High-performance liquid chromatographic determination of phospholipid peroxidation products of rat liver after carbon tetrachloride administration

A method to detect and determine phospholipid peroxidation products in a biological system was developed using reversed-phase high performance liquid chromatography and normal-phase HPLC. Reversed-phase HPLC could separate phosphatidylcholine (PC) hydroperoxides and phosphatidylethanolamine (PE) hydroperoxides of rat liver from the respective phospholipids. A linear relationship was observed between these hydroperoxides and their peak areas on the chromatogram. In the experiment with rats administered CCl4, reversed-phase HPLC gave prominent, large peaks attributable to the peroxidation of phospholipids, and the peroxide level of the liver phospholipids was tentatively determined. Normal-phase HPLC analysis confirmed that both PC and PE in the liver phospholipids were peroxidized after CCl4 treatment. Neither the thiobarbituric acid value of the liver homogenate nor the fatty acid composition of the liver phospholipid fraction showed any significant difference between CCl4-treated and control rats. It is concluded that normal-phase HPLC and reversed-phase HPLC can complement each other to serve as a direct and sensitive method for the determination of lipid peroxide levels in a biological source. However, it was difficult to distinguish phospholipid hydroperoxides from their hydroxy derivatives.     

Lipid hydroperoxide levels in the heart and liver of rats of various ages

The level of lipid hydroperoxides and secondary products of peroxide oxidation of lipids reacting with 2-thiobarbituric acid (TBA) has been studied in the heart and liver of Wistar male rats aged 1, 3, 12 and 24 months. It is shown that the both indices in the heart lower in the pubertal period of ontogenesis and do not vary with the further ageing. The content of hydroperoxides in the liver is unchanged and that of secondary TBA-active products decreases with the age.     

Lipid peroxidation: mechanisms, inhibition, and biological effects

In the last 50 years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipids are oxidized by three distinct mechanisms; enzymatic oxidation, non-enzymatic, free radical-mediated oxidation, and non-enzymatic, non-radical oxidation. Each oxidation mechanism yields specific products. The oxidation of linoleates and cholesterol is discussed in some detail. The relative susceptibilities of lipids to oxidation depend on the reaction milieu as well as their inherent structure. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. Phospholipid hydroperoxides, for example, are reduced to the corresponding hydroxides by selenoproteins in vivo. Various kinds of antioxidants with different functions inhibit lipid peroxidation and the deleterious effects caused by the lipid peroxidation products. Furthermore, the biological role of lipid peroxidation products has recently received a great deal of attention, but its physiological significance must be demonstrated in future studies.     

Lipid oxidation by hypochlorous acid: chlorinated lipids in atherosclerosis and myocardial ischemia

Leukocytes, containing myeloperoxidase (MPO), produce the reactive chlorinating species, HOCl, and they have important roles in the pathophysiology of cardiovascular disease. Leukocyte-derived HOCl can target primary amines, alkenes and vinyl ethers of lipids, resulting in chlorinated products. Plasmalogens are vinyl ether-containing phospholipids that are abundant in tissues of the cardiovascular system. The HOCl oxidation products derived from plasmalogens are α-chlorofatty aldehyde and unsaturated molecular species of lysophosphatidylcholine. α-chlorofatty aldehyde is the precursor of both α-chlorofatty alcohol and α-chlorofatty acid. Both α-chlorofatty aldehyde and α-chlorofatty acid accumulate in activated neutrophils and have disparate chemotactic properties. In addition, α-chlorofatty aldehyde increases in activated monocytes, human atherosclerotic lesions and rat infarcted myocardium. This article addresses the pathways for the synthesis of these lipids and their biological targets.     

