Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation

Initiation of lipid peroxidation and the formation of bioactive eicosanoids are pivotal processes in inflammation and atherosclerosis. Currently, lipoxygenases, cyclooxygenases, and cytochrome P450 monooxygenases are considered the primary enzymatic participants in these events. Myeloperoxidase (MPO), a heme protein secreted by activated leukocytes, generates reactive intermediates that promote lipid peroxidation in vitro. For example, MPO catalyzes oxidation of tyrosine and nitrite to form tyrosyl radical and nitrogen dioxide ((.)NO(2)), respectively, reactive intermediates capable of initiating oxidation of lipids in plasma. Neither the ability of MPO to initiate lipid peroxidation in vivo nor its role in generating bioactive eicosanoids during inflammation has been reported. Using a model of inflammation (peritonitis) with MPO knockout mice (MPO(-/-)), we examined the role for MPO in the formation of bioactive lipid oxidation products and promoting oxidant stress in vivo. Electrospray ionization tandem mass spectrometry was used to simultaneously quantify individual molecular species of hydroxy- and hydroperoxy-eicosatetraenoic acids (H(P)ETEs), F(2)-isoprostanes, hydroxy- and hydroperoxy-octadecadienoic acids (H(P)ODEs), and their precursors, arachidonic acid and linoleic acid. Peritonitis-triggered formation of F(2)-isoprostanes, a marker of oxidant stress in vivo, was reduced by 85% in the MPO(-/-) mice. Similarly, formation of all molecular species of H(P)ETEs and H(P)ODEs monitored were significantly reduced (by at least 50%) in the MPO(-/-) group during inflammation. Parallel analyses of peritoneal lavage proteins for protein dityrosine and nitrotyrosine, molecular markers for oxidative modification by tyrosyl radical and (.)NO(2), respectively, revealed marked reductions in the content of nitrotyrosine, but not dityrosine, in MPO(-/-) samples. Thus, MPO serves as a major enzymatic catalyst of lipid peroxidation at sites of inflammation. Moreover, MPO-dependent formation of (.)NO-derived oxidants, and not tyrosyl radical, appears to serve as a preferred pathway for initiating lipid peroxidation and promoting oxidant stress in vivo.     

Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.     

Cholesteryl ester oxidation products in atherosclerosis

Lipid oxidation products are formed at sites of increased oxidant stress and have been shown to accumulate in atherosclerotic lesions. Although recent studies have focused on the formation and metabolism of oxidized lipids, very little is known about their biological activities and possible (patho)physiological functions. Oxidation of cholesteryl esters containing unsaturated fatty acids leads to the formation of hydroperoxides that are either reduced to alcohols or degrade into biologically active "core-aldehydes". In this review, the mechanisms of formation and metabolic fate of oxidized cholesteryl esters, their occurrence, as well as possible biological activities are discussed. Based on the current knowledge, cholesteryl ester oxidation leads to the formation of biologically active substances, which could actively contribute to the progression of atherosclerotic lesions and their resulting complications.     

The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation

Detergent-insoluble membrane domains, enriched in saturated lipids and cholesterol, have been implicated in numerous biological functions. To understand how cholesterol promotes domain formation, the effect of various sterols and sterol derivatives on domain formation in mixtures of the saturated lipid dipalmitoylphosphatidylcholine (DPPC) and a fluorescence quenching analogue of an unsaturated lipid was compared. Quenching measurements demonstrated that several sterols (cholesterol, dihydrocholesterol, epicholesterol, and 25-hydroxycholesterol) promote formation of DPPC-enriched domains. Other sterols and sterol derivatives had little effect on domain formation (cholestane and lanosterol) or, surprisingly, strongly inhibit it (coprostanol, androstenol, cholesterol sulfate, and 4-cholestenone). The effect of sterols on domain formation was closely correlated with their effects on DPPC insolubility. Those sterols that promoted domain formation increased DPPC insolubility, whereas those sterols that inhibit domain formation decreased DPPC insolubility. The effects of sterols on the fluorescence polarization of diphenylhexatriene incorporated into DPPC-containing vesicles were also correlated with sterol structure. These experiments indicate that the effect of sterol on the ability of saturated lipids to form a tightly packed (i.e., tight in the sense that the lipids are closely packed with one another) and ordered state is the key to their effect on domain formation. Those sterols that promote tight packing of saturated lipids promote domain formation, while those sterols that inhibited tight packing of saturated lipids inhibited domain formation. The ability of some sterols to inhibit domain formation (i.e., act as "anti-cholesterols") should be a valuable tool for examining domain formation and properties in cells.     

