Subjects. One hundred and eighty healthy blood donors, aged between 18 and 60 years (94 males, 86 fe­males), recruited from the Republic Institute of Transfu­siology, Skopje, Republic of Macedonia, were included in the study. A detailed medical and family history was obtained from all the subjects. All subjects gave written informed consent.
The K3EDTA plasma was obtained through venipunc­ture after 14 hours of overnight fasting. Plasma was prepared by low speed centrifugation at 1800 rpm/m for 15 min. and stored at –80°C as aliquots in segments of plastic tubes until assayed [23].
Electrophoresis. Denaturing 3-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a vertical Bio-Rad Mini Protean II System (Bio-Rad Laboratories, Hercules, CA, USA) was used for Apo(a) isoforms separation. Two peristaltic pumps (Mas­terflex L/S, Cole Parmer Instrument Co., Vernon Hills, IL, USA) that were controlled by Cole Parmer software, were used to convey the gradient gel characteristics. Sample preparation, electrophoresis and immunoblotting have been presented in previous publications [24,25].
Twenty µL of plasma was mixed with 80 µL of reducing buffer (containing b-mercapthoethanol, bromphenol blue in glycerol, SDS) to give a total volume of 100 µL and boiled in a water bath for 5 min. For the stacking gel, 3.3% polyacrylamide gel [0.5 M Tris-HCl buffer pH 6.8, 10% SDS, TEMED and 10% APS (Bio-Rad Laboratories)] was prepared. A commercial standard, purchased from Immuno Ag, Vienna, Austria, that contained the B, S1, S3, S4, >S4 isoforms was used. Ten µL of treated plasma samples and 5 µL of the standard were resolved in 3-15% gradient gels. A maximum of eight samples/gel was applied to avoid edge effects. Electrophoresis was performed in a Tris-glycine buffer, for 90 min. (until the dye front was just out of the gels), at 100 V and at room temperature.
Immunochemistry. The proteins separated on poly­acrylamide gels were transferred to nitrocellulose membranes (BA 83, 0.2 µm, NC.; S & S Protran, Dassel, Germany) by electroblotting using a Hoefer TE22 Transfer Unit (Amersham Pharmacia Biotech, Vienna, Austria), in Tris-glycine methanol buffer, for 17 hours at 50 V. The apo(a) isoforms were visualized immunochemically with an Lp(a) phenotyping kit (Immuno Ag, Vienna, Austria) containing primary antibody [a polyclonal antihuman Lp(a) (sheep)] and secondary antibody [an alkaline phos- phatase-conjugated antisheep Ig G (rabbit)] that was diluted with 5% Blotto solution (non fat dry milk, Tween 20, 10X PBS and antifoam A (Sigma Chemical Co., St. Louis, MO, USA)].
Five apo(a) isoforms were designated according to their relative electrophoretic mobility compared with apo B-100, using the terminology proposed in [14]. B is a band with the same mobility as apo B-100 and S1, S3, S4, >S4 denoting larger isoforms with progressively lower mobility than apo B-100. The Mr of each apo(a) band was calculated by comparison with the standards located in the adjacent lane [26]. Gels were scanned with a Pharmacia LKB Ultra Scan XL laser (Pharmacia LKB Biotechnology, Uppsala, Sweden) controlled from a computer running the Image Master Software (Pharmacia LKB Biotechnology).
Lipoprotein(a) Assay. The Lp(a) concentration was measured at the Department of Immunobiology and Human Genetics, Medical Faculty, Skopje, Republic of Macedonia, on a Behring Nephelometer Analyzer using immu­nonephelometrical kits (Dade Behring Marburg GmbH, Marburg, Germany).
Statistical Methods. Allele frequencies were estimated by maximum likelihood method using EM algorithm [27,28]. A chi-square test was used to test observed versus the expected type frequencies assuming Hardy-Weinberg equilibrium. All other statistical procedures were carried out using the STATWIN statistical software package (version 5.0 A; Statsoft Inc., 1984-95, Tulsa, OK, USA). Results were presented as mean ± SD (standard deviation).