Covalent binding of hydroxy-alkenals 4-HDDE, 4-HHE,and 4-HNE to ethanolamine phospholipid subclasses

Lipid oxidation is implicated in a wide range of pathophysiogical disorders, and leads to reactive compounds such as fatty aldehydes, of which the most well known is 4-hydroxy-2E-nonenal (4-HNE) issued from 15-hydroperoxyeicosatetraenoic acid (15-HpETE), an arachidonic acid (AA) product. In addition to 15-HpETE, 12(S)-HpETE is synthesized by 12-lipoxygenation of platelet AA. We first show that 12-HpETE can be degraded in vitro into 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), a specific aldehyde homologous to 4-HNE. Moreover, 4-HDDE can be detected in human plasma. Second, we compare the ability of 4-HNE, 4-HDDE, and 4-hydroxy-2E-hexenal (4-HHE) from n-3 fatty acids to covalently modify different ethanolamine phospholipids (PEs) chosen for their biological relevance, namely AA- (20: 4n-6) or docosahexaenoic acid- (22:6n-3) containing diacyl-glycerophosphoethanolamine (diacyl-GPE) and alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) molecular species. The most hydrophobic aldehyde used, 4-HDDE, generates more adducts with the PE subclasses than does 4-HNE, which itself appears more reactive than 4-HHE. Moreover, the aldehydes show higher reactivity toward alkenylacyl-GPE compared with diacyl-GPE, because the docosahexaenoyl-containing species are more reactive than those containing arachidonoyl. We conclude that the different PE species are differently targeted by fatty aldehydes: the higher their hydrophobicity, the higher the amount of adducts made. In addition to their antioxidant potential, alkenylacyl-GPEs may efficiently scavenge fatty aldehydes.     

Hydroxy-alkenals from the peroxidation of n-3 and n-6 fatty acids and urinary metabolites

4-Hydroxy-2E-hexenal (4-HHE) and 4-hydroxy-2E-nonenal (4-HNE) have been characterized as prominent by-products of n-3 and n-6 hydroperoxy derivatives of n-3 and n-6 fatty acids, respectively. We also have characterized the homolog 4-hydroxy-2E,6Z-dodecadienal (4-HDDE) as a specific by-product of the 12-lipoxygenase product of arachidonic acid 12-hydroperoxy-eicosatetraenoate (12-HpETE). The three hydroxy-alkenals have been found in human plasma with 4-HHE being the most prominent followed by 4-HNE. They were found increased in tissues submitted to oxidative stress, according to the fatty acid characteristic of those tissues, e.g., 4-HNE and 4-HDDE in blood platelets and 4-HHE in the retina. We have shown they covalently bind to the primary amine moiety of ethanolamine phospholipids (PE), especially the plasmalogen subclass, with the highest hydrophobic alkenal (4-HDDE) being the most reactive. Their carboxylic acid metabolites, 4-hydroxy-2E-hexenoic acid (4-HHA), 4-hydroxy-2E-nonenoic acid (4-HNA) and 4-hydroxy-2E,6Z-dodecadienoic acid (4-HDDA), respectively, were found in human urine and measured in higher amounts in situations in which oxidative stress has been reported such as aging and diabetes. As reported above with their hydroxy-alkenals precursors, 4-HHA proved to be the most prominent followed by 4-HNA. Altogether, the three hydroxy-alkenals, either in their free form or bound to membrane PE, may be considered as specific markers of lipid peroxidation able to discriminate between n-3 and n-6 fatty acids. This is corroborated by the measurement of their urinary carboxylic acid metabolites.     

Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals

Hydroxy-alkenals, such as 4-hydroxy-2(E)-nonenal (4-HNE; from n-6 fatty acids), are degradation products of fatty acid hydroperoxides, including those generated by free radical attack of membrane polyunsaturated fatty acyl moieties. The cytotoxic effects of hydroxy-alkenals are well known and are mainly attributable to their interaction with different molecules to form covalent adducts. Indeed, ethanolamine phospholipids (PEs) can be covalently modified in a cellular system by hydroxy-alkenals, such as 4-HNE, 4-hydroxy-2(E)-hexenal (4-HHE; from n-3 fatty acids), and 4-hydroxy-dodecadienal (4-HDDE; from the 12-lipoxygenase product of arachidonic acid), to form mainly Michael adducts. In this study, we describe the formation of PE Michael adducts in human blood platelets in response to oxidative stress and in retinas of streptozotocin-induced diabetic rats. We have successfully characterized and evaluated, for the first time, PEs coupled with 4-HHE, 4-HNE, and 4-HDDE by gas chromatography-mass spectrometry measurement of their ethanolamine moieties. We also report that aggregation of isolated human blood platelets enriched with PE-4-hydroxy-alkenal Michael adducts was altered. These data suggest that these adducts could be used as specific markers of membrane disorders occurring in pathophysiological states with associated oxidative stress and might affect cell function.     

Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology

4-Hydroxynonenal (4-HNE) is a cytotoxic alpha,beta-unsaturated acyl aldehyde that is naturally produced from lipid peroxidation and cleavage in response to oxidative stress and aging. Such reactive lipids covalently modify cellular target proteins, thereby affecting biological structure and function. Herein we report the identification of the epithelial fatty acid-binding protein (E-FABP) as a molecular target for 4-HNE modification both in vitro and in vivo. 4-HNE covalently modified (t(12) < 60 s) E-FABP in vitro, as revealed by a combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry and immunochemical reactivity using antibodies directed to 4-HNE-protein conjugates. Identification of Cys-120 as the major site of modification was determined through tandem mass spectral sequencing of tryptic peptides, as well as analysis of E-FABP mutants C120A, C127A, and C120A/C127A. The in vitro modification of Cys-120 by 4-HNE was relatively insensitive to pH (6.4-8.4), and temperature (4-37 degrees C) but was markedly potentiated by noncovalently bound fatty acids. 4-HNE-modified E-FABP was more stable than unmodified E-FABP to chemical denaturation by guanidine hydrochloride, as assessed by changes in intrinsic tryptophan fluorescence. Analysis of soluble protein extracts from rat retina with antibodies directed to 4-HNE-protein conjugates revealed immunoreactivity with a 15-kDa protein that was identified by electrospray ionization and matrix-assisted laser desorption ionization-time of flight mass spectrometry as E-FABP. Evaluation of retinal pigment epithelial cell extracts derived from E-FABP null mice by two-dimensional gel electrophoresis using anti-4-HNE antibodies revealed increased modification in the null cells relative to those from wild type cells. These results indicate that E-FABP is a molecular target for 4-HNE modification and the hypothesis that E-FABP functions as an antioxidant protein by scavenging reactive lipids through covalent modification of Cys-120.     

Phospholipid fatty acid composition, vitamin E content and susceptibility to lipid peroxidation of duck spermatozoa

Recent studies on chicken semen have suggested that the lipid and fatty acid composition of spermatozoa may be important determinants of fertility. Phospholipid fatty acid composition, vitamin E content and in vitro susceptibility to lipid peroxidation of duck spermatozoa were investigated using GC-MS and HPLC based methods. The total phospholipid fraction of duck spermatozoa was characterized by high proportions of the n-6 polyunsaturated fatty acids arachidonic (20:4n-6), docosatetraenoic (22:4n-6) and docosapentaenoic (22:5n-6) acids but a substantial proportion of the n-3 fatty acid docosahexaenoic (22:6n-3) acid was also present. Palmitic (16:0) and stearic (18:0) fatty acids were the major saturates in sperm phospholipids. Among the phospholipid classes, phosphatidylserine (PS) had the highest degree of unsaturation due to very high proportions of 22:6n-3, 22:5n-6, 22:4n-6 and 20:4n-6, comprising together more than 75% of total fatty acids in this fraction. Phosphatidylethanolamine (PE) also contained high proportions of these four C(20-22) polyunsaturates, which together formed 60% of total fatty acids in this phospholipid. Spermatozoa and seminal plasma of duck semen were characterized by unexpectedly low content of vitamin E, being more than 4-fold lower than in chicken semen. In duck semen the major proportion of the vitamin E (>70%) was located in the spermatozoa. The very high proportion of 22:6n-3 in PS and PE fractions of duck sperm lipids and the comparatively low levels of vitamin E could predispose semen to lipid peroxidation. Nevertheless the in vitro susceptibilities to Fe2+-stimulated lipid peroxidation of duck and chicken spermatozoa were very similar. The results of the study suggest that increased superoxide dismutase and glutathione peroxidase activity and increased antioxidant activity of seminal plasma may compensate for the low levels of vitamin E to help protect the membranes of duck spermatozoa, which exhibit a high degree of unsaturation from oxidative stress.     

