Chemical modification of proteins during peroxidation of phospholipids

Chemical modification of proteins by advanced glycation and lipoxidation end products is implicated in the pathogenesis of macrovascular disease in aging and diabetes. To identify biomarkers of the lipoxidative modification of protein, we studied the oxidation of phospholipids in the presence of the model protein RNase A and compared protein-bound products formed in these reactions with those formed during oxidation of plasma proteins. Metal-catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylcholine or 1-palmitoyl-2-linoleoyl-phosphatidylcholine in the presence of RNase led to the loss of amino groups in RNase and the incorporation of phosphate, hexanoate, pentanedioate, nonanedioate, and palmitate into protein. Protein-bound palmitate and phosphate correlated strongly with one another, and protein-bound pentanedioate and nonanedioate, derived from arachidonate and linoleate, respectively, accounted for approximately 20% of the cross-linking of lipid phosphorus to protein. Similar results were obtained on oxidation of total plasma or isolated LDL. We conclude that alkanedioic acids are quantitatively important linkers of oxidized phospholipids to proteins and that measurement of protein-bound phosphate and long-chain fatty acids may be useful for assessing long-term lipid peroxidative damage to proteins in vivo. Analyses of plasma proteins from control and diabetic patients indicated significant increases in lipoxidative modification of protein in diabetic compared with control subjects.     

Aldehyde adducts generated during lipid peroxidation modification of proteins

Abstract

Various lines of evidence indicate that an important part in the pathogenesis of atherosclerosis is the modification of the plasma low-density lipoproteins (LDLs). A large number of pro-inflammatory and pro-atherogenic properties have been ascribed to the oxidatively modified LDLs and their components. There is considerable evidence to support the role of lipid peroxidation products, reactive aldehydes in particular, originating from the oxidized LDL as important signaling molecules in the context of the atherosclerotic lesion. These aldehydes generated during the peroxidation of LDL exhibit a facile reactivity with proteins, generating a variety of intra- and intermolecular covalent adducts on the apolipoprotein B-100 particle in LDL. Characterization of the aldehyde adducts generated in the protein is therefore critical in understanding the nature of the oxidized LDL. However, the majority of adducts generated during the oxidative modification of LDL have not yet been chemically characterized. In this review, the current status of aldehyde adducts quantitatively analyzed in the Cu²⁺-oxidized LDL is reviewed.     

Liposomes as membrane model for study of lipid peroxidation

This article describes the properties, production and characterization of liposomes with special reference to their use as membrane model for the study of lipid peroxidation. It presents briefly the methods that can be used for the assay of liposomal lipid peroxidation and brings out the special advantages these liposomes provide in elucidating the mechanism of lipid peroxidation by different physical and chemical agents. Studies involving liposomal lipid peroxidation by different agents and the consequent changes in the structure and function of liposomal membrane have been reviewed briefly.     

7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: Difference between cellular and plasma lipid peroxidation

Polyunsaturated fatty acids and their esters are known to be susceptible to free radical-mediated oxidation, whereas cholesterol is thought to be more resistant to oxidation. In fact, it has been observed that in the case of plasma lipid peroxidation, the amount of oxidation products of polyunsaturated fatty acids such as linoleic acid was higher than that of cholesterol. In contrast, during oxidative stress-induced cellular lipid peroxidation, oxidation products of cholesterol such as 7-hydroxycholesterol (7-OHCh) were detected in greater amounts than those of linoleates such as hydroxyoctadecadienoic acid (HODE). There are several forms of oxidation products of cholesterol and linoleates in vivo, namely, hydroperoxides, as well as the hydroxides of both the free and ester forms of cholesterol and linoleates. To evaluate these oxidation products, a method used to determine the lipid oxidation products after reduction and saponification was developed. With this method, several forms of oxidation products of cholesterol and linoleates are measured as total 7-OHCh (t7-OHCh) and total HODE (tHODE), respectively. During free radical-mediated lipid peroxidation in plasma, the amount of tHODE was 6.3-fold higher than that of t7-OHCh. In contrast, when Jurkat cells were exposed to free radicals, the increased amount of cellular t7-OHCh was 5.7-fold higher than that of tHODE. Higher levels of t7-OHCh than those of tHODE have also been observed in selenium-deficient Jurkat cells and glutamate-treated neuronal cells. These results suggest that, in contrast to plasma oxidation, cellular cholesterol is more susceptible to oxidation than cellular linoleates. Collectively, cholesterol oxidation products at the 7-position may be a biomarker of cellular lipid peroxidation.     