Oxidative risk for atherothrombotic cardiovascular disease

In the vasculature, reactive oxidant species, including reactive oxygen, nitrogen, or halogenating species, and thiyl, tyrosyl, or protein radicals may oxidatively modify lipids and proteins with deleterious consequences for vascular function. These biologically active free radical and nonradical species may be produced by increased activation of oxidant-generating sources and/or decreased cellular antioxidant capacity. Once formed, these species may engage in reactions to yield more potent oxidants that promote transition of the homeostatic vascular phenotype to a pathobiological state that is permissive for atherothrombogenesis. This dysfunctional vasculature is characterized by lipid peroxidation and aberrant lipid deposition, inflammation, immune cell activation, platelet activation, thrombus formation, and disturbed hemodynamic flow. Each of these pathobiological states is associated with an increase in the vascular burden of free radical species-derived oxidation products and, thereby, implicates increased oxidant stress in the pathogenesis of atherothrombotic vascular disease.     

Specific oxygenation of plasma membrane phospholipids by Pseudomonas aeruginosa lipoxygenase induces structural and functional alterations in mammalian cells

Pseudomonas aeruginosa is a gram-negative pathogen, which causes life-threatening infections in immunocompromized patients. These bacteria express a secreted lipoxygenase (PA-LOX), which oxygenates free arachidonic acid to 15S-hydro(pero)xyeicosatetraenoic acid. It binds phospholipids at its active site and physically interacts with lipid vesicles. When incubated with red blood cells membrane lipids are oxidized and hemolysis is induced but the structures of the oxygenated membrane lipids have not been determined. Using a lipidomic approach, we analyzed the formation of oxidized phospholipids generated during the in vitro incubation of recombinant PA-LOX with human erythrocytes and cultured human lung epithelial cells. Precursor scanning of lipid extracts prepared from these cells followed by multiple reaction monitoring and MS/MS analysis revealed a complex mixture of oxidation products. For human red blood cells this mixture comprised forty different phosphatidylethanolamine and phosphatidylcholine species carrying oxidized fatty acid residues, such as hydroxy-octadecadienoic acids, hydroxy- and keto-eicosatetraenoic acid, hydroxy-docosahexaenoic acid as well as oxygenated derivatives of less frequently occurring polyenoic fatty acids. Similar oxygenation products were also detected when cultured lung epithelial cells were employed but here the amounts of oxygenated lipids were smaller and under identical experimental conditions we did not detect major signs of cell lysis. However, live imaging indicated an impaired capacity for trypan blue exclusion and an augmented mitosis rate. Taken together these data indicate that PA-LOX can oxidize the membrane lipids of eukaryotic cells and that the functional consequences of this reaction strongly depend on the cell type.     

The proteasomal system and HNE-modified proteins

Metabolic processes and environmental conditions cause the constant formation of oxidizing species over the lifetime of cells and organisms. This leads to a continuous oxidation of intracellular components, including lipids, DNA and proteins. During the extensively studied process of lipid peroxidation, several reactive low-molecular weight products are formed, including reactive aldehydes as 4-hydroxynonenal (HNE). These aldehydic lipid peroxidation products in turn are able to modify proteins. The degradation of oxidized and oxidatively modified proteins is an essential part of the oxidant defenses of cells. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasomal system. The proteasomal system by itself is a multicomponent system responsible for the degradation of the majority of intracellular proteins. It has been shown that some, mildly cross-linked, HNE-modified proteins are preferentially degraded by the proteasome, but extensive modification with this cross-linking aldehyde leads to the formation of protein aggregates, that can actually inhibit the proteasome. This review summarizes our knowledge of the interactions between lipid peroxidation products, proteins, and the proteasomal system.     

