A two-step homogeneous assay for apolipoprotein E-containing high-density lipoprotein-cholesterol

Area of Science:

• Lipid metabolism and cardiovascular research
• Development of diagnostic biochemical assays
• Biochemistry of lipoproteins

Background:

• Apolipoprotein E-containing high-density lipoprotein (ApoE-HDL) exhibits antiatherogenic properties in vitro.
• A need exists for a homogeneous assay to quantify ApoE-HDL concentrations for in vivo studies.
• Current methods for ApoE-HDL measurement lack the homogeneity required for widespread in vivo application.

Purpose of the Study:

• To develop and validate a homogeneous assay for the selective measurement of apolipoprotein E-containing high-density lipoprotein cholesterol.
• To assess the assay's performance in distinguishing physiological from pathological ApoE-HDL cholesterol levels.
• To enable the study of ApoE-HDL's role in physiological conditions using standard autoanalyzers.

Main Methods:

• A two-step homogeneous assay was designed, involving initial elimination of interfering lipoproteins.
• Apolipoprotein E-containing high-density lipoprotein cholesterol was quantified in the second step.
• Assay performance was validated against a 13% polyethylene glycol precipitation assay (control assay).

Main Results:

• The homogeneous assay demonstrated good performance in validation studies, correlating significantly with the control assay (r=0.824) in subjects with normal liver function (n=78).
• The assay successfully recovered cholesterol from physiological ApoE-HDL in healthy individuals (n=12) but not from pathological ApoE-HDL in cholestatic patients (n=6).
• Median ApoE-HDL cholesterol levels were 0.10 mmol/L (homogeneous) vs. 0.05 mmol/L (control) in healthy individuals and 0.02 mmol/L (homogeneous) vs. 0.03 mmol/L (control) in cholestatic patients.

Conclusions:

• The developed two-step homogeneous assay allows for the selective measurement of physiological ApoE-HDL cholesterol using common autoanalyzers.
• This assay facilitates further investigation into the role of ApoE-HDL in various physiological states.
• The assay's ability to differentiate between physiological and pathological ApoE-HDL provides a valuable tool for clinical and research applications.