Investigation of Lipid Peroxidation in Human Low Density Lipoprotein

Human plasma low density lipoprotein (LDL) exposed to oxygen saturated buffer becomes depleted of alpha-tocopherol within 3 to 6 hours. Thereafter, lipid peroxidation commences as evidenced by the loss of 18:2 (67nmol/mg LDL) and 20:4 (12nmol/mg LDL) and the concomitant formation of 4-hydroxy-nonenal (0.28 nmol/mg LDL) and fluorescent compounds. The major fluorophor in apo B of oxidized LDL has an excitation maximum at 355 nm and an emission maximum at 430 nm. A fluorophor with the same spectral properties is produced in apo B, if LDL is incubated with 4-hydroxynonenal, whereas malonal-dehyde gives a fluorophor with excitation and emission maxima at 400/470nm. Three-dimensional fluorescence spcetroscopy proved to be an useful tool in analysing the complex fluorescence of apo B.     

Lipid Peroxidation and Biogenic Aldehydes: From the Identification of 4-Hydroxynonenal to Further Achievements in Biopathology

The formation, reactivity and toxicity of aldehydes originating from lipid peroxidation of cellular membranes are reviewed. Very reactive aldehydes, namely 4-hydroxyalkenals, were first shown to be formed in autoxidizing chemical systems. It was subsequently shown that 4-hydroxyalkenals are formed in biological conditions, i.e. during lipid peroxidation of liver microsomes incubated in the NADPH-Fe systems. Our studies carried out in collaboration with Hermann Esterbauer which led to the identification of 4-hydroxynonenal (4-HNE) are reported. 4-HNE was the most cytotoxic aldehyde and was then assumed as a model molecule of oxidative stress. Many other aldehydes (alkanals, alk-2-enals and dicarbonyl compounds) were then identified in peroxidizing liver microsomes or hepatocytes. The in vivo formation of aldehydes in liver of animals intoxicated with agents that promote lipid peroxidation was shown in further studies. In a first study, evidence was forwarded for aldehydes (very likely alkenals) bound to liver micro-somal proteins of CCl₄ or BrCCl₃-intoxicated rats. In a second study, 4-HNE and a number of other aldehydes (alkanals and alkenals) were identified in the free (non-protein bound) form in liver extracts from bromoben-zene or ally-1 alcohol-poisoned mice. The detection of free 4-HNE in the liver of CCl₄ or BrCCl₃-poisoned animals was obtained with the use of an electrochemical detector, which greatly increased the sensitivity of the HPLC method. Furthermore, membrane phospho-lipids bearing carbonyl groups were demonstrated in both in vitro (incubation of microsomes with NADPH-Fe) and in vivo (CCl₄ or BrCCl₃ intoxication) conditions. Finally, the results concerned with the histochemical detection of lipid peroxidation are reported. The methods used were based on the detection of lipid peroxidation-derived carbonyls. Very good results were obtained with the use of fluorescent reagents for carbonyls, in particular with 3-hydroxy-2-naphtoic acid hydrazide (NAH) and analysis with confocal scanning fluorescence microscopy with image video analysis. The significance of formation of toxic aldehydes in biological membranes is discussed.     

Assessment of growth inhibition by aldehydic lipid peroxidation products and related aldehydes by Saccharomyces cerevisiae

The effect of pretreatment with aldehydes on the subsequent colony forming efficiency (CFE) of Saccharomyces cerevisiae was investigated. All 21 aldehydes tested inhibited CFE in a dose-dependent manner. The effective doses, however, differed markedly from 300 mM to 0·07 mM depending on the functional groups and chain length of the aldehydes. Amongst the nine representatives of n-alkanals, formaldehyde was the most potent inhibitor, reducing CFE to 50 per cent at a dose of 0·3 mM (IC₅₀). In the series of 2-trans-alkenals, acrolein was most effective with an IC₅₀ of 0·08 mM and amongst the 4-hydroxy 2-trans-alkenals, 4-hydroxynonenal was most effective with IC₅₀ of 0·07 mM . In general, effectiveness decreased in the order: 4-hydroxyalkenals > 2-alkenals ? n-alkenals. It is proposed that S. cerevisiae is a promising target cell to elucidate further the molecular mechanisms by which aldehydes, particularly the lipid peroxidation product 4-hydroxynonenal, inhibits cell proliferation.     