Role of myeloperoxidase in the neutrophil-induced oxidation of low density lipoprotein as studied by myeloperoxidase-knockout mouse

Low density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of a-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of a-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.     

Cellular sterol ester synthesis in plants is performed by an enzyme (phospholipid:sterol acyltransferase) different from the yeast and mammalian acyl-CoA:sterol acyltransferases

A gene encoding a sterol ester-synthesizing enzyme was identified in Arabidopsis. The cDNA of the Arabidopsis gene At1g04010 (AtPSAT) was overexpressed in Arabidopsis behind the cauliflower mosaic virus 35S promoter. Microsomal membranes from the leaves of overexpresser lines catalyzed the transacylation of acyl groups from phosphatidylethanolamine to sterols. This activity correlated with the expression level of the AtPSAT gene, thus demonstrating that this gene encodes a phospholipid:sterol acyltransferase (PSAT). Properties of the AtPSAT were examined in microsomal fractions from the tissues of an overexpresser. The enzyme did not utilize neutral lipids, had the highest activity with phosphatidylethanolamine, had a 5-fold preference for the sn-2 position, and utilized both saturated and unsaturated fatty acids. Various sterols and sterol intermediates, including triterpenic precursors, were acylated by the PSAT, whereas other triterpenes were not. Sterol selectivity studies showed that the enzyme is activated by end product sterols and that sterol intermediates are preferentially acylated by the activated enzyme. This indicates that PSAT both regulates the pool of free sterols as well as limits the amount of free sterol intermediates in the membranes. Two T-DNA insertion mutants in the AtPSAT gene, with strongly reduced (but still measurable) levels of sterol esters in their tissues, had no detectable PSAT activity in the microsomal fractions, suggesting that Arabidopsis possess other enzyme(s) capable of acylating sterols. The AtPSAT is the only intracellular enzyme found so far that catalyzes an acyl-CoA-independent sterol ester formation. Thus, PSAT has a similar physiological function in plant cells as the unrelated acyl-CoA:sterol acyltransferase has in animal cells.     

GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis

Oxygen is necessary for aerobic metabolism but can cause the harmful oxidation of lipids and other macromolecules. Oxidation of cholesterol and phospholipids containing polyunsaturated fatty acyl chains can lead to lipid peroxidation, membrane damage, and cell death. Lipid hydroperoxides are key intermediates in the process of lipid peroxidation. The lipid hydroperoxidase glutathione peroxidase 4 (GPX4) converts lipid hydroperoxides to lipid alcohols, and this process prevents the iron (Fe²⁺)‐dependent formation of toxic lipid reactive oxygen species (ROS). Inhibition of GPX4 function leads to lipid peroxidation and can result in the induction of ferroptosis, an iron‐dependent, non‐apoptotic form of cell death. This review describes the formation of reactive lipid species, the function of GPX4 in preventing oxidative lipid damage, and the link between GPX4 dysfunction, lipid oxidation, and the induction of ferroptosis.     

Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.

