Comparative 5-doxylstearoyllecithin and 3-doxylcholestane EPR spin labeling study of phospholipid bilayer perturbation by different oxidized lecithin species

A 3-doxylcholestane spin label was employed in addition to 5-doxylstearoyl lecithin for a more detailed study of the different effects exerted by variously oxidized lecithins on fatty acid alignment in phospholipid planar bilayers. Either spin label was enclosed in oriented PLPC planar samples also containing in turn a variety of conjugated-dienes lecithins and cleaved chain lecithins, in order to monitor EPR spectral angular dependence loss. Data obtained by use of arachidonoyl-hydroxystearoyl-PC and palmitoyl-hydroxylinoleoyl-PC confirm that the 5-DSPC nitroxide ring almost completely retains its orientation in CD-PCs-containing planar membranes, in contrast with angular dependence loss observed in the presence of the CC-PC molecular species palmitoyl-oxononanoyl-PC and palmitoyl-oxovaleroyl-PC, already seen with cleaved-chain palmitoyl-glutaroyl-PC and palmitoyl-azelaoyl-PC. However, the 3-DC nitroxide ring also loses its orientation with CD-PCs, in addition to being disoriented by cleaved chain-lecithins, similarly to 5DSPC. Joint information from the two spin labels will help to clarify whether OXPC-related disordering involved the whole bilayer structure or only the hydrophobic core. In addition, the propensity of different OXPCs to form bilayer vesicles in water suspension was also determined by Sepharose 4B gel-chromatography. The results suggest that CD-PCs might yield SPB bilayer structures with a disordered hydrophobic core, while pure CC-PC more probably forms non-bilayer disordered structures, possibly micelles or mixed micelle/bilayers.     

Naturally occurring human plasminogen,like genetically related apolipoprotein(a), contains oxidized phosphatidylcholine adducts

Human apolipoprotein(a) (apo(a)), synthesized in the liver, contains oxidized phosphatidylcholine (oxPtdPC) adducts probably generated at the hepatic site. Since plasminogen (Plg), also synthesized in the liver, is genetically related and structurally homologous to apo(a), we wanted to determine whether it contains oxPtdPCs and their location. We used Plg isolated from fresh or frozen normal human plasma and several commercial preparations. Some were freed of non-covalently bound lipids by organic solvent extraction. By immunoblot analyses, all products reacted against T15, a natural IgM monoclonal antibody specific for phosphorylcholine -containing oxidized phospholipids (ox-PLs). This immunoreactivity was retained in urokinase type plasminogen activator -generated plasmin and was abrogated in Plg previously digested with lipoprotein-associated phospholipase A₂ (Lp-PLA₂), a reaction that generated predominantly C16:0 lysophosphatidylcholine species as determined by mass spectrometry. Lyso derivatives were also generated upon the cleavage by Lp-PLA2 of a model ox-PL chemically linked to a lysine-containing pentapeptide. From inorganic phosphorous analyses, we found 2 mol of oxPtdPC/mole of Plg distributed between the kringles 1–4 and mini-Plg domain. OxPtdPCs were also present in the Plg isolated from the serum-free medium of cultured human HepG2 cells. In conclusion, our results provide strong evidence that naturally occurring Plg contains oxPtdPC probably linked by a Schiff base and also suggest that the linkage occurs at the hepatic site. Given the emerging evidence for the cardiovascular pathogenicity of oxPtdPCs, we speculate that they may impart athero-thrombogenic properties to Plg under inflammatory conditions.     

Properties of apolipoprotein E derived peptide modulate their lipid-binding capacity and influence their anti-inflammatory function

