Plasma high density lipoproteins (HDL) are small, dense, protein-rich particles compared with other lipoprotein classes; roughly half of total HDL mass is accounted for by lipid components. Phospholipids predominate in the HDL lipidome, accounting for 40–60% of total lipid, with lesser proportions of cholesteryl esters (30–40%), triglycerides (5–12%), and free cholesterol (5–10%). Lipidomic approaches have provided initial insights into the HDL lipidome with identification of over 200 individual molecular lipids species in normolipidemic HDL. Plasma HDL particles, however, reveal high levels of structural, compositional, and functional heterogeneity. Establishing direct relationships between HDL structure, composition, and atheroprotective functions bears the potential to identify clinically relevant HDL subpopulations. Furthermore, development of HDL-based therapies designed to target beneficial subspecies within the circulating HDL pool can be facilitated using this approach. HDL lipidomics can equally contribute to the identification of biomarkers of both normal and deficient HDL functionality, which may prove useful as biomarkers of cardiovascular risk. However, numerous technical issues remain to be addressed in order to make such developments possible. With all technical questions resolved, quantitative analysis of the molecular components of the HDL lipidome will contribute to expand our knowledge of cardiovascular and metabolic diseases.

Objective—: High-density lipoprotein (HDL) displays multiple atheroprotective activities and is highly heterogeneous in structure, composition, and function; the molecular determinants of atheroprotective functions of HDL are incompletely understood. Because phospholipids represent a major bioactive lipid component of HDL, we characterized the phosphosphingolipidome of major normolipidemic HDL subpopulations and related it to HDL functionality. Approach and Results—: Using an original liquid chromatography–mass spectrometry/mass spectrometry methodology for phospholipid and sphingolipid profiling, 162 individual molecular lipid species were quantified across the 9 lipid subclasses, in the order of decreasing abundance, phosphatidylcholine>sphingomyelin>lysophosphatidylcholine>phosphatidylethanolamine>phosphatidylinositol>ceramide>phosphatidylserine>phosphatidylglycerol>phosphatidic acid. When data were expressed relative to total lipid, the contents of lysophosphatidylcholine and of negatively charged phosphatidylserine and phosphatidic acid increased progressively with increase in hydrated density of HDL, whereas the proportions of sphingomyelin and ceramide decreased. Key biological activities of HDL subpopulations, notably cholesterol efflux capacity from human THP-1 macrophages, antioxidative activity toward low-density lipoprotein oxidation, antithrombotic activity in human platelets, cell-free anti-inflammatory activity, and antiapoptotic activity in endothelial cells, were predominantly associated with small, dense, protein-rich HDL3. The biological activities of HDL particles were strongly intercorrelated, exhibiting significant correlations with multiple components of the HDL phosphosphingolipidome. Specifically, the content of phosphatidylserine revealed positive correlations with all metrics of HDL functionality, reflecting enrichment of phosphatidylserine in small, dense HDL3. Conclusions—: Our structure–function analysis thereby reveals that the HDL lipidome may strongly affect atheroprotective functionality.

Uptake of low-density lipoprotein (LDL) particles by macrophages represents a key step in the development of atherosclerotic plaques, leading to the foam cell formation. Chemical modification of LDL is however necessary to induce this process. Proatherogenic LDL modifications include aggregation, enzymatic digestion and oxidation. LDL oxidation by one-electron (free radicals) and two-electron oxidants dramatically increases LDL affinity to macrophage scavenger receptors, leading to rapid LDL uptake and fatty streak formation. Circulating high-density lipoprotein (HDL) particles, primarily small, dense, protein-rich HDL3, provide potent protection of LDL from oxidative damage by free radicals, resulting in the inhibition of the generation of pro-inflammatory oxidized lipids. HDL-mediated inactivation of lipid hydroperoxides involves their initial transfer from LDL to HDL and subsequent reduction to inactive hydroxides by redox-active Met residues of apolipoprotein A-I. Several HDL-associated enzymes are present at elevated concentrations in HDL3 relative to large, light HDL2 and can be involved in the inactivation of short-chain oxidized phospholipids. Therefore, HDL represents a multimolecular complex capable of acquiring and inactivating proatherogenic lipids. Antioxidative function of HDL can be impaired in several metabolic and inflammatory diseases. Structural and compositional anomalies in the HDL proteome and lipidome underlie such functional deficiency. Concomitant normalization of the metabolism, circulating levels, composition and biological activities of HDL particles, primarily those of small, dense HDL3, can constitute future therapeutic target.

A molecular understanding of high-density lipoprotein (HDL) will allow a more complete grasp of its interactions with key plasma remodelling factors and with cell-surface proteins that mediate HDL assembly and clearance. However, these particles are notoriously heterogeneous in terms of almost every physical, chemical and biological property. Furthermore, HDL particles have not lent themselves to high-resolution structural study through mainstream techniques like nuclear magnetic resonance and X-ray crystallography; investigators have therefore had to use a series of lower resolution methods to derive a general structural understanding of these enigmatic particles. This chapter reviews current knowledge of the composition, structure and heterogeneity of human plasma HDL. The multifaceted composition of the HDL proteome, the multiple major protein isoforms involving translational and posttranslational modifications, the rapidly expanding knowledge of the HDL lipidome, the highly complex world of HDL subclasses and putative models of HDL particle structure are extensively discussed. A brief history of structural studies of both plasma-derived and recombinant forms of HDL is presented with a focus on detailed structural models that have been derived from a range of techniques spanning mass spectrometry to molecular dynamics.

Previous studies indicate that reduced concentrations of circulating high-density lipoprotein (HDL) particles can be superior to HDL-cholesterol (HDL-C) levels as a predictor of cardiovascular disease. Measurements of HDL particle numbers therefore bear a potential for the improved assessment of cardiovascular risk. Furthermore, such measurement can be relevant for the evaluation of novel therapeutic approaches targeting HDL. Modern in-depth analysis of HDL particle profile may further improve evaluation of cardiovascular risk. Although clinical relevance of circulating concentrations of HDL subpopulations to cardiovascular disease remains controversial, the negative relationship between the number of large HDL particles and cardiovascular disease suggests that assessment of HDL particle profile can be clinically useful. Reduced mean HDL size is equally associated with cardiovascular disease in large-scale clinical studies. Since HDL-C is primarily carried in the circulation by large, lipid-rich HDL particles, the inverse relationship between HDL size and cardiovascular risk can be secondary to those established for plasma levels of HDL particles, HDL-C and large HDL. The epidemiological data thereby suggest that HDL particle number may represent a more relevant therapeutic target as compared to HDL-C.

Low plasma levels of HDL-cholesterol (HDL-C) represent a strong and independent risk factor for cardiovascular disease. HDL particles display a wide spectrum of atheroprotective activities, which include effluxing cellular cholesterol, diminishing cellular death, decreasing vascular constriction, reducing inflammatory response, protecting from pathological oxidation, combating bacterial infection, lessening platelet activation, regulating gene expression by virtue of microRNAs, and improving glucose metabolism. It remains presently indeterminate as to whether some biological activities of HDL are more relevant for the protection of the endothelium from atherogenesis when compared with others. The multitude of such activities raises the question of a proper assay to assess HDL functionality ex vivo . Together with clear understanding of molecular mechanisms underlying atheroprotective properties of HDL, such assay will provide a basis to resolve the ultimate question of the HDL field to allow the development of efficient HDL-targeting therapies.