Substituted p-hydroquinones as inhibitors of lipid peroxidation

The technique based on monitoring oxygen consumption was applied to study 12 alkyl- and methoxy-substituted p-hydroquinones (QH(2)) as a chain-breaking antioxidant during the oxidation of styrene and methyl linoleate (ML) in bulk as well as ML oxidation in micellar solution of sodium dodecyl sulfate (SDS) at 37 degrees C. The antioxidant activities of QH(2) were characterized by two parameters: the rate constant k(1) for reaction of QH(2) with the peroxy radical LO(2)*: QH(2)+LO(2)*-->QH*+LOOH and the stoichiometric factor of inhibition, f, which shows how many kinetic chains may be terminated by one molecule of QH(2). In the case of styrene and ML oxidation in bulk, f values never exceed two; for the majority of QH(2), f was found to be significantly less than two due to the interaction of QH* with molecular oxygen. In the absence of superoxide dismutase (SOD), all the studied QH(2) displayed a very moderate if any antioxidant capability during ML oxidation in SDS micelles. When 20U/ml SOD was added, the majority of QH(2) showed a pronounced ability to inhibit ML oxidation, f parameter being ca. one. The features of QH(2) as an antioxidant in aqueous environment are suggested to associate with the reactivity of semiquinone (Q*(-)). Q*(-) reacts readily with molecular oxygen with formation of superoxide (O(2)*(-)); further reactions of O(2)*(-) result in fast depleting QH(2) and chain propagation. The addition of SOD results in purging a reaction mixture from O(2)*(-) and, as a corollary, in depressing undesirable reactions with the participation of O(2)*(-). With all the oxidation models, QH(2) were found to be very reactive to LO(2)*. The rate constants k(1) decreased progressively when going from the oxidation of styrene to ML oxidation in bulk and further to ML oxidation in SDS micelles.     

Protein modification by oxidized phospholipids and hydrolytically released lipid electrophiles: Investigating cellular responses

Oxygen is essential for the growth and function of mammalian cells. However, imbalances in oxygen or abnormalities in the ability of a cell to respond to oxygen levels can result in oxidative stress. Oxidative stress plays an important role in a number of diseases including atherosclerosis, rheumatoid arthritis, cancer, neurodegenerative diseases and asthma. When membrane lipids are exposed to high levels of oxygen or derived oxidants, they undergo lipid peroxidation to generate oxidized phospholipids (oxPL). Continual exposure to oxidants and decomposition of oxPL results in the formation of reactive electrophiles, such as 4-hydroxy-2-nonenal (HNE). Reactive lipid electrophiles have been shown to covalently modify DNA and proteins. Furthermore, exposure of cells to lipid electrophiles results in the activation of cytoprotective signaling pathways in order to promote cell survival and recovery from oxidant stress. However, if not properly managed by cellular detoxification mechanisms, the continual exposure of cells to electrophiles results in cytotoxicity. The following perspective will discuss the biological importance of lipid electrophile protein adducts including current strategies employed to identify and isolate protein adducts of lipid electrophiles as well as approaches to define cellular signaling mechanisms altered upon exposure to electrophiles. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.     