大蒜素对运动大鼠心肌脂质过氧化反应的干预作用研究

研究大蒜素对大鼠大负荷运动后心肌脂质过氧化反应的干预作用及机制。将成年雄性SD大鼠48只,随机分为安慰剂组(A组)和用药组(B组),B组每日定时经口腔灌胃给予大蒜素30 mg/kg体重,A组给予相同体重比例的安慰剂(蒸馏水)。两周后观察各组在安静时及完成一次性大负荷游泳运动后即刻、运动后24 h心肌超微结构变化,并测定心肌组织SOD、MDA、TAC和血清CK-MB等指标。结果显示运动后两组大鼠心肌超微结构都出现改变和损伤,B组表现较轻微。B组在运动后两个时相心肌SOD和TAC均显著高于A组;心肌MDA和血清CK-MB非常显著低于A组。提示大蒜素有助于减轻大负荷运动导致的心肌脂质过氧化反应。     

Effect of Ascorbate on Red Blood Cell Lipid Peroxidation

The present study investigates the effect of ascorbate on red cell lipid peroxidation. At a concentration between 0.2 mmol-20 mmol/l ascorbic acid reduces hydrogen peroxide-induced red blood cell lipid peroxidation resulting in a marked decrease in ethane and pentane production as well as in haemolysis. Ascorbic acid also shows an antioxidant effect on chelated iron-catalyzed hydrogen peroxide-induced peroxidation of erythrocyte membranes. At a concentration of 10 mmol/l ascorbic acid totally inhibits oxidative break-down of polyunsaturated fatty acids by radicals originating from hydrogen peroxide.

Our results indicate that ascorbate at the chosen concentration has an antioxidant effect on red blood cell lipid peroxidation.     

Lipid Peroxidation and Antioxidant Systems in Rat Brain: Effect of Chronic Alcohol Consumption

The effect of chronic ethanol exposure, in a liquid diet, on lipid peroxidation and some antioxidant systems of rat brain was investigated. Chronic ethanol administration induced a greater susceptibility to iron/ascorbate-induced lipid peroxidation, estimated as thiobarbituric reactive substances (TBARS) production, in the microsomal fraction, but a lower lipid peroxidation in the total homogenate. Glutathione (GSH) levels as well as GSH peroxidase and GSH reductase were unaffected, while the activity of Cu-Zn superoxide dismutase was decreased and that of catalase increased. Lipid peroxidation experiments performed in the presence of some hydroxyl radical scavengers suggested that a greater OH^· generation may be responsible of the greater TBARS production in the microsomal fraction of ethanol treated rats; differently, in total homogenate of control and ethanol rats a relationship was found between the redox state of iron and TBARS production, suggesting that the lower lipid peroxidation in treated rats may depend on a different modulation of the iron redox state.     

Inhibitory Effect of Dipyridamole and its Derivatives on Lipid Peroxidation in Mitochondria

Dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidino-[5,4-d]pyrimidine, is a coronary vasodilator widely used in clinics. It has also been reported to have coactivator activity for a number of antitumour drugs and antioxidant activity in membrane systems. In recent years we have been studying the spectroscopic properties of this drug and several of its derivatives as well as their interaction with charged micelles and phospholipid monolayers. A strong interaction of DIP and DIP derivatives with these model membrane systems and a dependence of the strength of the interaction upon the chemical structure of the DIP derivative was observed. Here, the antioxidant effect of DIP and the derivatives, RA14, RA47, and RA25, was compared. We observed that although it strongly inhibits the iron-induced lipoperoxidation on mitochondria (IC₅₀ = 1 μM), it shows no protection against an organic oxidant, cumene hydroperoxide. The order of hydrophobicity of the DIP derivatives, DIP > RA14 > RA47 > RA25, correlates very well with both the values of the association constants of these derivatives to micelles, their localization in the micelles, and phospholipid films and their antioxidant effect on mitochondria. So, a very good correlation of the structure of the drug in regarded to the nature of its substituents with the biological activity is observed. Essentially the same result was observed either measuring the lipid peroxidation or the membrane fluidity by ESR, suggesting that the effect of DIP and DIP derivatives is probably associated to their binding to the lipid bilayer and not to interaction with membrane proteins.     

