A Fluorescence-Based Histidine-Imidazole Polyacrylamide Gel Electrophoresis (HI-PAGE) Method for Rapid and Practical...

Background: Polyacrylamide gel electrophoresis (PAGE) has long been used for lipoprotein analysis, enabling the separation and profiling of lipoprotein fractions such as LDL and HDL. However, conventional disc PAGE systems are limited by low throughput and inability to directly compare multiple samples under identical conditions. Alternative methods, including high-performance liquid chromatography and agarose gel electrophoresis, require specialized equipment and expertise, limiting their clinical utility. Methods: We present a colorimetric and fluorescence-based histidine-imidazole PAGE (HI-PAGE) system that provides rapid, cost-effective, and reproducible separation and profiling of lipoproteins in human serum. By combining electrophoretic separation with lipid-specific fluorescent staining using Nile Red, the fluorescence-based HI-PAGE (fHI-PAGE) not only visualizes distinct migration patterns of lipoprotein fractions, but also enables the quantification of LDL-cholesterol (LDL-C). Clear resolution of LDL and other lipoprotein fractions was achieved within 1 h without band distortion, allowing for direct comparison of multiple samples on a single gel. Results: We validated fHI-PAGE using serum from healthy individuals and patients, demonstrating that its fluorescence-based detection was more sensitive than conventional Sudan Black B staining while providing LDL-C estimates concordant with values calculated by the Friedewald formula. Moreover, fHI-PAGE proved advantageous in cases of hypertriglyceridemia, where Friedewald calculations are unreliable. Conclusions: These findings establish fHI-PAGE as a practical and clinically applicable platform for simultaneous lipoprotein profiling and LDL-C quantification.