Shortcomings in methodology complicate measurements of serum retinol binding protein (RBP4) in insulin-resistant human subjects

Abstract

Aims/hypothesis 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. Subjects and 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 insulinresistant 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 fulllength RBP4, the predominant form in serum. Conclusions/interpretations 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.

Retinol-binding protein 4 is associated with components of the metabolic syndrome, but not with insulin resistance, in men with type 2 diabetes or coronary artery disease

“…Hence, we used this method to validate the results obtained by sandwich ELISA in our patients and control subjects. Consistent with the previous data [8], serum RBP4 levels detected by western blot were significantly higher than the concentrations obtained by ELISA in all patient groups (Fig. 1b).…”

“…Yet we found close correlations between the two techniques in all patient subgroups studied. Interestingly, in accordance with the previous data [8], RBP4 levels detected by immunoblot were significantly higher than in ELISA (Fig. 1b).…”

“…A comparison of commercially available multiwell immunoassays with quantitative western blotting using full-length RBP4 protein as standard suggested that the latter is superior [8]. Yet we found close correlations between the two techniques in all patient subgroups studied.…”

“…Compared with control subjects, CAD patients were older and had lower mean HDLcholesterol and adiponectin levels, as well as increased HOMA-IR and insulin levels. Serum RBP4 levels were significantly lower in patients with CAD than in control subjects and patients with type 2 diabetes (CAD: 27.8 [22.5- Comparison of RBP4 measurement by ELISA and quantitative Western blot analyses Quantitative Western blots were previously described as the 'gold standard' for quantification of RBP4 [8]. Hence, we used this method to validate the results obtained by sandwich ELISA in our patients and control subjects.…”

“…Quantitative Western blotting of serum RBP4 Modified immunoblotting was performed in 40 patients included in this study (10 patients with type 2 diabetes, 10 non-insulinresistant CAD patients, 10 insulin-resistant CAD patients and 10 control subjects) as previously described in detail [8] using human recombinant full-length RBP4 (3378-LC; R&D Systems, Wiesbaden, Germany) as standard. Standard solutions of 15, 30, 60 or 120 μg/ml RBP4 were prepared in Tris-buffered saline containing 1% NP-40.…”