脂质过氧化引起的DNA损伤研究进展

脂质过氧化可以引起各种碱基损伤、DNA链断裂和各种荧光产物生成,并对DNA分子鸟嘌呤碱基具有选择性损伤.过渡金属离子可以明显加深脂质过氧化对DNA的损伤程度.多种抗氧化剂、活性氧自由基清除剂对脂质过氧化引起的DNA损伤有一定程度的保护作用.具有致突、致癌作用的8－羟基鸟嘌呤已经观察到．脂质过氧化的致突变、致癌变作用机制引起了人们的极大兴趣．     

Lipid Peroxidation in Choline-Methionine Deficiency

A deficiency of choline and methionine is hepatocarcinogenic and is associated with an apparent increase in lipid peroxidation. In this study the susceptibility of microsomes and nuclei to ferritin-dependent lipid peroxidation is examined together with the status of the peroxidation- protective systems. Choline-methionine deficiency caused an increase in Se-independent GSH peroxidases (GSH transferase subunit 2) and membrane vitamin E but a decrease in Se-dependent GSH peroxidase and microsomal GSH peroxidase activity. Choline-methionine deficient microsomes and nuclei were 4-fold more susceptible to lipid peroxidation induced in vitro by physiological concentrations of ferritin/ascorbate/ADP; and the peroxidation was less effectively inhibited by GSH and soluble GSH peroxidases than controls. The results indicate that a decreased level of Se-dependent and membrane GSH peroxidases is involved in the increase in lipid peroxidation observed in choline-methionine deficiency.     

Comparison of the Inactivation of Microsomal Glucose-6-Phosphatase by in Situ Lipid Peroxidation-Derived 4-Hydroxynonenal and Exogenous 4-Hydroxynonenal

1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied.

2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes.

3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E.

4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5μM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150μM or 300μM was needed with a preincubation time of 30 and 60 min, respectively.

5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynbnenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.     

Aluminum Enhances Melanin-Induced Lipid Peroxidation

The present study was conducted to characterize the possible interaction of Al³⁺ and Fe²⁺ with synthetic melanin in the potentiation of lipid peroxidation in liposomes and rat caudate-putamen homogenates. Al³⁺ stimulated melanin-initiated lipid peroxidation as measured by the production of 2-thiobarbituric acid-reactive substances (TBARS) and conjugated dienes. The effect of Al³⁺ was dependent on melanin (10–100 g/ml) and Al³⁺ (2.5–250 M) concentrations and no synergism between Fe²⁺ and Al³⁺ was observed. The prooxidant effect of Al³⁺ was partially inhibited by superoxide dismutase indicating the involvement of O ₂ ^- . Ga³⁺ and Be²⁺ which can increase NADH oxidation in the presence of O ₂ ^- , also were shown to stimulate melanin-initiated TBARS production. Based on the effect of Al³⁺ and other non redox metals, we suggest that Al³⁺ does not act through either the induction of melanin free radicals, or the induction of changes in membrane physical properties. Results show that Al³⁺ enhances melanin-initiated lipid peroxidation in part through an interaction with O ₂ ^- generated from the autoxidation of melanin. We speculate that Al³⁺ contributes to neuromelanin-mediated oxidative damage in dopaminergic neurons and subsequent neuronal degeneration and death in Parkinson's disease.     

Effect of Low-Intensity Laser Radiation on the Lipid Peroxidation in Wheat Callus Culture

The effect of low-intensity laser radiation on the accumulation of secondary products of lipid peroxidation was studied in wheat (Triticum aestivum L.) tissue culture. A five-min-long callus irradiation by the helium-neon laser light with the wavelength = 632.8 nm and the intensity of 10 mW resulted in an increase in the accumulation of the products of reaction with thiobarbituric acid (TBA-reactive products). The effect was less pronounced within two days after laser treatment, but even in this case the content of TBA-reactive products was greater than in the control. The data obtained confirm that the low-intensity laser radiation can induce lipid peroxidation processes in plant tissues.     

