High density lipoproteins: Measurement techniques and potential biomarkers of cardiovascular risk

Abstract

Plasma high density lipoprotein cholesterol (HDL) comprises a heterogeneous family of lipoprotein species, differing in surface charge, size and lipid and protein compositions. While HDL cholesterol (C) mass is a strong, graded and coherent biomarker of cardiovascular risk, genetic and clinical trial data suggest that the simple measurement of HDL-C may not be causal in preventing atherosclerosis nor reflect HDL functionality. Indeed, the measurement of HDL-C may be a biomarker of cardiovascular health. To assess the issue of HDL function as a potential therapeutic target, robust and simple analytical methods are required. The complex pleiotropic effects of HDL make the development of a single measurement challenging. Development of laboratory assays that accurately HDL function must be developed validated and brought to high-throughput for clinical purposes. This review discusses the limitations of current laboratory technologies for methods that separate and quantify HDL and potential application to predict CVD, with an emphasis on emergent approaches as potential biomarkers in clinical practice.

Keywords: 2D-PAGGE, two dimensional polyacrylamide gradient gel electrophoresis; ApoA-I, apolipoprotein A-I; Apolipoprotein A-I; Atherosclerosis; Biomarkers of cardiovascular risk; CHD, coronary heart disease; CVD, cardiovascular disease; Cellular cholesterol efflux; Coronary artery disease; HDL, high density lipoprotein; HPLC, High Performance Liquid Chromatography; High density lipoproteins; LCAT, lecithin–cholesterol acyltransferase; LDL, low density lipoprotein; MALDI, matrix-assisted laser desorption/ionization; MOP, myeloperoxidase; MS/MS, tandem-mass spectrometry; ND-PAGGE, non-denaturant polyacrylamide gradient gel electrophoresis; NMR, nuclear magnetic resonance; PEG, polyethylene glycol; PON1, paraoxonase 1; SELDI, surface enhanced laser desorption/ionization; TOF, time-of-flight; UTC, ultracentrifugation; Vascular endothelial function.

Fig. 1

Separation of HDL-species by ND-PAGGE. The left panel (A) shows the apoA-I containing HDL subpopulations separated by ND-PAGGE (5–35%) of a normolipidemic, healthy male subject (left) and healthy woman subject (right). Plasma samples were transferred to nitrocellulose membrane, and probed by radiolabeled-I¹²⁵ apoA-I radio imaging. Molecular markers are indicated on the gel. Panel (B) is a schematic diagram of all the apoA-I containing α-HDL species.

Fig. 2

Separation of HDL-species by 2D-PAGGE and techniques for measurement. Panel (A) shows the apoA-I containing HDL subpopulations separated by 2D-PAGGE (3–24%) of a normolipidemic, healthy male subject. The plasma was subjected to 2-dimensional agarose/native PAGGE; samples were transferred to nitrocellulose membrane, and probed for radiolabeled-I¹²⁵ apoA-I. Molecular markers are indicated on the gel. Panel (B) is a schematic diagram of all the apoA-I containing HDL particles. Nomenclatures of HDL subclasses determined by different methods are shown: ND-PAGGE and 2D-PAGGE (mass: charge); UTC (density) separation; and NMR (size), FPLC (size). The HDL particle images were created by using the Autodesk 3ds Max 2014 software.

Fig. 3

Schematic representation of HDL functional assays in RCT pathway. Hepatocytes, enterocytes and macrophages express ATP-binding cassette (ABC) transporter A1 (ABCA1), which effluxes phospholipids and cholesterol (assay 8) and thereby lipidates apoA-I extracellularly (assays 2–3). Effluxed (FC) is modified by the HDL enzyme (LCAT) into (CE) (assays 6–7). The initially smaller HDL₃ (assays 1–2–3–4–5) particles grow in size by ongoing lipid efflux, and cholesterol esterification. The resulting HDL₂ (assays 1–2–3–4–5) particles deliver lipids to the liver, either directly via SR-BI and indirectly via CETP mediated transfer of CE to VLDL and LDL (assays 1–4–5). The RCT is finalized by the biliary excretion of cholesterol from the liver into the intestine either directly via ABCG5 and ABCG8 to bile acids via the bile salt export pump ABCB11 (assays 10–11). The actions of hepatic lipase (HL), and endothelial lipase (EL) on HDL₃, as well as of PLTP on HDL₂, liberate lipid-free apoA-I (assays 2–3). Lipid free apoA-I is either used for de novo formation of mature HDL particles or is filtrated through the renal glomerulus for tubular uptake and degradation (dotted arrows). Numbers in rectangles refer to Table 2, Table 3.

Fig. 4

Mechanisms of vascular effects of normal HDL and associated functional assays. Circulating monocytes attach to endothelial cells by cell adhesion molecules (assay 20) that are induced in response to inflammatory signals, which is facilitated by endothelial adhesion molecules, including ICAM1/VCAM1 (assay 20). HDL causes membrane-initiated signaling, which stimulates eNOS activity (assay 19). Monocytes migrate through the endothelial layer into the intima, where they differentiate further into macrophages in response to locally produced factors such as monocyte colony-stimulating factor (assays 13–14). The recruited monocytes differentiate into macrophages or dendritic cells in the intima, where they interact with atherogenic lipoproteins (LDL) (assay 13). LDL penetrates into the artery wall where it can adhere to proteoglycans. These interactions are thought to trap the LDL particles and increase their susceptibility to oxidation. Enzymes contributing to LDL oxidation include lipoxygenases, MPO and eNOS that induce NO release in the endothelium (assays 17, 18, 19). HDL-associated PON1 (assay 16) inhibits macrophage cholesterol biosynthesis and enhances HDL-mediated cholesterol efflux. Numbers in rectangles refer to Table 3.