Effect of partial ischemia on phospholipids and postischemic lipid peroxidation in rabbit spinal cord

Rabbit spinal cord, subjected to severe partial ischemia induced by abdominal aorta ligation tightly below the renal arteries, was analyzed for phospholipid composition and levels of lipid peroxidation products after 10, 20, and 40 min of the insult. Under conditions when spinal cord blood flow was decreased below 5% of control, concentrations of inositol and ethanolamine phospholipids were decreased by 30% and 10%, respectively. Phosphatidic acid concentration was also altered during ischemia. No accumulation of thiobarbituric acid reactive substances (TBA-RS), conjugated dienes and fluorescent lipid soluble material was found throughout the ischemic period. Pattern of TBA-RS, conjugated diene, and fluorophore formation during postischemic in vitro incubation without and with a peroxidation couple (Fe²⁺, ascorbic acid) showed increased susceptibility to postischemic lipid peroxidation in tissues after 20 and 40 min of ischemia.     

An antioxidant-like action for non-peroxidisable phospholipids using ferrous iron as a peroxidation initiator

The degradation of phospholipids containing polyunsaturated fatty acids, termed peroxidation, poses a constant challenge to membranes lipid composition and function. Phospholipids with saturated (e.g. PC 16:0/16:0) and monounsaturated fatty acids (e.g. PC 16:0/18:1) are some of the most common phospholipids found in membranes and are generally not peroxidisable. The present experiments show that these non-peroxidisable phospholipids, when present in liposomes with peroxidisable phospholipids (i.e. those containing polyunsaturated fatty acids) such as PC 16:0/18:2 and Soy PC, produce an inhibitory effect on rates of peroxidation induced by ferrous-iron. This inhibitory effect acts to extend the duration of the lag phase by several-fold. If present in natural systems, this action could enhance the capacity of conventional antioxidant mechanisms in membranes. The results of this preliminary work suggest that non-peroxidisable phospholipids may exert an antioxidant-like action in membranes.     

Oxidative modification of low-density lipoprotein: lipid peroxidation by myeloperoxidase in the presence of nitrite

Oxidative modification of low-density lipoprotein (LDL) is a pivotal process in early atherogenesis and can be brought about by myeloperoxidase (MPO), which is capable of reacting with nitrite, a NO metabolite. We studied MPO-mediated formation of conjugated dienes in isolated human LDL in dependence on the concentrations of nitrite and chloride. This reaction was strongly stimulated by low concentrations (5-50 microM) of nitrite which corresponds to the reported concentration in the arterial vessel wall. Under these conditions no protein tyrosine nitration occurred; this reaction required much higher nitrite concentrations (100 microM-1 mM). Chloride neither supported lipid peroxidation alone nor was its presence mandatory for the effect of nitrite. We propose a prominent role of lipid peroxidation for the proatherogenic action of the MPO/nitrite system, whereas peroxynitrite may be competent for protein tyrosine nitration of LDL. Monomeric and oligomeric flavan-3-ols present in cocoa products effectively counteracted, at micromolar concentrations, the MPO/nitrite-mediated lipid peroxidation of LDL. Flavan-3-ols also suppressed protein tyrosine nitration induced by MPO/nitrite or peroxynitrite as well as Cu2+-mediated lipid peroxidation of LDL. This multi-site protection by (-)-epicatechin or other flavan-3-ols against proatherogenic modification of LDL may contribute to the purported beneficial effects of dietary flavan-3-ols for the cardiovascular system.     

青藤碱磷脂复合物纳米结构脂质载体的制备、表征及药动学研究

为制备青藤碱磷脂复合物纳米结构脂质载体,并进行体外和SD大鼠体内评价。实验采用溶剂挥发法制备青藤碱磷脂复合物,乳化超声法制备青藤碱磷脂复合物纳米结构脂质载体。考察其粒径分布、Zeta电位,包封率,载药量及体外释药等基本理化性质。SD大鼠分别灌胃给予青藤碱混悬液和青藤碱磷脂复合物纳米结构脂质载体,比较药动学行为及生物利用度。结果显示,青藤碱磷脂复合物纳米结构脂质载体的平均粒径为201.32±5.05 nm,Zeta电位为-22.2±1.5 mV,包封率为80.31±1.01%,载药量为4.42±0.28%,体外释药具有明显的缓释特征,体外释药模型符合Weibull释药模型,拟合方程为:LnLn(1/1-Mt/M∞)=0.576 6Lnt-1.478 1(r=0.988 8)。体内药动学研究结果表明,磷脂复合物纳米结构脂质载体改变了青藤碱的药动学行为,增强了体内吸收,延长了青藤碱在体内滞留时间,相对生物利用度提高到了1.75倍。因此,青藤碱磷脂复合物纳米结构脂质载体可显著促进青藤碱体内吸收,提高其口服生物利用度。     

