Interference by pro-apolipoprotein A-I in apolipoprotein E phenotyping using chemical precipitation procedures

Area of Science:

• Biochemistry
• Lipid Metabolism
• Protein Analysis

Background:

• Apolipoprotein E (ApoE) isoforms are crucial in lipoprotein metabolism and cardiovascular disease.
• Standard methods for lipoprotein isolation and phenotyping can be subject to artifacts.
• Isoelectric focusing is a common technique for ApoE phenotyping.

Purpose of the Study:

• To investigate discrepancies in apolipoprotein E isoform phenotyping between precipitation and ultracentrifugation methods.
• To identify the cause of observed differences in ApoE isoform patterns.
• To recommend improved methods for accurate lipoprotein phenotyping.

Main Methods:

• Isolation of lipoproteins (d < 1.063 g/mL) using polyanion-cation precipitation.
• Isoelectric focusing by rod-gel electrophoresis for ApoE isoform analysis.
• Two-dimensional gel electrophoresis (SDS-PAGE) and immunoelectrophoresis against anti-ApoA-I.
• Comparison with ultracentrifugally-derived very low-density lipoproteins (VLDL).

Main Results:

• Precipitation methods yielded different ApoE isoform patterns compared to ultracentrifugation.
• The most frequent observation was an increased frequency of the E3 isoform in precipitated lipoproteins.
• Two-dimensional analysis revealed contamination with pro-apolipoprotein A-I as the cause of the artifact.
• Contamination was identified using SDS-PAGE and immunoelectrophoresis.

Conclusions:

• Polyanion-cation precipitation of lipoproteins can lead to inaccurate ApoE phenotyping.
• Contamination with pro-apolipoprotein A-I is responsible for the observed increase in the E3 isoform frequency.
• Two-dimensional techniques are essential for accurate ApoE phenotyping when using precipitated lipoproteins.