Endothelial cells, macrophages, neutrophils, and neuronal cells generate superoxide (O2-) and nitric oxide (.NO) which can combine to form peroxynitrite anion (ONOO-). Peroxynitrite, known to oxidize sulfhydryls and to yield products indicative of hydroxyl radical (.OH) reaction with deoxyribose and dimethyl sulfoxide, is shown herein to induce membrane lipid peroxidation. Peroxynitrite addition to soybean phosphatidylcholine liposomes resulted in malondialdehyde and conjugated diene formation, as well as oxygen consumption. Lipid peroxidation was greater at acidic and neutral pH, with no significant lipid peroxidation occurring above pH 9.5. Addition of ferrous (Fe+2) or ferric (Fe+3) iron did not enhance lipid peroxide formation over that attributable to peroxynitrite alone. Diethylenetetraminepentacetic acid (DTPA) or iron removal from solutions by ion-exchange chromatography decreased conjugated diene formation by 25-50%. Iron did not play an essential role in initiating lipid peroxidation, since DTPA and iron depletion of reaction systems were only partially inhibitory. In contrast, desferrioxamine had an even greater concentration-dependent inhibitory effect, completely abolishing lipid peroxidation at 200 microM. The strong inhibitory effect of desferrioxamine on lipid peroxidation was due to direct reaction with peroxynitrous acid in addition to iron chelation. We conclude that the conjugate acid of peroxynitrite, peroxynitrous acid (ONOOH), and/or its decomposition products, i.e., .OH and nitrogen dioxide (.NO2), initiate lipid peroxidation without the requirement of iron. These observations demonstrate a potential mechanism contributing to O2-(-)and .NO-mediated cytotoxicity.     

Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations

Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer—the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.     

Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reaction in bile acid biosynthesis.

The enzyme sterol 27-hydroxylase catalyzes the first step in the oxidation of the side chain of sterol intermediates in the bile acid synthesis pathway. Human sterol 27-hydroxylase cDNAs were isolated from a liver cDNA library by cross-hybridization with a previously cloned rabbit cDNA probe. DNA sequence analysis of hybridization-positive clones predicted a human sterol 27-hydroxylase consisting of a 33-amino-acid mitochondrial signal sequence followed by a mature protein of 498 amino acids. RNA blotting experiments demonstrated sterol 27-hydroxylase mRNAs of approximately 1.8 to 2.2 kilobases in liver and fibroblast cells. The steady state levels of the mRNA did not change when cultured cells were grown in the presence or absence of sterols. Introduction of the sterol 27-hydroxylase cDNA into Simian COS cells resulted in the expression of active enzyme capable of catalyzing multiple oxidation reactions (R-CH3----R-CH2OH----R-COOH) at carbon 27 of sterol intermediates of the bile acid synthesis pathway.     

Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis

The oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. alpha-Tocopherol (alpha-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of alpha-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the alpha-TOH radical and exporting the radical from the lipoprotein particle, alpha-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of alpha-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis.     

Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.

Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844-849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.     

Enzymatic lipid peroxidation

Biosynthesis of certain biologically active substances (prostaglandins, thromboxanes, prostacyclins and leukotrienes) in animal tissues occurs with participation of cyclooxygenases and lipoxygenases, enzymic systems of lipid peroxidation. In normal physiological and pathological processes the enzymic lipid peroxidation by microsomal dioxygenases is considerably more active than the nonenzymic one in the same membrane structures. The molecular structure of the products of the enzymic and nonenzymic peroxidation of lipids also differs essentially. An assumption is advanced that cytosol lipoxygenase may be an easily dissociating component of the cyclooxygenase multienzymic complex and its transition from the biomembrane to the cell cytoplasm is accompanied by changes in the enzyme conformation and chemical nature of the products resulted from polyenic lipids oxidation catalyzed by the enzyme.     

Light-induced oxidation of photoreceptor outer segment phospholipids generates ligands for CD36-mediated phagocytosis by retinal pigment epithelium: a potential mechanism for modulating outer segment phagocytosis under oxidant stress conditions