Influence of tidal volume on pulmonary NO release,tissue lipid peroxidation and surfactant phospholipids

Mechanical stress during ventilation may cause or aggravate acute lung injury. This study investigates the influence of low vs. high tidal volume (V(t)) on factors known to play key roles in acute lung injury: nitric oxide release, eNOS and iNOS gene expression, lipid peroxidation (LPO), and surfactant phospholipids (PL). Isolated rabbit lungs were subjected to one of three ventilation patterns for 135 min (V(t)-PEEP): 6 ml/kg-0 cm H(2)O. 12 ml/kg-0 cm H(2)O 6 ml/kg-5 cm H(2)O, 12 ml/kg-0 cm H(2)O, and 6 ml/kg-5 cm H(2)O resulted in comparable peak inspiratory pressure (PIP). This allowed comparing low and high V(t) without dependence on PIP. Ventilatory patterns did not induce changes in pulmonary artery pressure, vascular permeability (K(f,c)), PIP or pulmonary compliance. High V(t) in comparison with both of the low V(t) groups caused an increase in BALF-nitrite (30.6+/-3.0* vs. 21.4+/-2.2 and 16.2+/-3.3 microM), BALF-PL (1110+/-19* vs. 750+/-68 and 634+/-82 microg/ml), and tissue LPO product accumulation (0.62+/-0.051* vs. 0.48+/-0.052 and 0.43+/-0.031 nmol/mg), *P<0.05 each. Perfusate nitrite and BALF-PL composition (assessed by use of 31P-NMR spectroscopy and MALDI-TOF mass spectrometry) did not differ among the groups. High V(t) ventilation reduced eNOS gene expression but did not affect iNOS expression. The increased release of NO and the accumulation of LPO products may represent early lung injury while elevated BALF-PL may reflect distension-induced surfactant secretion.     

Effect of liposomes on lipid peroxidation and total phospholipids in rabbit ischemic spinal cord model

The effect of spinal cord ischemia (induced by abdominal aorta ligation for 20 minutes) on lipid peroxidation and TPL composition was investigated and discussed in our previous articles. It is known, that partially reduced species of oxygen can be formed under aerobic conditions. For that reason, the effect of ligation release for 60 minutes was observed in experimental animals treated with the selected liposomes. Administration of CP, (CP+SA) and (CP+Chol) liposomes applied 30 minutes before 20 minutes ischemia revealed an ameliorating effect on in vivo and in vitro Fe-dependent peroxidation manifested by TBA-RS accumulation. Combined use of (CP+SA) liposomes with lipophylic form of stobadine (DP 1031) was not more effective. Application of CP liposomes directly before the ligation release slightly increased the antiradical capacity in spinal cord homogenates comparing with not-treated animals. Accumulation of TBA-RS was accompanied by TPL degradation during recirculation period but values of TPL after liposomal treatment were unaffected.     

The role of lipid peroxidation and antioxidants in oxidative modification of LDL.

The purpose of this study is to provide a comprehensive survey on the compositional properties of LDL (e.g., lipid classes, fatty acids, antioxidants) relevant for its susceptibility to oxidation, on the mechanism and kinetics of LDL oxidation, and on the chemical and physico-chemical properties of LDL oxidized by exposure to copper ions. Studies on the occurrence of oxidized LDL in plasma, arteries, and plaques of humans and experimental animals are discussed with particular focus on the use of poly- and monoclonal antibodies for immunochemical demonstration of apolipoprotein B modifications characteristic for lipid peroxidation. Apart from uptake of oxidized LDL by macrophages, studies describing biological effects of heavily or minimally oxidized LDL are only briefly addressed, since several reviews dealing with this subject were recently published. This article is concluded with a section on the role of natural and synthetic antioxidants in protecting LDL against oxidation, as well as some previously unpublished material from our laboratories.     

