2.P.99 A novel kringle-4 number-based recombinant apo(a) standard for human Lp(a) phenotyping

Abstract

Apolipoprotein(a) phenotyping is a critically im- portant method to explore the role of kringle-4 repeat num- ber as a modulator of lipoprotein(a)-associated cardiovas- cular risk. The availability of a kringle-4 number-based reference standard is therefore necessary for a reliable and generally accepted classification of apo(a) phenotypes. We propose here a battery of recombinant apo(a) isoforms that may be used as the reference standard in various gel sys- tems. Five plasmids encoding for r-apo(a) containing a known number (n 5 9, 13, 17, 25, 33) of plasminogen-like kringle-4 copies were constructed, and transfected into the human embryonic kidney cell line 293. The electrophoretic mobility of the recombinant apo(a) isoforms expressed by these cells in a hollow-fiber bioreactor was determined after reduction by SDS-gel (agarose, acrylamide or a mixture of both) electrophoresis and immunoblotting using an anti- body specific for human apo(a). The equation of the linear relationship between log r-apo(a) kringle number and rela- tive migration was used to determine the isoform size of apo(a) in normal human plasma. A very good correlation ( r 5 0.97) was found with the genotype (pulsed-field gel eletrophoresis of kpn I-digested restriction fragments of ge- nomic DNA) and among electrophoretic methods. The pro- posed recombinant standard offers the possibility to iden- tify apo(a) isoforms within a large range of molecular sizes, 9 to 33 kringle-4 copies, using simple electrophoretic tech- niques and a nomenclature based on its molecular struc- ture, i.e., the number of kringle-4 repeats.— Angles-Cano, E., S. Loyau, G. Cardoso-Saldana, R. Couderc, and P. Gillery. A novel kringle-4 number-based recombinant apo(a) stan- dard for human apo(a) phenotyping. J. Lipid Res. 1999. 40: 354-359.