Graham T E, Wason C J, Blüher M, Kahn B B
Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center, 99 Brookline Avenue/Research North, Rm. 380C, Boston, MA 02215, USA.
Diabetologia. 2007 Apr;50(4):814-23. doi: 10.1007/s00125-006-0557-0. Epub 2007 Feb 9.
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.
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.
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/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.
目的/假设：在显性糖尿病发生之前，胰岛素抵抗的人类受试者血清中视黄醇结合蛋白（RBP4）水平就已升高。RBP4水平与胰岛素抵抗、体重指数、腰臀比、血脂异常和高血压相关。运动训练改善胰岛素敏感性与血清RBP4水平降低有关。因此，血清RBP4可能有助于胰岛素抵抗的早期诊断以及监测胰岛素敏感性的改善情况。我们试图确定用于该应用的检测方法的性能。
我们比较了定量蛋白质免疫印迹法和三种市售的多孔免疫分析法，平行测量了胰岛素敏感受试者以及糖耐量受损或2型糖尿病的胰岛素抵抗受试者血清中的RBP4浓度。
这些检测方法得出的血清RBP4绝对值和升高幅度不同。蛋白质免疫印迹法和夹心酶联免疫吸附测定法报告的RBP4浓度与通过正常血糖高胰岛素钳夹法测得的胰岛素敏感性高度负相关。然而，蛋白质免疫印迹法得出的浓度具有更大的动态范围，且对照组和胰岛素抵抗组受试者之间的重叠较少。两种竞争性酶联免疫分析法低估了胰岛素抵抗受试者的血清RBP4浓度，这可能是由于检测饱和所致。稀释的线性不佳也限制了检测方法的实用性。所有检测的方法对尿中（C端蛋白水解的）RBP4的免疫反应性均高于血清中主要形式的全长RBP4。
结论/解读：这些发现支持将标准化为全长RBP4蛋白的定量蛋白质免疫印迹法用作在胰岛素抵抗状态下测量血清RBP4的“金标准”方法。其他检测方法应使用全长RBP4，并使用其他方法进行广泛的交叉验证。