Apolipoprotein-derived peptides are promising candidates for the treatment of various inflammatory conditions. The beneficial effects of these peptides are based on multiple mechanisms; prominent among them being high-affinity binding to pro-inflammatory oxidized phospholipids (Ox-PLs) and facilitating their sequestration/metabolism/clearance in the body. This indicates that peptides which can bind exclusively to Ox-PLs without recognizing normal, non-oxidized phospholipids (non-Ox-PLs) will be more potent anti-inflammatory agent than that of the peptides that bind to both Ox-PLs and non-Ox-PLs. In order to develop such Ox-PL-specific peptides, the knowledge about the properties (molecular determinants) of peptides that govern their Ox-PL preference is a must. In this study we have synthesized eleven peptides corresponding to the conserved regions of human apolipoprotein E and compared their biochemical properties, lipid-binding specificities, and anti-inflammatory properties. Our results show that these peptides exhibit considerably different specificities towards non-Ox-PL and different species of Ox-PLs. Some of these peptides bind exclusively to the Ox-PLs and inhibit the pro-inflammatory function of Ox-PLs in human blood. Biochemical characterization revealed that the peptides possess substantially different properties. Our results suggest that physicochemical properties of peptides play an important role in their lipid-binding specificity.     

Cellular phospholipid uptake: Flexible paths to coregulate the functions of intracellular lipids

Mammalian and arthropod cells acquire phospholipids by protein-mediated pathways that comprise selective and whole particle uptake routes. Phospholipid uptake critically supports cellular incorporation of nutrition-derived polyunsaturated fatty acids. It can occur jointly with cholesterol uptake, but intracellular processing of phospholipids is distinctively different from sterol processing. The newly imported phospholipids are utilized for production of bioactive lipids, such as thromboxane A₂ and lyso phosphatidic acid, and for synthesis of triacylglycerol. Class B scavenger receptor BI (SR-BI) represents a major mediator of the uptake of various phospholipids. The related scavenger receptor CD36, as shown here, also facilitates cellular phospholipid uptake. CD36 supports import of the choline phospholipids phosphatidylcholine (PC) and sphingomyelin (SM), but not of phosphatidylethanolamine (PE). Other transferases trigger cellular uptake of selective phospholipids, such as phosphatidic acid (PA) phosphatases that facilitate PA import and thereby modify cell survival and synaptic transmission. Phospholipid uptake depends on the activation status of cells. Activation of blood platelets indeed increases PE uptake. This is mediated by the serpin protein C inhibitor (PCI) and enhances thrombin formation. Exchange of phospholipids between blood cells and lipoproteins partially adjusts the lipid distribution pattern of blood cells to the one of lipoprotein particles. This in turn modifies the activities of cell membrane sodium transporters and could thereby contribute to sodium flux alterations in the metabolic syndrome. The in vivo relevance of phospholipid uptake in humans is indicated by comparable and reversible changes in the same phospholipid species in both lipoproteins and cells after rapid removal of low-density lipoproteins. Finally, cells also incorporate oxidized (pathogenic) phospholipids using partially overlapping entry pathways as native phospholipids which might support the ability of oxidized lipids to promote atherothrombosis.     

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.     

Reductive metabolism increases the proinflammatory activity of aldehyde phospholipids

The generation of oxidized phospholipids in lipoproteins has been linked to vascular inflammation in atherosclerotic lesions. Products of phospholipid oxidation increase endothelial activation; however, their effects on macrophages are poorly understood, and it is unclear whether these effects are regulated by the biochemical pathways that metabolize oxidized phospholipids. We found that incubation of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) with THP-1-derived macrophages upregulated the expression of cytokine genes, including granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-1β, IL-6, and IL-8. In these cells, reagent POVPC was either hydrolyzed to lyso-phosphatidylcholine (lyso-PC) or reduced to 1-palmitoyl-2-(5-hydroxy-valeroyl)-sn-glycero-3-phosphocholine (PHVPC). Treatment with the phospholipase A(2) (PLA(2)) inhibitor, pefabloc, decreased POVPC hydrolysis and increased PHVPC accumulation. Pefabloc also increased the induction of cytokine genes in POVPC-treated cells. In contrast, PHVPC accumulation and cytokine production were decreased upon treatment with the aldose reductase (AR) inhibitor, tolrestat. In comparison with POVPC, lyso-PC led to 2- to 3-fold greater and PHVPC 10- to 100-fold greater induction of cytokine genes. POVPC-induced cytokine gene induction was prevented in bone-marrow derived macrophages from AR-null mice. These results indicate that although hydrolysis is the major pathway of metabolism, reduction further increases the proinflammatory responses to POVPC. Thus, vascular inflammation in atherosclerotic lesions is likely to be regulated by metabolism of phospholipid aldehydes in macrophages.     

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.