exclusion chromatography to separate the ternary 158-kDa complex from the binary IGFBP-3-IGF (approx 50 kDa) complex. Fractions were then subjected to Western ligand blot (WLB), western immunoblot (WIB), and measurement of IGFBP-3 by immunoradiometric assay (IRMA), while the IGF distribution was measured by radioimmunoassay (RIA) following acidic size-exclusion chromatography. In both serum and plasma of a normal adult, WLB detected a 45-40 kDa IGFBP-3 doublet eluting primarily within the 158-kDa IGFBP region (i.e., ternary complex). 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 chromatography. Measurement of IGFBP-3 by IRMA confirmed these findings. No difference between serum and plasma was detected in either collection protocol. RIA of IGF-I revealed that the ternary complex carried the majority of the circulating IGF-I and that there was no difference between serum and plasma. Assessment of serum and plasma of a GHD child showed reduced serum concentrations of IGFBP-3 but no difference in the IGFBP profiles between serum and plasma. These data demonstrate that the collection of blood in heparinized tubes does not alter the molecular distribution of forms of IGFBP-3 and IGF-I.