Total hydroxyoctadecadienoic acid as a marker for lipid peroxidation in vivo

An improved method for the measurement of lipid peroxidation in vivo has been recently developed, where total hydroxyoctadecadienoic acid (HODE) and 7-hydroxycholesterol (FCOH) were determined by GC/MS analysis from physiological samples after reduction with sodium borohydride and saponification by potassium hydroxide. In this method, both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as HODE and FCOH, respectively. The ratio of stereo-isomer, (Z, E)-HODE/(E, E)-HODE, could be also measured. In the present study, in order to examine the effect of continuous, slow flux of free radicals in vivo, a water-soluble radical generator was administered to rats and mice and the amounts of HODE and 8-isoprostane in plasma and liver were measured. It was found that the administration of free radical-generating azo compound increased the level of HODE and decreased the (Z, E)-HODE/(E, E)-HODE ratio in both plasma and liver. The level of HODE was much higher than 8-isoprostane.     

Modulation of endoplasmic reticulum-bound cholesterol regulatory enzymes by iron/ascorbate-mediated lipid peroxidation

Mammalian sterol regulatory enzymes are integral membrane proteins of the endoplasmic reticulum. They play a critical role in liver cholesterol homeostasis and the maintenance of overall cholesterol balance in different species. Because lipid peroxidation has been implicated in hepatic dysfunction and atherosclerosis, we hypothesized that its occurrence could alter the composition and properties of the bilayer lipid environment, and thereby affect the functions of these membrane proteins. Preincubation of rat liver microsomes with iron (Fe)/ascorbate (50 microM/200 microM), known to induce peroxidation, resulted in a significant inhibition of (i) the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase (46%, p < .01), (ii) the crucial enzyme controlling the conversion of cholesterol in bile acids, cholesterol 7alpha-hydroxylase (48%, p < .001), and (iii) the central enzyme for cholesterol esterification: Acyl-CoA:cholesterol acyltransferase (ACAT, 80%, p < .0001). The disturbances of these key enzymes took place concomitantly with the high production of malondialdehyde (350%, p < .007) and the loss of polyunsaturated fatty acids (36.19 +/- 1.06% vs. 44.24 +/- 0.41 in controls, p < .0008). While alpha-tocopherol simultaneously neutralized lipid peroxidation, preserved microsomal fatty acid status, and restored ACAT activity, it was not effective in preventing Fe/ascorbate-induced inactivation of both HMG-CoA reductase (44%, p < .01) and cholesterol 7alpha-hydroxylase (71%, p < .0001). These results indicate that Fe/ascorbate alters the activity of the rate-determining steps in liver cholesterol metabolism, either directly or via lipid peroxidation, capable of modifying their membrane environment. The present data also suggest that the three regulatory enzymes respond differently when exposed to Fe/ascorbate or antioxidants, which may be due to dissimilar mechanisms.     

The effect of histidine modification on copper-dependent lipid peroxidation in human low-density lipoprotein

Summary

Lipid peroxidation and subsequent oxidative modification of low-density lipoprotein (LDL) have been implicated as causal events in atherosclerosis. Cu²⁺ may play an important role in LDL oxidation by binding to histidine residues of apolipoprotein B-100 (apo B) and initiating and propagating lipid peroxidation. To investigate the role of histidine residues, we used diethylpyrocarbonate (DEPC), a lipid-soluble histidine-specific modifying reagent. When LDL (0.1 mg protein/ml, or 0.2 µM) was incubated with DEPC (1 mM), at least 76 ± 7% of the histidine residues in apo B were modified. Treatment of LDL with DEPC led to an increase in the rate of Cu²⁺-induced initiation of lipid peroxidation (R_i), but a significant decrease in the rate of propagation. These changes resulted in an overall increased resistance of LDL to oxidation, with a significantly increased lag phase preceding the propagation phase of lipid peroxidation. In contrast to DEPC, ascorbate completely prevented the initiation of LDL oxidation (R_i = 0). Our data indicate that there are two types of copper/histidine binding sites on apo B: those facing the lipid core of the LDL particle, which mediate the propagation of lipid peroxidation and are modified by DEPC; and those found on the surface of the LDL particle exposed to the aqueous environment, which are responsible for mediating the initiation of lipid peroxidation and are modifiable by ascorbate in the presence of Cu²⁺.     