Preferential formation of the hydroperoxide of linoleic acid in choline glycerophospholipids in human erythrocytes membrane during peroxidation with an azo initiator

The formation of phospholipid hydroperoxides was monitored in human red blood cell (RBC) membranes that had been peroxidized with an azo initiator. Peroxidation of RBC membranes caused a profound decrease in the amount of polyunsaturated fatty acids and concomitantly hydroperoxides, as primary products of peroxidation, appeared in the phospholipids. Hydroperoxides were predominantly generated in choline glycerophospholipid (CGP), while the extent of formation of ethanolamine glycerophospholipid (EGP) hydroperoxides was low and their presence was transient. Hydroxy and hydroperoxy moieties in CGP were identified as 9-hydroxy and 13-hydroxy octadecanoic acid, derived from linoleic acid, by gas chromatography-mass spectrometric analysis. No consistent generation of hydroperoxide from arachidonic acid was evident in CGP. The CGP-hydroperoxide accounted for approximately 76% of linoleic acid consumed during peroxidation of RBC membranes. The prominent generation of phospholipid hydroperoxides was observed in the linoleic acid-rich membranes from rabbit RBC, indicating that the level of linoleic acid in phospholipids determins, in part, the extent of formation of phospholipid hydroperoxides. Aldehydic phospholipids, as secondary products of peroxidation, were detected in oxidized membranes. EGP was the most prominent aldehydic phospholipid, while negligible amounts of aldehydic CGP were formed. This study indicates that the process of oxidation of individual phospholipids clearly differs among phospholipids and depends on the structure of each.     

Interactions of plasmalogens and their diacyl analogs with singlet oxygen in selected model systems

Plasmalogens are phospholipids containing a vinyl-ether linkage at the sn-1 position of the glycerophospholipid backbone. Despite being quite abundant in humans, the biological role of plasmalogens remains speculative. It has been postulated that plasmalogens are physiological antioxidants with the vinyl-ether functionality serving as a sacrificial trap for free radicals and singlet oxygen. However, no quantitative data on the efficiency of plasmalogens at scavenging these reactive species are available. In this study, rate constants of quenching of singlet oxygen, generated by photosensitized energy transfer, by several plasmalogens and, for comparison, by their diacyl analogs were determined by time-resolved detection of phosphorescence at 1270nm. Relative rates of the interactions of singlet oxygen with plasmalogens and other lipids, in solution and in liposomal membranes, were measured by electron paramagnetic resonance oximetry and product analysis using HPLC-EC detection of cholesterol hydroperoxides and iodometric assay of lipid hydroperoxides. The results show that singlet oxygen interacts with plasmalogens significantly faster than with the other lipids, with the corresponding rate constants being 1 to 2 orders of magnitude greater. The quenching of singlet oxygen by plasmalogens is mostly reactive in nature and results from its preferential interaction with the vinyl-ether bond. The data suggest that plasmalogens could protect unsaturated membrane lipids against oxidation induced by singlet oxygen, providing that the oxidation products are not excessively cytotoxic.     

The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids

Myeloperoxidase (MPO), an abundant enzyme in phagocytes, has been implicated in the pathogenesis of various inflammatory diseases including atherosclerosis. The major oxidant produced by MPO, hypochlorous acid (HOCl), is able to modify a great variety of biomolecules by chlorination and/or oxidation. In this paper the reactions of lipids (preferentially unsaturated fatty acids and cholesterol) with either reagent HOCl or HOCl generated by the MPO-hydrogen peroxide-chloride system are reviewed. One of the major issues has been whether the reaction of HOCl with lipids of low density lipoprotein (LDL) yields predominantly chlorohydrins or lipid hydroperoxides. Electrospray mass spectrometry provided direct evidence that chlorohydrins rather than peroxides are the major products of HOCl- or MPO-treated LDL phosphatidylcholines. Nevertheless lipid peroxidation is a possible alternative reaction of HOCl with polyunsaturated fatty acids if an additional radical source such as pre-formed lipid hydroperoxides is available. In phospholipids carrying a primary amino group such as phosphatidylethanolamine chloramines are the preferred products compared to chlorohydrins. Cholesterol can be converted by HOCl to great variety of oxysterols besides three isomers of chlorohydrins. For the situation in vivo it appears that the type of reaction occurring between HOCl and lipids would very much depend on the circumstances, e.g. the pH and the presence of radical initiators. The biological effects of lipid chlorohydrins are not yet well understood. It has been shown that chlorohydrins of both unsaturated fatty acids as well as of cholesterol may cause lysis of target cells, possibly by disruption of membrane structures.     

