Direct determination of lipoprotein(a) cholesterol by ultracentrifugation and agarose gel electrophoresis with enzymatic staining for cholesterol

Abstract

Lipoprotein(a) [(Lp(a)], a low-density lipoprotein (LDL)-like particle, contains in addition to LDL a specific protein component, apolipoprotein(a) [apo(a)]. Conventionally, Lp(a) has been measured by immunological methods that distinguish between Lp(a) and LDL by dealing with apo(a) as an antigen. We describe a new method to determine Lp(a) on the basis of its cholesterol content. Very-low-density lipoproteins were removed from serum by preparative ultracentrifugation at a density of 1.006 kg/L. The infranate was subjected to agarose gel electrophoresis to separate Lp(a) and LDL. Lp(a) cholesterol was then determined by direct enzymatic staining for cholesterol. On electrophoresis of the > 1.006 kg/L (bottom) fraction, Lp(a) migrates to the pre-beta position, regardless of the genetic apo(a) isoform. The interassay CVs of Lp(a) cholesterol determinations ranged from 6.9% to 11.5%, and the results correlated well with the Lp(a) concentrations measured by immunonephelometry (r = 0.937). There was an inverse relation between the molecular mass of the genetically determined apo(a) isoforms and Lp(a) cholesterol concentrations. Patients with angiographically proven coronary artery disease (CAD) had significantly more Lp(a) cholesterol than healthy controls did. The ratio of Lp(a) cholesterol to immunologically determined Lp(a) tended to be lower in CAD patients, suggesting that Lp(a) particles contained less cholesterol than apo(a). In addition, the new method allows determination of LDL cholesterol without contamination by Lp(a).

Novel method for quantification of lipoprotein(a)-cholesterol: implications for improving accuracy of LDL-C measurements

“…This Lp(a)-C assay is a high-throughput one and can be performed on 96-well plates or adapted for use with existing clinical analyzers using magnetic beads. The entire assay is completed within 1 h. Other Lp(a)-C assays have been described, including those using electrophoretic ( 17 , 18 ), single-density gradient ultracentrifugation ( 19 , 20 ), and affinity based on porous matrices using wheat germ agglutinin (which can bind glycoproteins such as apo(a)) ( 21 ) or polyclonal anti-Lp(a) serum ( 22 ). However, neither a gold-standard assay nor reference materials to standardize or harmonize Lp(a)-C assays currently exist.…”

Cholesterol Levels of Six Fractionated Serum Lipoproteins and its Relevance to Coronary Heart Disease Risk Scores

“…Several methods to measure Lp (a)-C were previously reported. Nauck et al reported direct determination of Lp (a)-C by ultracentrifugation and agarose gel electrophoresis 25 ) . Sheman et al reported a measurement method with lectin affinity to isolate Lp (a) from other lipoproteins 26 ) .…”

Identification of recurrent and novel mutations in the LDL receptor gene in German patients with familial hypercholesterolemia

“…Plasma lipoproteins were separated by preparative ultracentrifugation and precipitation and cholesterol and triglycerides were assayed by enzymatic methods (Roche Diagnostics, Mannheim, Germany) (LRC Program, 1982). Lp (a) was determined with polyclonal antibodies (Inkstar, Stillwater, MN, USA) on a Behring nephelometer as described in detail by Nauck et al (Nauck et al, 1995). Apo E phenotyping was performed by agarose gel by isoelectric focusing and immunofixation as described previously (Luley et al, 1991).…”