|Abstract Title:||Apolipoproteins, non-polar lipids, polar lipids and lipoprotein particle numbers. Can we measure them all on large number of samples?|
|Session Choice:||Clinical Hyphenations|
|Presenter Name:||Dr Zsuzsanna Kuklenyik|
|Co-authors:||Mr Feffery Jones|
Mr Michael Gardner
Mr Toth Christopher
Dr Bryan Parks
Dr John Barr
|Company/Organisation:||Centers for Disease Control and Prevention|
Abstract Information :
High and low density lipoproteins (HDL and LDL) are complex molecular assemblies that are key participants in extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. A lipid/protein composition based conceptual framework of lipoproteins and the clinical measurement of an extended panel of lipids and proteins has been advocated for many years, however the development of high throughput multiplexed mass spectrometry (MS) based analytical techniques allowed effective lipoprotein composition analysis to become a reality only recently.
In this study we developed two simplified, high throughput "one-pot" extraction protocols in well-plate format, for polar lipids and non-polar lipids, and implemented an automated on-line protein digestion platform. These three high throughput sample preparation methods were coupled with liquid chromatography (UPLC) and multiplexed tandem mass spectrometry (MS/MS) detections, and quantified non-polar lipids (FC, CE, TG), polar lipids (PC, SM, LPC, PE and PI) and a panel of apolipoproteins (apos A-I, A-II, A-IV, B-100, C-I, C-II, C-III and E). We also applied a preparative size fractionation technique, asymmetric flow field-flow fractionation (AF4) to separate lipoproteins in 1-2 nm size increments, measuring hydrodynamic size by dynamic light scattering. Lipids and proteins were measured in each size fraction and in the whole serum, using a total of 0.1 ml of each serum sample (656 measurements per sample).
We demonstrate the applicability of this workflow to the analysis of 125 serum samples, from individuals with normal lipid profiles, hyperlipidemia, hypercholesterolemia, and hypertriglyceridemia. The hyphenation of methods provided an extensive amount of quantitative information for varying sizes of HDL and LDL (8 to 36 nm). The quantitative measurement of all major lipoprotein constituents and particle size allowed volumetric calculation of particle numbers and composition in number of molecules per particle. The comprehensive composition data provided clear evidence of sub-HDL and sub-LDL particle populations with distinctly different composition that can be interpreted by lipid-lipid and lipid-protein interactions. Statistical analysis of molar ratios, protein/protein (apoE/apoC-III, apoC-II/apoC-III, and apoA-I/apoA-II) and lipid/lipid (FC/PC, SM/PC, PE/PC, and PI/PC) allowed differentiation of dyslipidemic versus normolipidemic subjects. These differences can be linked to underlying irregularities in HDL and LDL particle functions, and clinically relevant metabolic conditions that causes accumulation of atherogenic lipoprotein particles.
For the amount of clinically relevant information, our hyphenation of multiplexed, high throughput LC-MS/MS methods is highly cost effective, especially for analysis of typically limited volumes of archived samples. In the past with traditional single-analyte approaches, elucidation of such depth of quantitative information required substantial amount of sample volume and combinations of sequential ultracentrifugation, immunofractionation, cross-linking, and gradient gel electrophoresis techniques. Our results demonstrate how combination of high throughput sample preparation and multiplexed LC-MS/MS based methods can reveal compositional characteristics of lipoprotein particles and underlying irregularities in lipoprotein functions in lipid metabolism pathways that lead to cardiovascular disease. The ability of these comprehensive measurements to predict risk of coronary artery disease will be the subject of planned future investigations.