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, xi 2, xi 3, xi 4 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 micrograms of protein in the sample. The results of 403 normo-or hyperlipidemic patients are shown. In the group of 191 normolipidemic patients (cholesterol less than 6.40 mmol/L triglycerides less than 2 mmol/L), the relative frequency of the xi 3 allele (0.83) is higher than in other reports on Caucasians (about 0.77) whereas the xi 4 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.
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Service de Biologie, Hôpital Neurologique et Neuro-Chirurgical Pierre Wertheimer, Lyon Montchat, France.
Sassolas
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D000178:Acrylamides; D000483:Alleles; D001057:Apolipoproteins E / Q000235:genetics*; D003543:Cysteamine; D014644:Genetic Variation / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007525:Isoelectric Focusing; D009439:Neuraminidase; D010641:Phenotype; D016622:Silver Staining
D000178:Acrylamides; D001057:Apolipoproteins E; D003543:Cysteamine; C059413:Immobiline; D009439:Neuraminidase