Abstract 399: High Throughput Comprehensive Volumetric Approach for Determination of Particle Number and Composition of Lipoproteins in Normolipidemic and Dyslipidemic Populations

Abstract

Lipoproteins (Lps) are large molecular assembles formed by lipid and apolipoprotein constituents. The physical metric of Lps as metabolically functional entities is particle concentration in serum or plasma (Lp-P). However, the metabolic functions of Lp particles is determined by their lipid/protein composition and structure. To be able to determine both Lp composition and Lp-P, a volumetric approach is required, as demonstrated by Segrest et al and other groups. In this work the volumetric approach was implemented but with applicability to population studies. The workflow included size based separation of Lps by asymmetric flow field-flow fractionation while collecting fractions with 1-1.5 nm increments in the range of 7-15 nm (HDL), 20-30 nm (LDL) and >30 nm Lps (40 fractions in total from 0.1 mL serum aliquots). The average particle size in each fraction was measured by dynamic light scattering. Three high throughput, parallel LC-MS/MS based methods were developed to quantify main non-polar lipids (FC, CE and TG), phospholipids (PC, SM, PE, PI and LPC), and apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E). Quantification of particle size and all major Lp components was achieved with 4-15% CVs. Overall accuracy of the methods was demonstrated by ApoB-100/LDL-P molar ratios of 0.7-1.3 (vs. 1 expected) in the 22-26 nm maximum LDL size range. In the 7.5-13 nm size range, ApoA-I/HDL-P was 0.7-3.5 and ApoA-II/HDL-P of 0.5-2.5. Using the calculated Lp-P values, average individual analyte/Lp-P molar ratios were calculated in each fraction. The workflow was applied to 120 patient samples with wide range of Total-C and Total-TG levels. Multivariate response surface modeling was used to show significant correlations among individual lipid/Lp-P and apolipoprotein/Lp-P molar ratios. For example, with correction for particle size, the correlations of apoC-III/Lp-P and FC/Lp-P with SM/Lp-P, PC/Lp-P, TG/Lp-P and CE/Lp-P were determined, showing the effect of surface lipid and core lipid composition on apoC-III and FC binding to HDL and LDL particles, while also revealing significant cross effects among Lp components. By using <0.1 mL serum or plasma, the workflow is applicable to archived samples collected in large cohort studies.