Formation of N-(hexanoyl)ethanolamine, a novel phosphatidylethanolamine adduct, during the oxidation of erythrocyte membrane and low-density lipoprotein

The primary amino groups of biomolecules such as aminophospholipids, as well as proteins, are the potential targets of covalent modifications by lipid peroxidation products; however, little attention has been paid to the modification of aminophospholipids such as phosphatidylethanolamine (PE). The purpose of this study is to characterize the formation of a novel modified phospholipid, N-(hexanoyl)phosphatidylethanolamine (HEPE), in the reaction of PE with lipid hydroperoxides using mass spectrometric analyses. Upon reaction of egg PE with 13-hydroperoxyoctadecadienoic acid or other oxidized polyunsaturated fatty acids followed by phospholipase D-mediated hydrolysis, the formation of N-(hexanoyl)ethanolamine (HEEA), a head group of HEPE, was confirmed by isotope dilution liquid chromatography/tandem mass spectrometry. Moreover, increasing HEEA was detected in the hydrolysates of oxidized erythrocyte ghosts and low-density lipoprotein with their increasing lipid peroxidation levels. Collectively, these results suggest that the N-hexanoylated product of phospholipid, HEPE, can be generated during lipid peroxidation and may serve as one mechanism for the covalent modification of aminophospholipids in vivo.     

Covalent modifications of aminophospholipids by 4-hydroxynonenal

Lipid oxidation is implicated in a wide range of pathophysiological disorders, which leads to reactive compounds such as aldehydes. Among them 4-hydroxynonenal (4-HNE) reacts strongly with the NH₂ groups of amino acids and forms mainly Michael adducts and minor Schiff-base adducts. Such reactions occur also with compounds containing thiol groups. No data are available describing 4-HNE interactions with amino-phospholipids. To investigate such a possibility, 4-HNE was incubated with either phosphatidylethanolamine (PE) or phosphatidylserine (PS) in an aqueous-organic biphasic system and the resulting products were identified by liquid chromatography-mass spectrometry (LC-MS). Our study points out the potential capacity of 4-HNE to react with phospholipids containing amino groups and particularly PE. The main resulting compounds found were a Michael adduct plus a minor Schiff base adduct, which was partly cyclized as a pyrrole derivative via a loss of water. Its stabilization as a pyrrole derivative allows to differentiate 4-HNE from the other aldehydes generated via lipid oxidation (e.g., malondialdehyde, 2-nonenal) that lack the 4-hydroxyl group. Their formation seems not to be affected when the pH varies from 6.5 to 8.5. Surprisingly, PS reacted poorly producing only a small amount of Michael adduct, the Schiff-base adduct being nondetectable. We conclude that such adducts, if they are formed in cell membranes, could alter the phospholipase-dependent cell signaling.     

N-3 and n-6 fatty acids stimulate restitution by independent mechanisms in the IEC-6 model of intestinal wound healing

We have shown that intestinal epithelial restitution is stimulated by n-3 and n-6 fatty acids. The current studies were undertaken to elucidate the mechanistic pathway(s) involved in this fatty acid modulation of restitution. Inhibition of phospholipase A(2) and eicosanoid synthesis and its effect on fatty acid stimulation of cellular migration in confluent, wounded IEC-6 monolayers was examined. The production of prostaglandin E(2) and transforming growth factor beta(1) were also measured in fatty acid supplemented cultures. Inhibition of phospholipase A(2) attenuated the effect of fatty acid stimulation of restitution in both n-3 and n-6 supplemented cultures. The lipoxygenase inhibitor, nordihydorguaretic acid (2 &mgr;mol/L), had no effect on stimulation of migration by fatty acids. The cyclooxygenase inhibitor piroxicam (5 &mgr;mol/L) and cyclooxygenase-2 specific inhibitors dexamethasone (2 &mgr;mol/L) and NS-398 (10 &mgr;mol/L) all attenuated the fatty acid stimulation of migration by n-6 fatty acids but had no effect on n-3 stimulated restitution. Prostaglandin E(2) production in n-6 supplemented cultures was significantly greater than in control and n-3 supplemented cultures and was partially inhibited by dexamethasone and NS-398. Latent transforming growth factor beta(1) production in n-3 supplemented cultures was significantly higher than baseline and n-6 supplemented cultures. Docosapentaenoic acid supplementation significantly enhanced the restitution process and NS-398 treatment had no effect on this stimulation of cellular migration. The liberation of fatty acid from the sn-2 position of phospholipid appears to be necessary for both n-3 and n-6 fatty acid stimulation of restitution. N-6 fatty acid modulation of restitution appears to be mediated through the production of eicosanoid products, however, prostaglandin E(2) does not appear to be the sole prostanoid involved. N-3 supplementation elevates the production of latent transforming growth factor beta(1) and may be responsible for n-3 mediated stimulation of restitution. These results further emphasize that n-3 and n-6 fatty acids convey their effects through unique pathways.     