Effect of mercury chloride on the level of lipids and products of their peroxidation in rat vital organs

The influence of mercury chloride on peroxidation processes of lipids and level of common lipids, phospholipids and spectrum of neutral lipids in liver, heart, lung and kidney of rats has been investigated. Administration of mercury chloride in a dose 0.7 mg/100 g of body weight to animals has invoked accumulation of lipids peroxide products in fractions of neutral lipids and phospholipids so it testifies the development of oxidative stress. Decrease of the most sensitive to oxidation fractions in the early stages of oxidative stress development and increase of free cholesterol and its ethers content in kidney and free cholesterol in the heart in more later terms as a result of mercury chloride administration have been revealed.     

Oxidation of lipids in low density lipoprotein particles

This study was undertaken to understand further the mechanisms and dynamics of the oxidation of lipids in low density lipoprotein (LDL) particles, aiming specifically at elucidating the material balance between oxygen uptake and products found and also the relative susceptibilities to oxidation of cholesteryl ester in the core and phosphatidylcholine in the outer monolayer in the LDL particles. It was found that considerable amount of oxygen uptake could not be accounted for by conjugated diene or total peroxides. Total peroxide was measured from the phosphine oxide formed from triphenylphosphine or diphenylpyrenylphosphine by reduction of peroxides. Cholesteryl ester hydroperoxides and phosphatidylcholine hydroperoxides were the major peroxides formed in LDL oxidation, but they accounted for about 60% of total peroxide. Cholesterol was also oxidized, but its oxidation was significant only at the later stages of the reaction. It was also found that the oxidizability of cholesteryl ester relative to phosphatidylcholine was larger within the LDL particle than in homogeneous solution and this was interpreted in the context of the physical properties of LDL particle.     

Lipid Hydroperoxides Inhibit Reacylation of Phospholipids in Neuronal Membranes

The unsaturated fatty acids that rapidly accumulate during ischemia are thought to participate in inducing irreversible brain injury, especially because they are highly susceptible to peroxidation when the tissue is reoxygenated. Our hypothesis was that peroxidation products of unsaturated fatty acids interfere with the reacylation of synaptic phospholipids, a process essential to membrane repair. To test this hypothesis, we have examined the effect of fatty acid hydroperoxides on incorporation of [1-14C]arachidonic acid into synaptosomal phospholipids. Rat forebrain synaptosomes were incubated with arachidonic or linoleic acid hydroperoxides and [14C]arachidonate, and then lipids were extracted and separated by TLC. Both hydroperoxides inhibited [14C]arachidonate incorporation into phospholipids in a concentration-dependent manner, with 50% inhibition occurring at less than 25 microM hydroperoxide, in both the absence and presence of exogenous lysophospholipids. The inhibition was of the non-competitive type. It is concluded that (a) low levels of fatty acid hydroperoxides inhibit the reacylation of synaptosomal phospholipids, and (b) this inhibition may constitute an important mechanism whereby peroxidative processes contribute to irreversible brain damage.     

Preparative high-performance liquid chromatography purification of polyunsaturated phospholipids and characterization using ultraviolet derivative spectroscopy

A preparative reversed-phase HPLC system utilizing an isocratic mobile phase to purify up to 10-mg quantities of phospholipids is described. The method was developed to separate oxidation products of polyunsaturated phospholipids from intact, parent lipids. The method is useful for phosphatidylcholine and phosphatidylethanolamine on a preparative scale and for phosphatidylserine and phosphatidic acid on an analytical scale. Both intact phospholipids and oxidized phospholipids were monitored by absorbance at 206 nm. The oxidation products were simultaneously monitored at 234 nm where the intact phospholipids have only a very slight end absorption. Second-derivative uv spectroscopy proved to be extremely useful to identify the presence or to verify the absence of oxidation products in phospholipid samples. For autoxidized docosahexaenoic acid containing phospholipids, the absorbance maximum of diene oxidation products is 237 nm for the trans,trans (t,t) isomer and 246 nm for the cis,trans (c,t) isomers. Similarly, five classes of triene oxidation product stereoisomers have distinct absorbance maxima detected by second-derivative spectroscopy ranging from 269 to 292 nm.     