Clearance by the retinal pigment epithelium (RPE) of shed photoreceptor outer segments (OSs), a tissue with one of the highest turnover rates in the body, is critical to the maintenance and normal function of the retina. We hypothesized that there is a potential role for photo-oxidation in OS uptake by RPE via scavenger receptor-mediated recognition of structurally defined lipid peroxidation products. We now demonstrate that specific structurally defined oxidized species derived from arachidonyl, linoleoyl, and docosahexanoyl phosphatidylcholine may serve as endogenous ligands on OSs for uptake by RPE via the scavenger receptor CD36. Mass spectrometry studies of retinal lipids recovered from dark-adapted rats following physiological light exposure demonstrate in vivo formation of specific oxidized phosphatidylcholine molecular species possessing a CD36 recognition motif, an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy(or oxo)-alpha,beta-unsaturated carbonyl. Cellular studies using RPE isolated from wild-type versus CD36 null mice suggest that CD36 plays a role in engulfment, but not initial binding, of OSs via these oxidized phospholipids. Parallel increases in OS protein-bound nitrotyrosine, a post-translational modification by nitric oxide (NO)-derived oxidants, were also observed, suggesting a possible role for light-induced generation of NO-derived oxidants in the initiation of OS lipid peroxidation. Collectively, these studies suggest that intense light exposure promotes "oxidative tagging" of photoreceptor outer segments with structurally defined choline glycerophospholipids that may serve as a physiological signal for CD36-mediated phagocytosis under oxidant stress conditions.     

A role for reduced coenzyme Q in atherosclerosis?

Substantial evidence implicates oxidative modification of low density lipoprotein (LDL) as an important event contributing to atherogenesis. As a result, the elucidation of the molecular mechanisms by which LDL is oxidized and how such oxidation is prevented by antioxidants has been a significant research focus. Studies on the antioxidation of LDL lipids have focused primarily on alpha-tocopherol (alpha-TOH), biologically and chemically the most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. In addition to alpha-TOH, plasma LDL also contains low levels of ubiquinol-10 (CoQ10H2; the reduced form of coenzyme Q10). Recent studies have shown that in oxidizing plasma lipoproteins alpha-TOH can exhibit anti- or pro-oxidant activities for the lipoprotein's lipids exposed to a vast array of oxidants. This article reviews the molecular action of alpha-TOH in LDL undergoing "mild" radical-initiated lipid peroxidation, and discusses how small levels of CoQ10H2 can represent an efficient antioxidant defence for lipoprotein lipids. We also comment on the levels alpha-TOH, CoQ10H2 and lipid oxidation products in the intima of patients with coronary artery disease and report on preliminary studies examining the effect of coenzyme Q10 supplementation on atherogenesis in apolipoprotein E knockout mice.     

Effects of oxidation on the structure and stability of human low-density lipoprotein

Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mechanisms and to determine the effects of oxidation on the protein secondary structure and lipoprotein fusion in vitro, we analyzed LDL oxidized by nonenzymatic (Cu2+, H2O2, and HOCl) or enzymatic methods (myeloperoxidase/H2O2/Cl- and myeloperoxidase/H2O2/NO2-). Far-UV circular dichroism spectra showed that LDL oxidation induces partial unfolding of the secondary structure rather than folding into cross-beta amyloid conformation. This unfolding correlates with increased negative charge of oxidized LDL and with a moderate increase in thioflavin T fluorescence that may result from electrostatic attraction between the cationic dye and electronegative LDL rather than from dye binding to amyloid. These and other spectroscopic studies of low- and high-density lipoproteins, which encompass amyloid-promoting conditions (high protein concentrations, high temperatures, acidic pH), demonstrate that in vitro lipoprotein oxidation does not induce amyloid formation. Surprisingly, turbidity, near-UV circular dichroism, and electron microscopic data demonstrate that advanced oxidation inhibits heat-induced LDL fusion that is characteristic of native lipoproteins. Such fusion inhibition may result from the accumulation of anionic lipids and lysophospholipids on the particle surface and/or from protein cross-linking upon advanced lipoprotein oxidation. Consequently, oxidation alone may prevent rather than promote LDL fusion, suggesting that additional factors, such as albumin-mediated removal of lipid peroxidation products and/or LDL binding to arterial proteoglycans, facilitate fusion of oxidized LDL in vivo.