Selective Inhibition of Insulin-Degrading Enzyme Eliminates Hemolysis Interference in Serum Insulin Measurements

Objectives. Hemolysis significantly interferes with insulin measurements in clinical settings, leading to inaccurate results. Although the activity of insulin-degrading enzyme (IDE) is assumed to be the primary mechanism, the potential involvement of additional mechanisms remains unclear. This study aims to determine if IDE is the sole cause of this interference by using a selective IDE inhibitor, 6bK, and to explore whether this inhibition can completely prevent hemolysis-related inaccuracies in insulin assays. Methods. The effects of 6bK on insulin degradation were evaluated in hemolyzed and non-hemolyzed serum samples at room temperature, following the CLSI guidelines EP07-A2 and C56-A. Insulin levels were measured using chemiluminescent immunoassays. Additional assessments included the impact of 6bK on serum C-peptide, proinsulin, and standard biochemical parameters. The effects of 6bK were also evaluated at 4 °C and after 21 days of storage at room temperature prior to use. Results. Hemolysis caused a significant decrease in insulin concentrations, dependent on hemolysate levels and incubation time. The addition of 10 µM 6bK completely reversed hemolysis-induced insulin degradation in serum across a broad range of insulin baseline concentrations and degrees of hemolysis. Furthermore, 6bK did not affect insulin levels in non-hemolyzed samples or alter the quantification of C-peptide, proinsulin, or standard biochemical parameters. Conclusions. The decrease in serum insulin concentration due to hemolysis is exclusively attributed to the action of IDE. Selective inhibition of IDE by 6bK effectively eliminates hemolysis-induced interference in insulin measurements, providing a novel and reliable solution for accurate insulin quantification in hemolyzed clinical samples.