A method for lipoprotein (a) Isolation from a small volume of plasma with applications for clinical research

Abstract

High levels of circulating Lipoprotein (a) [Lp(a)] are an independent risk factor for CVD. One of the major limitations to investigating Lp(a) biology is the need for large volumes of plasma (4–10 mL) for its isolation. We developed an isolation technique requiring only 0.4 mL of plasma yielding an enriched Lp(a) fraction suitable for compositional and functional studies. We collected plasma from patients (n = 9) in EDTA presenting to our Center for Preventive Cardiology for CVD risk management and with circulating Lp(a) > 66 mg/dL. 0.4 mL of plasma was added to 90 µL of potassium bromide (1.33 g/mL) and subjected to our two-step density-gradient ultracentrifugation method. The first step separates VLDL and LDL from the Lp(a) and HDL fractions and the second step further separates VLDL from LDL and Lp(a) from HDL. Lp(a) is then dialyzed for up to 24 h in potassium phosphate buffer. We performed cholesterol gel electrophoresis, immunoblotting and LC-MS/MS proteomics on isolated lipoprotein fractions to confirm fraction enrichment. Functional studies including Lp(a)-dependent induction of macrophage gene expression and cholesterol efflux inhibition were performed on isolated Lp(a) to confirm its preserved bioactivity. Lp(a) yields (264 ± 82.3 µg/mL on average) correlated with Lp(a) plasma concentrations (r² = 0.75; p < 0.01) and represented the relative distribution of circulating apo(a) isoforms. Proteomic analyses confirm lipoprotein fraction separation. Functional integrity was confirmed by the findings that isolated Lp(a) inhibited plasminogen-dependent cholesterol efflux in HEK293T cells expressing ABCA1 and increased expressions of Il1b, Nos2 and Ccl2. We developed a small-volume isolation technique for Lp(a) suited for a range of applications used in biomedical research. The use of this technique circumvents volume-dependent limitations and expands our ability to investigate the mysteries of this deleterious lipoprotein.