SIMPLIFIED METHOD FOR THE DETECTION OF Apo(a) ISOFORMS

Abstract

Apolipoprotein a, is a high molecular weight glycoproteic component of Lp(a), a molecule associated with coronary arterial disease. Apo(a) exhibits considerable size heterogeneity due to variable repetitions of the carbohydrate-containing structural unit, termed kringle. There are five different kringle forms and 10 different kringle 4 types. Apo(a) polymorphism and molecular weight depend on the number of copies of kringle 4 type 2. In this paper we describe a modified 3.75% and 6% discontinuous polyacrylamide gel system and Western-blot technique that shortness the assay time and improves the identification of apo(a) isoforms with a theoretical error of less than 1 kringle. The assay uses a standard curve prepared with five different recombinant apo(a) molecules, detected up to 50 ng of protein in Lp(a), showed a maximal resolution of 2 kringles and, with the use of third degree polynominal regression analysis, had an error of 0.01275. The inter-assay coefficient of variation was 1.7, 2, and 1.4 for the 14 K, 18 K, and 22 K phenotypes, whereas the intra-assay coefficient of variation was 0.32%, 0.18%, and 0.17%, respectively. It is possible that this modified method will diminish the number of putative null alleles so far detected in various studies, but most of all, we are certain that it can be of use in epidemiological studies due to its ease of use, speed, low cost, and enhanced number of samples that can be tested.

Comprehensive Analysis of Genomic Variation in the LPA Locus and Its Relationship to Plasma Lipoprotein(a) in South Asians, Chinese, and European Caucasians

“…14 -16 Recently, a large Mendelian randomization study reported association between low KIV-2 copy number and myocardial infarction in a sample of European Caucasians but did not include single-nucleotide polymorphism (SNP) genotypes. 17 To date, KIV-2 copy number generally has been identified either at the biochemical level by examining the protein size using electrophoresis and immunoblotting 18,19 or at the genomic level using pulse-field gel electrophoresis with Southern blotting of unamplified genomic DNA. 19 Because of the requirement of either large amounts of fresh plasma or genomic DNA, respectively, for these methods, candidategene and genome-wide association studies of Lp(a) concentration have not included the apo(a) KIV-2 copy number as a covariate or independent variable, nor has there been any opportunity to study association or linkage disequilibrium between LPA SNPs and KIV-2 copy number.…”