A homogeneous assay to determine high-density lipoprotein subclass cholesterol in serum

Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL₂-C and HDL₃-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL₂, HDL₃, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL₃-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL₂, LDL, and VLDL increased, but the size of HDL₃ fraction particles remained constant, allowing only HDL₃-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.