High-fat diet induces changes in adipose tissue trans-4-oxo-2-nonenal and trans-4-hydroxy-2-nonenal levels in a depot-specific manner

Protein carbonylation is the covalent modification of proteins by α,β-unsaturated aldehydes produced by nonenzymatic lipid peroxidation of polyunsaturated fatty acids. The most widely studied aldehyde product of lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE), is associated with obesity-induced metabolic dysfunction and has demonstrated reactivity toward key proteins involved in cellular function. However, 4-HNE is only one of many lipid peroxidation products and the lipid aldehyde profile in adipose tissue has not been characterized. To further understand the role of oxidative stress in obesity-induced metabolic dysfunction, a novel LC–MS/MS method was developed to evaluate aldehyde products of lipid peroxidation and applied to the analysis of adipose tissue. 4-HNE and trans-4-oxo-2-nonenal (4-ONE) were the most abundant aldehydes present in adipose tissue. In high fat-fed C57Bl/6J and ob/ob mice the levels of lipid peroxidation products were increased 5- to 11-fold in epididymal adipose, unchanged in brown adipose, but decreased in subcutaneous adipose tissue. Epididymal adipose tissue of high fat-fed mice also exhibited increased levels of proteins modified by 4-HNE and 4-ONE, whereas subcutaneous adipose tissue levels of these modifications were decreased. High fat feeding of C57Bl/6J mice resulted in decreased expression of a number of genes linked to antioxidant biology selectively in epididymal adipose tissue. Moreover, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 while upregulating the expression of SOD2. These results suggest that inflammatory cytokines selectively downregulate antioxidant gene expression in visceral adipose tissue, resulting in elevated lipid aldehydes and increased protein carbonylation.     

Fatty Acid Composition of Human Brain Phospholipids During Normal Development

Abstract: The fatty acid composition of phosphatidylethanolamine (PE), ethanolamine plasmalogens (EPs), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin was studied in 22 human forebrains, ranging in age from 26 prenatal weeks to 8 postnatal years. Phospholipids were separated by two-dimensional TLC, and the fatty acid methyl esters studied by capillary column GLC. Docosahexaenoic acid (22:6n-3) increased with age in PE and PC, whereas arachidonic acid (20:4n-6) remained quite constant. In EP, 22:6n-3 increased less markedly than 20:4n-6, adrenic (22:4n-6) and oleic (18:1n-9) acids being the predominant fatty acids during postnatal age. In PS, 18:1n-9 increased dramatically throughout development, and 20:4n-6 and 22:4n-6 increased only until ∼6 months of age. Although 22:6n-3 kept quite constant during development in PS, its percentage decreased due to the accretion of other polyunsaturated fatty acids (PUFAs). As a characteristic myelin lipid, sphingomyelin was mainly constituted by very long chain saturated and monounsaturated fatty acids. Among them, nervonic acid (24:1n-9) was the major very long chain fatty acid in Sp, followed by 24:0, 26:1n-9, and 26:0, and its accretion after birth was dramatic. As myelination advanced, 18:1n-9 increased markedly in all four glycerophospholipids, predominating in EP, PS, and PC. In contrast, 22:6n-3 was the most important PUFA in PE in the mature forebrain.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.     

