A Simple Electrophoretic Method for Phenotyping Apo(a): Phenotype Frequency in Healthy Subjects from Paris, France

Lipoprotein(a) [Lp(a)] is an LDL-like particle whose apo B100 is disulfide-linked to an apolipoprotein A [apo(a)]. Apo(a) is a glycoprotein containing 23% carbohydrate by weight (1). Apo(a) possesses a high degree of polymorphism because of a variable number (11–52) of the repetitive structural unit called “kringle” IV (K IV ; M r ≈ 17 kDa), which leads to a wide range of molecular weights, from almost 200 kDa to 800 kDa (2). The apo(a) K IV shares a strong sequence homology with plasminogen K IV, and apo(a) has been shown to inhibit fibrinolysis in vitro. (3) A high Lp(a) plasma concentration is an independent risk factor for cerebro- and cardiovascular atherosclerosis; furthermore, low molecular mass isoforms are more frequent in patients with high Lp(a) concentrations (4). In addition, the antifibrinolytic effect of Lp(a) is inversely correlated with the size of these isoforms (5). Accurately determining the number of apo(a) Ks is, therefore, important. The molecular weight determination of apo(a) is complicated by its anomalous mobility in SDS gels, which differs from the mobility of most commercially available molecular weight markers (1). In addition, because of the relatively small size of a K, electrophoretic techniques for apo(a) phenotyping must provide high resolution. The aims of our study were to develop an accurate and simple method for apo(a) phenotyping by measuring the number of Ks, to validate this method by comparison with a genotyping method, and to determine, through this technique, the frequency of occurrence of the common apo(a) isoforms in healthy subjects. We mixed EDTA-treated plasma samples from 238 healthy Caucasian subjects with antiproteases (1 μmol/L d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone, Calbiochem 520 222; 0.5 mmol/L aminoethyl-benzenesulfonyl fluoride from Sigma diluted in isopropanol; 100 000 U/L aprotinin, Bayer Pharma 92807) and stored them frozen at −80 °C …