Apolipoprotein E phenotyping by isoelectric focusing in immobilized pH gradients and silver staining

A simple and high resolution procedure of apoprotein E (apo E) phenotyping by isoelectric focusing with immobilized pH gradients and silver staining is described. This method needs delipidated very low density lipoproteins (isolated from 1 mL of serum) but obviates immunoblotting as well as neuraminidase treatment in routine applications because the sialylated forms are clearly separated. Immunoblotting (with polyclonal and monoclonal anti-apo E antiserum), cysteamine and neuraminidase treatment, and pI markers allowed the localization of three main alleles, ξ2, ξ3, ξ4 and the detection of variants or rare alleles (6/450 determinations). The serum amyloid A (SAA) apolipoproteins (SAA1, SAA2) could be characterized unequivocally (especially with E3 and E4). Silver staining proved more sensitive than Coomassie Brilliant Blue and needs only 5 μg of protein in the sample. The results of 403 normo-or hyperlipidemic patients are shown. In the group of 191 normolipidemic patients (cholesterol < 6.40 mmol/L triglycerides < 2 mmol/L), the relative frequency of the ξ3 allele (0.83) is higher than in other reports on Caucasians (about 0.77) whereas the ξ4 allele is lower. As previously described, we find a high frequency of the 4/3 phenotype in hypercholesterolemia and 3/2 in hypertriglyceridemia. The high frequency of the E2/E2 phenotype, usually associated with hyperlipidemia, and variants in complex hypertriglyceridemia makes the apo E phenotyping necessary in many cases of dyslipidemias.