Analysis of lipids in the salivary glands of Amblyomma americanum (L.): Detection of a high level of arachidonic acid

Analysis of lipids in salivary glands of the lone star tick, Amblyomma americanum, demonstrated that arachidonic acid (20:4, n-6) comprises 8% of all fatty acids identified by gas chromatography. The occurrence of arachidonic acid and other C₂₀ polyunsaturated fatty acids in tick salivary glands was confirmed by gas chromatography-mass spectrometry. Arachidonate is located entirely in the phospholipid fraction and is associated exclusively with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Salivary glands stored and frozen for several months had a similar lipid composition as freshly dissected salivary glands, with the exception of a small amount of free arachidonic acid and an increase in lysophosphatidylcholine. Incubation of salivary gland homogenates with snake venom phospholipase A₂ showed that most saturated fatty acids are esterified in the sn-1 position of PC and PE, with the unsaturated fatty acids in the sn-2 position. Approximately 75% of arachidonic acid is in the sn-2 position of PC and PE, adding support to the hypothesis that arachidonic acid is released into the cytoplasm after activation of a phospholipase A₂ for subsequent metabolism to prostaglandins and/or other eicosanoids. © 1993 Wiley-Liss, Inc.     

Studies on polyenoic acid incorporation into human platelet lipid stores: interactions with linoleic and arachidonic acids

Several polyunsaturated fatty acids (C18-C22 acids) have been compared in their uptake by human platelets and their acylation into glycerophospholipid subclasses. This was also studied in the presence of linoleic and/or arachidonic acids, the main fatty acids of plasma free fatty acid pool. Amongst C20 fatty acids, dihomogamma linolenic acid (20:3(n-6)), 5,8,11-icosatrienoic acid (20:3(n-9)) and arachidonic acid (20:4(n-6)) were better incorporated. The uptake of 5,8,11,14,17-icosapentaenoic acid (20:5(n-3)) was significantly lower and comparable to that of C22 fatty acids (7,10,13,16-docosatetraenoic acid (22:4(n-6)) and 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) and linoleic acid (18:2(n-6)). In this respect, linolenic acid (18:3(n-3)) appeared the poorest substrate. The bulk of each acid was acylated into glycerophospholipids although the presence of linoleic and/or arachidonic acids diverted a part towards neutral lipids. This was prominent for 18:3(n-3) and C22 fatty acids. The glycerophospholipid distribution of each acid differed substantially and was not affected by the presence of linoleic and or arachidonic acids, except for 18:3(n-3) and 22:6(n-3) that were strongly diverted towards phosphatidylethanolamine (PE) at the expense of phosphatidylcholine (PC). The main features were an efficient acylation of 20:3(n-9) into phosphatidylinositol (PI) followed by 20:3(n-6) and 20:4(n-6), then by 20:5(n-3) and 22:4(n-6), and finally 22:6(n-3) and C18 fatty acids. This was reciprocal to the acylation into PE and to a lesser extent into PC which remained the main storage species in all cases. We conclude that human platelets may exhibit a certain specificity for taking up polyunsaturated fatty acids both in terms of total uptake and glycerophospholipid subclass distribution. Also the presence of polyunsaturated fatty acids of normal plasma, like linoleic and arachidonic acids, may interact specifically with such an uptake and distribution.     

Docosahexaenoic and arachidonic acid peroxidation: It's a within molecule cascade

Peroxidation is a well-known natural phenomenon associated with both health and disease. We compared the peroxidation kinetics of phosphatidylcholine (PC) molecules with different fatty acid compositions (i.e. 18:0, 18:1n-9, 18:2n-6, 20:4n-6 and 22:6n-3 at the sn-2 and 16:0 at sn-1 position) either as molecules free in solution or formed into liposomes. Fatty acid levels, oxygen consumption plus lipid hydroperoxide and malondialdehyde production were measured from the same incubations, at the same time during maximal elicitable peroxidation. PCs with highly peroxidizable fatty acids (i.e. 20:4n-6 and 22:6n-3) in the same incubation were found to be either fully peroxidized or intact. Rates of peroxidation of PCs with multiple bisallylic groups (i.e. 20:4n-6 and 22:6n-3) peroxidized at 2-3 times the rate per bisallylic bond than the same phospholipid with 18:2n-6. The results suggest that propagation of peroxidation (H-atom transfer) is firstly an intramolecular process that is several-fold faster than intermolecular peroxidation. PCs in solution peroxidized twice as fast as those in liposomes suggesting that only half of the phospholipids in liposomes were available to peroxidize i.e. the outer leaflet. Experiments on liposomes suggest that even after heavy peroxidation of the outer leaflet the inner leaflet is unaffected, indicating how cells may protect themselves from external peroxidation and maintain control over internal peroxidation. Intramolecular peroxidation may produce highly concentrated, localized sites of peroxidation product that together with internal control of peroxidation of the inner leaflet of membranes provide new insights into how cells control peroxidation at the membrane level.     

