Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Area of Science:

• Clinical Chemistry
• Biochemistry
• Lipid Metabolism

Background:

• Traditional sequential salt density flotation for lipoprotein separation is time-consuming (approx. 72 hours) and yields variable recovery.
• Iodixanol-based self-generating density gradients offer a faster alternative for lipoprotein fractionation.

Purpose of the Study:

• To evaluate a novel three-layered iodixanol gradient for rapid and high-resolution separation of lipoprotein fractions.
• To compare the efficacy of iodixanol fractionation with traditional sequential flotation ultracentrifugation.

Main Methods:

• A three-layered iodixanol density gradient was developed and tested.
• Lipoprotein fractions from 63 subjects with dyslipidemia were analyzed using the iodixanol gradient.
• Measurements included cholesterol, triglycerides, and apolipoproteins in 21 successive fractions.

Main Results:

• The iodixanol gradient demonstrated high reproducibility (CV 0.29%) and excellent total lipid recovery (95.4% cholesterol, 84.7% triglyceride).
• Recoveries for VLDL, LDL, and HDL components were approximately 10% higher with iodixanol compared to sequential flotation.
• Improved resolution was observed, enabling discrimination of classic lipoproteins and subfractions, including small, dense LDL particles correlated with density >1.028 g/dl.

Conclusions:

• The novel iodixanol density gradient provides rapid (approx. 4 hours) separation of lipoproteins with enhanced resolution and recovery.
• This method offers significant advantages over traditional ultracentrifugation, minimizing in-vitro modification.
• It yields valuable information on LDL phenotype and subfractions in a single centrifugation step.