Pathways of phospholipid oxidation by HOCl in human LDL detected by LC-MS

A wealth of evidence now indicates that low-density lipoprotein (LDL) must be modified to promote atherosclerosis, and that this may involve oxidants released by phagocytes. Many studies of oxidative damage in atherosclerosis previously have concentrated on damage by nonhalogenated oxidants, but HOCl is a highly toxic oxidant produced by myeloperoxidase in phagocytes, which is also likely to be important in the disease pathogenesis. Currently some controversy exists over the products resulting from reaction of HOCl with LDL lipids, in particular regarding whether predominantly chlorohydrins or lipid peroxides are formed. In this study LC-MS of phosphatidylcholines in human LDL treated either with HOCl or the myeloperoxidase system was used as a specific method to detect chlorohydrin and peroxide formation simultaneously, and with comparable sensitivity. Chlorohydrin products from lipids containing oleic, linoleic and arachidonic acids were detected, but no hydroperoxides of linoleoyl or arachidonoyl lipids could be observed. This study provides the first direct evidence that lipid chlorohydrins rather than peroxides are the major products of HOCl- or myeloperoxidase-treated LDL phospholipids. This in turn provides important information required for the study of oxidative damage in vivo which will allow the type and source of oxidants involved in the pathology of atherosclerosis to be investigated.     

Analysis of oxidized and chlorinated lipids by mass spectrometry and relevance to signalling

Oxidized and chlorinated phospholipids are generated under inflammatory conditions and are increasingly understood to play important roles in diseases involving oxidative stress. MS is a sensitive and informative technique for monitoring phospholipid oxidation that can provide structural information and simultaneously detect a wide variety of oxidation products, including chain-shortened and -chlorinated phospholipids. MSn technologies involve fragmentation of the compounds to yield diagnostic fragment ions and thus assist in identification. Advanced methods such as neutral loss and precursor ion scanning can facilitate the analysis of specific oxidation products in complex biological samples. This is essential for determining the contributions of different phospholipid oxidation products in disease. While many pro-inflammatory signalling effects of oxPLs (oxidized phospholipids) have been reported, it has more recently become clear that they can also have anti-inflammatory effects in conditions such as infection and endotoxaemia. In contrast with free radical-generated oxPLs, the signalling effects of chlorinated lipids are much less well understood, but they appear to demonstrate mainly pro-inflammatory effects. Specific analysis of oxidized and chlorinated lipids and the determination of their molecular effects are crucial to understanding their role in disease pathology.     

Bioactive phospholipid oxidation products

Oxidation of phospholipids results in chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl residues, generating a myriad of phospholipid products. Certain oxidation products of phosphatidylcholine bind to and activate the human receptor for PAF, and these PAF-like lipids are potent, selective inflammatory mediators. Formation of PAF-like lipids is nonenzymatic and so their accumulation is unregulated. PAF-like lipids are produced in vivo in response to oxidative stresses and are responsible for attendant acute inflammatory responses. PAF-like lipids almost exclusively contain an ether-linked alkyl residue at the sn-1 position of the phosphatidylcholine backbone and molecular identification of these is facilitated by phospholipase A₁ treatment to remove the bulk of the inactive phospholipids. The identity of biologically active species generated by oxidative fragmentation and oxidation can be elucidated by understanding relevant reactions leading to the formation of PAF-like lipids, and then their structure can be established by tandem mass spectrometry and chemical synthesis.     

Oxidation of low density lipoprotein and plasma by 15-lipoxygenase and free radicals

It is generally accepted that the oxidation of pentadiene structures of polyunsaturated lipids by lipoxygenase (LOX) is regio- and enantio-specific, while the free radical-mediated lipid peroxidation gives stereo-random racemic products. It was confirmed that the oxidation of human low density lipoprotein (LDL) by 15-LOX from rabbit reticulocytes gave phosphatidylcholine (PC) and cholesteryl ester (CE) hydroperoxides regio-, stereo- and enantio-specifically. 15-LOX also oxidized human plasma to give specific PC and CE hydroperoxides in spite of the presence of high concentrations of antioxidants. More CE hydroperoxides were formed than PC hydroperoxides from LDL, but the reverse order was observed for plasma oxidation. The S/R ratio of the hydroperoxides decreased during long time incubation but remained significantly larger than one, while free radical-mediated oxidation of LDL and plasma gave racemic products.     