Apparent relative retention of the phosphatidylethanolamine molecular species 18:0-20:5(n-3), 16:0-22:6(n-3) and the sum 16:0-20:4(n-6) plus 16:0-20:3(n-9) in the liver microsomes of pig on an essential fatty acid deficient diet.

Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of chromatographic techniques and fast-atom bombardment-mass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn-1 position and (iii) fatty acids at the sn-2 position. The highest apparent relative retentions were displayed by the 18:0-20:5(n-3)-PE and 16:0-22:6(n-3)-PE. It is noteworthy that the behavior of 20:3 n-9--which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n-6--was very similar to that of 20:4 n-6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n-6) and 20:3(n-9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment-mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum.     

Liver and heart mitochondria obtained from Adelie penguin (Pygoscelis adeliae) offers high resistance to lipid peroxidation

Lipid peroxidation is generally thought to be a major mechanism of cell injury in aerobic organisms subjected to oxidative stress. All cellular membranes are especially vulnerable to oxidation due to their high concentration of polyunsaturated fatty acids. However, birds have special adaptations for preventing membrane damage caused by reactive oxygen species. This study examines fatty acid profiles and susceptibility to lipid peroxidation in liver and heart mitochondria obtained from Adelie penguin (Pygoscelis adeliae). The saturated fatty acids in these organelles represent approximately 40-50% of total fatty acids whereas the polyunsaturated fatty acid composition was highly distinctive, characterized by almost equal amounts of 18:2 n-6; 20:4 n-6 and 22:6 n-3 in liver mitochondria, and a higher proportion of 18:2 n-6 compared to 20:4 n-6 and 22:6 n-3 in heart mitochondria. The concentration of total unsaturated fatty acids of liver and heart mitochondria was approximately 50% and 60%, respectively, with a prevalence of oleic acid C18:1 n9. The rate C20:4 n6/C18:2 n6 and the unsaturation index was similar in liver and heart mitochondria; 104.33 +/- 6.73 and 100.09 +/- 3.07, respectively. Light emission originating from these organelles showed no statistically significant differences and the polyunsaturated fatty acid profiles did not change during the lipid peroxidation process.     

Effects of 4-hydroxynonenal, a lipid peroxidation product, on dopamine transport and Na+/K+ ATPase in rat striatal synaptosomes

Incubation of rat striatal synaptosomes in ascorbic acid induced the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and 4-hydroxynonenal (4-HNE), a lipid peroxidation aldehydic product. Incubations with 4-HNE, used at a range of concentrations comparable to those obtained during peroxidation, induced a simultaneous, dose-dependent decrease of dopamine (DA) uptake and Na⁺/K⁺ ATPase activity and a loss of sulfhydryl (SH) groups. Similar results were observed in a previous study when lipid peroxidation was induced after incubation of synaptosomes in ascorbic acid. Taken together, these data suggest that 4-HNE is an important mediator of oxidative stress and may alter DA uptake after binding to SH groups of the DA transporter and to Na⁺/K⁺ ATPase. These toxic events may contribute to the onset and progression of Parkinsons disease.     

Effect of culture in vitro with eicosatetraenoic (20:4(n-6) ) and eicosapentaenoic (20:5(n-3) ) acids on fatty acid composition, prostaglandin synthesis and chemiluminescence of rat peritoneal macrophages

Rat peritoneal macrophages were cultured in either eicosatetraenoic acid (20:4(n-6) ) or eicosapentaenoic acid (20:5(n-3) ) and the effects on phospholipid fatty acids, prostaglandin synthesizing capacity and the ability of the macrophages to show chemiluminescence were examined. Chemiluminescence is an activity resulting from the synthesis of reactive oxygen species. It has been reported that prostaglandins inhibit this activity. The fatty acid profile of the four major phospholipids reflected the fatty acid component of the medium. Macrophages cultured in 20:4(n-6) synthesized twice the prostaglandin produced by controls and those cultured in 20:5(n-3) synthesized 10% that of controls and 5% that of 20:4(n-6)-cultured cells. Macrophages cultured with 20:4(n-6) for 12 h showed half the chemiluminescence of those cultured with 20:5(n-3), while those cultured with 20:4(n-6) for 24 h showed 10% the chemiluminescence of 20:5(n-3)-cultured cells. Addition of the prostaglandin synthase inhibitor, indomethacin, had no effect on chemiluminescence.