pH dependence of lipid peroxidation and albumin oxidative modification: possible implications to the pH paradox

The hydrogen ion concentration may have an important influence in biological free radical reactions. We studied the effect of an acidic pH on two models of free radical-mediated damage: copper-induced lipid peroxidation in plasma and copper/hydrogen peroxide-induced oxidative modification of albumin. A reduction of pH from 7·4 to 6·6 decreased diene conjugation by 32%, thiobarbituric acid-reactive substances formation by 25% and fluorescence generation by 53% in plasma exposed to cupric chloride. At pH values lower than 6·6 an even greater inhibition of lipid peroxidation in plasma was obtained. Visible fluorescence development in albumin by exposure to site-specific generation of free radicals was also increasingly reduced by decreasing pH values. From pH 7·4 to 6·6 there was a 50% fluorescence generation inhibition. The observed partial protection of lipids and proteins against oxidative damage by an acidic pH alerts to the need for rigorously controlling the pH values when assaying compounds for antioxidant properties in vitro. It may also contribute to the explanation for the protective effect of an acidic pH against anoxic cell injury and for cell death that is precipitated by a rapid return to a normal pH following reperfusion (the ‘pH paradox’).     

Urinary aldehydes as indicators of lipid peroxidation in vivo

The excretion of malondialdehyde (MDA), lipophilic aldehydes and related carbonyl compounds in rat and human urine was investigated. MDA was found to be excreted mainly in the form of two adducts with lysine, indicating that its predominant reaction in vivo is with the lysine residues of proteins. Adducts with the phospholipid bases serine and ethanolamine and the nucleic acid bases guanine and deoxyguanosine also were found. Except for the adduct with deoxyguanosine (dG-MDA), the excretion of these compounds increased with peroxidative stress imposed in the form of vitamin E deficiency or the administration of iron or carbon tetrachloride. Marked differences in the concentration of dG-MDA in different tissues were correlated with their content of fatty acids having three or more double bonds, the putative source of MDA. Fourteen nonpolar and eleven polar lipophilic aldehydes and other carbonyl compounds were identified as their 2,4-diphenylhydrazine derivatives in rat urine. The excretion of five nonpolar and nine polar compounds was increased under conditions of peroxidative stress. The profile of lipophilic aldehydes obtained for human urine resembled that for rat urine. Except for a reported 4-hydroxynon-2-enal conjugate with mercapturic acid, the conjugated forms of the lipophilic aldehydes excreted in urine remain unidentified. Aldehyde excretion is influenced by numerous factors that affect the formation of lipid peroxides in vivo such as energy status, physical activity and environmental temperature, as well as by wide variations in the intake of peroxides in the diet. Consequently, urinalysis for aldehydic products of lipid peroxidation is an unreliable indicator of the general state of peroxidative stress in vivo.     

Protein-phosphate complexes as an inhibitor of iron-induced lipid peroxidation

Effect of phosphate buffer (pH 6.2) alone or in the presence of bovine serum albumin and other proteins on iron (II)-induced lipid peroxidation was studied. Phosphate buffer alone and in the presence of bovine serum albumin was found to inhibit lipid peroxidation. The inhibition was higher when bovine serum albumin was also present. Other proteins also inhibited lipid peroxidation in the presence of phosphate. Inhibition by proteins in the presence of phosphate seems to be due to binding of iron with phosphate and with protein-phosphate complexes. Reversal in inhibition was observed with an increase in iron concentration in reaction mixture. Equilibrium dialysis showed more binding of iron to protein in the presence of phosphate than in the presence of chloride ions.