Radiation-induced peroxidation of small unilamellar vesicles of phosphatidylcholine generated by sonication

The present study was aimed at determining the peroxidation of model membranes constituted of liposomes of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) submitted to hydroxyl free radicals (generated by gamma-radiolysis) attack. Liposomes of PLPC were prepared using the sonication technique, and dynamic light-scattering (DLS) measurements allowed characterization of the liposomal dispersions. Irradiation damages in sonication-generated liposomes were assessed by monitoring several oxidation products, such as conjugated dienes (by means of UV--visible spectrophotometry) and hydroperoxides (using reverse phase high-performance liquid chromatography (HPLC) associated with chemiluminescence detection). It has been shown that three different families of hydroperoxides are formed: the first one (at low radiation doses) results from HO. attack on the linoleyl chain of PLPC, giving phosphatidylcholine hydroperoxides possessing a conjugated dienic structure; the two others (at high radiation doses) are obtained by the secondary HO. attack on the primary hydroperoxide family. The quantification of these products associated with the comparison of their radiation-dose-dependent formation has provided valuable information concerning the mechanisms of their formation. Analysis by HPLC -- mass spectrometry has confirmed the presence of hydroperoxides and underlined various other products, like chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl chain residues. Structural assignment proposals of some oxidation products have been proposed.     

Activation of rat brain protein kinase C by lipid oxidation products

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Ka's for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.     

Detection of lipid hydroperoxides and hydrogen peroxide at picomole levels by an HPLC and isoluminol chemiluminescence assay

An isoluminol assay is utilized for the detection of hydrogen peroxide and lipid hydroperoxides in biological samples. The combination of this assay as a post-column detection for HPLC avoids interference of antioxidants and enables characterization of hydroperoxides at picomole levels. Two useful HPLC conditions for the separation of hydrogen peroxide, lipid hydroperoxides, antioxidants, and unoxidized lipids are described.     

Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol condensation.

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. The process involves nonspecific fragmentation, oxidative breakdown, and modification of the lipids and protein of LDL. The process yields a variety of bioactive products, including aldehyde-containing phospholipids, which can cross-react with primary amines (i.e. peptides or phospholipid head groups) to yield Schiff base products. We also demonstrate that such oxidized phospholipid products may further react through a post-oxidation chemical pathway involving aldol condensation. EO6, an IgM monoclonal autoantibody to oxidized phospholipids, blocks the uptake of oxidized LDL (OxLDL) by macrophages. Because the epitope(s) of EO6 also blocks the uptake of OxLDL, a series of oxidized phospholipids, their peptide complexes, and their aldol condensates have been synthesized and characterized, and their antigenicity has been determined. This study defines structural motifs of oxidized phospholipids responsible for antigenicity for EO6. Certain monomeric phospholipids containing short chain fatty acids were antigenic whether oxidized or not in the sn-2 position. However, oxidized phospholipids containing sn-1 long chain fatty acids were not antigenic unless the sn-2 oxidized fatty acid contained an aldehyde that first reacted with a peptide yielding a Schiff base or the sn-2 oxidized fatty acid underwent an aldol type self-condensation. Our data indicate that the phosphorylcholine head group is essential for antigenicity, but its availability depends on the oxidized phospholipid conformation. We suggest that upon oxidation, similar reactions occur in phospholipids on the surface of LDL, generating ligands for macrophage recognition. Synthetic imine adducts of oxidized phospholipids of this type are capable of blocking the uptake of OxLDL.     

Detection of lipid hydroperoxides and hydrogen peroxide at picamole levels by an HPLC and isoluminol chemiluminescence assay

An isoluminol assay is utilized for the detection. of hydrogen peroxide and lipid hydroperoxides in biological samples. The combination of this assay as a post-column detection for HPLC avoids interference of antioxidants and enables characterization of hydroperoxides at picomole levels. Two useful HPLC conditions for the separation of hydrogen peroxide, lipid hydroperoxides, antioxidants, and unoxidized lipids are described.