ReviewIsoprostanes: Novel Bioactive Products of Lipid Peroxidation

Isoprostanes, are a novel group of prostaglandin-like compounds that are biosynthesised from esterified polyunsaturated fatty acid (PUFA) through a non-enzymatic free radical-catalysed reaction. Several of these compounds possess potent biological activity, as evidenced mainly through their pulmonary and renal vasoconstrictive effects, and have short half-lives. It has been shown that isoprostanes act as full or partial agonists through thromboxane receptors. Both human and experimental studies have indicated associations of isoprostanes and severe inflammatory conditions, ischemia-reperfusion, diabetes and atherosclerosis. Reports have shown that F²-isoprostanes are authentic biomarkers of lipid peroxidation and can be used as potential in vivo indicators of oxidant stress in various clinical conditions, as well as in evaluations of antioxidants or drugs for their free radical-scavenging properties.

Higher levels of F²-isoprostanes have been found in the normal human pregnancy compared to non-pregnancy, but their physiological role has not been well studied so far. Since bioactive F²-isoprostanes are continuously formed in various tissues and large amounts of these potent compounds are found unmetabolised in their free acid form in the urine in normal basal conditions with a wide inter-individual variation, their role in the regulation of normal physiological functions could be of further biological interest, but has yet to be disclosed. Their potent biological activity has attracted great attention among scientists, since these compounds are found in humans and animals in both physiological and pathological conditions and can be used as reliable biomarkers of lipid peroxidation.     

Possible Sources of Iron for Lipid Peroxidation

Possible sources of iron for lipid peroxidation are described and discussed. In particular. evidence is presented that microsomes contain ferric nonheme iron which may participate in formation of lipid oxidants. provided reductants are available to favor its mobilization from membrane binding sites. Aging-and tumor-associated changes of this microsomal pool of nonheme iron are also described and discussed from biochemical and biomedical viewpoints.     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.     

Erythrocyte Lipid Peroxidation and Antioxidants in Gastric Cancer Patients

The present study has analysed the relationship between lipid peroxidation and antioxidant status in erythrocytes from 24 adult male gastic cancer patients and an equal number of age- and sex-matched normal subjects. Erythrocyte lipid peroxidation was markedly increased. Both enzymic and non-enzymic antioxidants were decreased in erythrocytes of gastric cancer patients. The present study highlights the occurrence of lipid peroxidation and possible breakdown of antioxidant status in patients with gastric carcinoma. © 1997 John Wiley & Sons, Ltd.     

Effect of Fish Oil and Coconut Oil on Antioxidant Defence System and Lipid Peroxidation in Rat Liver

Diets high in fish oil containing polyunsaturated fatty acids of the n-3 family. have been suggested to decrease the risk of cardiovascular disease. However these lipids are highly susceptible to oxidative deterioration. In order to investigate the influence of n-3 fatty acids on oxidative status, the effect of feeding rats with fish oil or cocunut oil diets was studied by measuring different parameters related to an oxidative free radical challenge. Synthetic diets containing 15% (w/v) fish oil or coconut oil were used to feed growing rats for 4 weeks. As compared to control diet, the fish oil containing diet produced a significant decrease of cholesterol and triglyceride concentration in serum. however there was a significant increase in lipid peroxidation products. In addition, in fish oil fed animals, there was also a decrease in vitamin E and A concentration. Furthermore, the rate of lipid peroxidation in isolated microsomes was three fold higher in rats fed fish oil as compared to rats with coconut oil diet. No significant differences between the two experimental groups were observed in superoxide dismutase (SOD) and phospholipid hydroperoxide glutathione peroxidase (PHGPX) activities. However, there was a decrease in glutathione peroxidase (GPX) activity. These results suggest that fish oil feeding at an amount compatible with human diet, although decreasing plasma lipids, actually challenge the antioxidant defence system, thus increasing the susceptibility of tissues to free radical oxidative damage.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

大豆下胚轴线粒体的衰老与膜脂的过氧化作用

离体的大豆下胚轴线粒体,在人工衰老条件下,产生了结构膨胀和细胞色素氧化酶活性的下降。衰老的线粒体也发生膜脂的过氧化作用——丙二醛、脂质的氢过氧化物和荧光脂褐色素明显增加。而且,线粒体衰老时产生的膜脂过氧化产物雨二醛,可能是膜脂的磷脂酰胆碱和磷脂酰乙醇胺中的亚麻酸发生过氧化反应的结果。     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.