title: Shortcomings in methodology complicate measurements of serum retinol binding protein (RBP4) in insulin-resistant human subjects.
abstract: OBJECTIVE
  Levels of retinol binding protein (RBP4) are increased in the serum of insulin-resistant human subjects even before overt diabetes develops. RBP4 levels correlate with insulin resistance, BMI, WHR, dyslipidaemia and hypertension. Improvement of insulin sensitivity with exercise training is associated with reduction in serum RBP4 levels. Therefore serum RBP4 may be useful for early diagnosis of insulin resistance and for monitoring improvements in insulin sensitivity. We sought to determine the performance of assays for this application.
  
  
  METHODS
  We compared quantitative western blotting and three commercially available multiwell immunoassays in parallel measurements of RBP4 concentrations in serum from insulin-sensitive subjects and from insulin-resistant subjects with impaired glucose tolerance or type 2 diabetes.
  
  
  RESULTS
  The assays yielded different absolute values and magnitudes of elevation of serum RBP4. Western blotting and a sandwich ELISA reported RBP4 concentrations that highly inversely correlated with insulin sensitivity measured by euglycaemic-hyperinsulinaemic clamp. However, western blotting yielded concentrations with a greater dynamic range and less overlap between control and insulin-resistant subjects. Two competitive enzyme-linked immunoassays undervalued serum RBP4 concentrations in insulin-resistant subjects, possibly due to assay saturation. Poor linearity of dilution also limited assay utility. All assays tested exhibited greater immunoreactivity with urinary (C-terminal proteolysed) RBP4 than with full-length RBP4, the predominant form in serum.
  
  
  CONCLUSIONS
  These findings support the use of quantitative western blotting standardised to full-length RBP4 protein as a 'gold standard' method for measuring serum RBP4 in insulin-resistant states. Other assays should use full-length RBP4 and be extensively cross-validated using other methods.
authors.affiliation: Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center, 99 Brookline Avenue/Research North, Rm. 380C, Boston, MA 02215, USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Graham
authors.forename: C J
authors.initials: CJ
authors.lastname: Wason
authors.forename: M
authors.initials: M
authors.lastname: Blüher
authors.forename: B B
authors.initials: BB
authors.lastname: Kahn
mesh_terms: D001786:Blood Glucose / Q000032:analysis; D015153:Blotting, Western; D003924:Diabetes Mellitus, Type 2 / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005951:Glucose Tolerance Test; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D007328:Insulin / Q000097:blood / Q000378:metabolism*; D007333:Insulin Resistance; D024821:Metabolic Syndrome / Q000378:metabolism; D009765:Obesity / Q000097:blood / Q000378:metabolism; D015203:Reproducibility of Results; D012177:Retinol-Binding Proteins / Q000032:analysis* / Q000096:biosynthesis*; D054839:Retinol-Binding Proteins, Plasma; D014801:Vitamin A / Q000378:metabolism
chemical_list: D001786:Blood Glucose; D007328:Insulin; C101326:RBP4 protein, human; D012177:Retinol-Binding Proteins; D054839:Retinol-Binding Proteins, Plasma; D014801:Vitamin A