title: Instability of IGF-IGFBP complex as a cause of the different performance of serum and EDTA-plasma after storage: EDTA-plasma is preferable for evaluating bioactive IGF especially in the mouse.
abstract: OBJECTIVE
  The insulin-like growth factor (IGF) signaling pathway is recognized as a potential target for treating several cancers, and strategies targeting the IGF type 1 receptor (IGF-1R) have been evaluated in many clinical trials. These suggested that the pretreatment level of circulating free IGF gives an estimate of IGF bioactivity and might be a predictive biomarker of the response to anti-IGF-1R antibodies. However, there is no defined protocol for measuring free and bioactive IGF concentrations, partly because the measurement procedures, including sample collection and handling, have not been standardized. We investigated the effects of sample collection methods and storage conditions on bioactive IGF measurement using a modified kinase receptor activation (KIRA) assay in human and mouse samples.
  
  
  METHODS
  Blood samples were obtained from healthy men and women, and from healthy male and female wild-type BALB/c mice. Serum and ethylenediaminetetraacetic acid (EDTA)-plasma samples were collected and used immediately or stored in small quantities at 4 °C or -80 °C for 3, 7, or 14 days. A bioassay directed against the phosphorylated IGF-1R using western blot analysis was developed as a modification of the KIRA assay, in which the level of phosphorylation of IGF-1R represented the IGF bioactivity in blood samples.
  
  
  RESULTS
  The levels of bioactive IGFs in mouse serum stored at 4 °C increased markedly in a time-dependent manner; the increase was slightly reduced in samples stored at -80 °C. Analysis of mouse EDTA-plasma stored at 4 °C showed a similar pattern, but the time-dependent increase was less than in the serum samples. By contrast, the levels of bioactive IGFs in EDTA-plasma stored at -80 °C were stable over 14 days. The levels of human bioactive IGFs in both serum and EDTA-plasma stored at 4 °C increased slightly with time, but the increases were much smaller than in mouse samples. The levels of human bioactive IGF in both serum and EDTA-plasma stored at -80 °C were stable over 14 days.
  
  
  CONCLUSIONS
  The use of EDTA-plasma avoids the problems with long-term storage. Therefore, EDTA-plasma should be used when measuring circulating IGF bioactivity, especially in mouse samples. All samples should be stored at -80 °C when long-term storage is unavoidable. Because of the large difference in the stability of the IGF-IGF-binding protein complex between the human and mouse in vitro, all samples should be handled carefully to ensure the accurate evaluation of IGF bioactivity, especially in mouse samples.
authors.affiliation: Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Setoyama
authors.affiliation: Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Electronic address: shmiyamo@kuhp.kyoto-u.ac.jp.
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Miyamoto
authors.affiliation: Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Nikaido
authors.affiliation: Department of Therapeutic Oncology, Kyoto University Graduate School of Medicine, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Muto
authors.affiliation: Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Chiba
mesh_terms: D000328:Adult; D000818:Animals; D001774:Blood Chemical Analysis / Q000379:methods*; D001800:Blood Specimen Collection / Q000379:methods; D004492:Edetic Acid; D005260:Female; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000097:blood*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D046912:Multiprotein Complexes / Q000097:blood; D011480:Protease Inhibitors / Q000032:analysis; D055550:Protein Stability; D013002:Somatomedins / Q000032:analysis*; D013045:Species Specificity
chemical_list: D018969:Insulin-Like Growth Factor Binding Proteins; D046912:Multiprotein Complexes; D011480:Protease Inhibitors; D013002:Somatomedins; D004492:Edetic Acid
keywords: Bioactive IGF; Free IGF; IGF-binding protein (IGFBP); IGF–IGFBP complex; Insulin-like growth factor (IGF); Kinase receptor activation (KIRA) assay