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

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.

Elevated Lipoprotein(a) Levels Lower ABCA1 Cholesterol Efflux Capacity.

CONTEXT: Elevated serum lipoprotein(a) [Lp(a)] levels are associated with increased cardiovascular disease risk. ABCA1-mediated cholesterol efflux from macrophages may be an antiatherogenic process. Plasminogen (PLG) is a driver of ABCA1-mediated cholesterol efflux, and its action is inhibited by purified human Lp(a). OBJECTIVE: To determine the effects of Lp(a) in human serum on ABCA1 cholesterol efflux. METHODS: Cholesterol efflux capacity (CEC) was measured with two different cell-culture models using serum from 76 patients with either low (<50 mg/dL) or high (>50 mg/dL) Lp(a) levels. RESULTS: Using cAMP-stimulated J774 macrophages or baby hamster kidney fibroblasts overexpressing human ABCA1, we show that CEC was lower in patients with high Lp(a) levels compared with patients with low levels (-30.6%, P = 0.002 vs -24.1%, P < 0.001, respectively). Total-serum CEC negatively correlated with Lp(a) levels (r = -0.433, P = 0.0007 vs r = -0.505, P = 0.0011, respectively). These negative associations persisted after adjusting for serum cholesterol, age, sex, and statin use in a multiple linear regression model (adjusted R2 = 0.413 or 0.405, respectively) and were strengthened when further adjusting for the interaction between Lp(a) and PLG levels (adjusted R2 = 0.465 and 0.409, respectively). Total-serum and isolated Lp(a) from patients with high Lp(a) inhibited PLG-mediated ABCA1 cholesterol efflux. CONCLUSION: Total-serum CEC is reduced in patients with high Lp(a) levels. This is in part due to the inhibition of PLG-mediated ABCA1 cholesterol efflux by Lp(a). Our findings suggest an atherogenic role for Lp(a) through its ability to inhibit CEC.