Lipid peroxidation during myocardial reperfusion

Reperfusion of heart muscle after prolonged ischaemia is associated with metabolic and functional abnormalities and eventual cell death. Free radical induced lipid peroxidation of cell membranes is thought to be a major mechanism in the evolution of reperfusion damage. The evidences in support for this kind of damage are based on tissue malondialdehyde quantitation by the thiobarbituric acid test (TBA-test). In an attempt to verify this topic we have subjected isolated and Langendorff perfused rabbit hearts to a period of 60 minutes of severe ischaemia plus 30 minutes of reperfusion. At appropriate time points malondialdehyde was determined in the tissue by means of TBA-test and directly by reversed phase, high pressure, liquid chromatography (HPLC).We have found no correlation between the two compared assays. During reperfusion, there was the formation of non-lipid related, malondialdehyde-like, TBA-reactive substance which leads to overstimations of the extent of lipid peroxidation.On the contrary, by direct HPLC quantitation, there was a decrease of tissue malondialdehyde during ischaemia and during the early phases of reperfusion. Our results demonstrate that TBA-test is not a reliable index of malondialdehyde accumulation in organ system.     

Lipid peroxidation in mitochondria

The present review article takes into consideration the most important aspects of lipid peroxidation in mitochondria. Firstly the various ways by which lipid peroxidation is induced and the relevant mechanisms are described and discussed. After examining the major effects of lipid peroxidation on mitochondrial enzymes and bioenergetic functions, some aspects of the pathophysiology of lipid peroxidation are considered in connection with maturation of reticulocytes, alternative oxidase of plant mitochondria, aging, and ischemia-reperfusion syndrome. The final part of the article is devoted to the regulation and control of lipid peroxidation in mitochondria with particular emphasis to the role of the respiratory substrates.     

Peroxidation of lipids from liver mitochondria during pneumonia

Inflammation of the respiratory system leads to an increase in the relative content of lipids in the liver mitochondria and to an activation of initial stages of their peroxidation. Antioxidative activity of the postmitochondrial liver fraction is in the inverse relation to the concentration of acyl residues with the conjugated diene structures. But the malonic dialdehyde level decreases during the lung inflammation development from 3 days to 3 months. These changes may be explained by respiratory mitochondria chain reconstruction due to an increase in the lipid utilization in the oxidative phosphorylation reactions.     

Antioxidants decreases the intensification of low density lipoprotein in vivo peroxidation during therapy with statins

The oxidative modification of low density lipoprotein (LDL) is thought to play an important role in atherogenesis. Drugs of -hydroxy--methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) family are usually used as a very effective lipid-lowering preparations but they simultaneously block biosynthesis of both cholesterol and ubiquinone Q₁₀ (coenzyme Q), which is an intermediate electron carrier in the mitochondrial respiratory chain. It is known that reduced form of ubiquinone Q₁₀ acts in the human LDL as very effective natural antioxidant. Daily per os administration of HMG-CoA reductase inhibitor simvastatin to rats for 30 day had no effect on high-energy phosphates (adenosin triphosphate, creatine phosphate) content in liver but decreased a level of these substances in myocardium. We study the Cu²⁺-mediated susceptibility of human LDL to oxidation and the levels of free radical products of LDL lipoperoxidation in LDL particles from patients with atherosclerosis after 3 months treatment with natural antioxidants vitamin E as well as during 6 months administration of HMG-CoA reductase inhibitors such as pravastatin and cerivastatin in monotherapy and in combination with natural antioxidant ubiquinone Q₁₀ or synthetic antioxidant probucol in a double-blind placebo-controlled trials. The 3 months of natural antioxidant vitamin E administration (400 mg daily) to patients did not increase the susceptibility of LDL to oxidation. On the other hand, synthetic antioxidant probucol during long-time period of treatment (3–6 months) in low-dose (250 mg daily) doesn't change the lipid metabolism parameters in the blood of patients but their high antioxidant activity was observed. Really, after oxidation of probucol-contained LDL by C-15 animal lipoxygenase in these particles we identified the electron spin resonance signal of probucol phenoxyl radical that suggests the interaction of LDL-associated probucol with lipid radicals in vivo. We observed that 6 months treatment of patients with pravastatine (40 mg daily) or cerivastatin (0.4 mg daily) was followed by sufficiently accumulation of LDL lipoperoxides in vivo. In contrast, the 6 months therapy with pravastatin in combination with ubiquinone Q₁₀ (60 mg daily) sharply decreased the LDL initial lipoperoxides level whereas during treatment with cerivastatin in combination with probucol (250 mg daily) the LDL lipoperoxides concentration was maintained on an invariable level. Therefore, antioxidants may be very effective in the prevention of atherogenic oxidative modification of LDL during HMG-CoA reductase inhibitors therapy.     

Role of dietary lipids in arteriosclerosis in experimental animals

Effects of dietary fats include the development of arteriosclerosis in humans and experimental animals, in addition to hypercholesterolemia. None of the preceding studies explicitly compared the effects of individual fatty acids. To address these issues, we chose exogenously hypercholesterolemic (ExHC) rats and apolipoprotein (apo) E deficient mice as a model for atherosclerosis and assessed the individual role of fatty acids in animals' susceptibility to atherosclerosis. The rats fed on the diet containing DHA or EPA, compared with those fed on the safflower oil (SO) diet, lowered serum cholesterol concentration, prevented platelet aggregation and slowed thickening in the ascending aorta. Apo E deficient mice developed hypercholesterolemia and severe lesion area in aortic root and arch, to a similar extent when they received DHA or SO. These results suggest a direct action of polyunsaturated fatty acids in the arterial wall, in addition to their effects on hypocholesterolemic and haemodynamic action.     

Lipid peroxidation in experimental myocardial infarct: the action of hyperbaric oxygenation

Rats with experimental myocardial infarction demonstrated decrease in the activity of superoxide dismutase and catalase and increase in the content of lipid peroxidation (LPO) products and Schiff bases both in and outside the area of necrosis. The combined ischemic damage and hyperbaric oxygenation resulted in the over additive effect of accumulation of LPO products in and outside the area of infarction. The data suggest that it is desirable to use antioxidants during hyperbaric oxygenation.     

Effects of cystamine and cysteamine on the peroxidation of lipids and the release of proteins from mitochondria

1. Cystamine slightly stimulated the peroxidation of lipids in mitochondria. Maximal effects were obtained at low concentrations (0.5mm). 2. Cysteamine, when allowed to autoxidize, had much stronger effects than cystamine. 3. Cysteamine and GSH did not induce peroxidation when their autoxidation was counteracted. 4. When kept reduced, cysteamine prevented the ascorbate-induced peroxidation of lipids. GSH was less efficient. 5. Cystamine as well as cysteamine prevented the loss of proteins from mitochondria induced by ascorbate, whereas cadaverine, GSSG and GSH were inefficient.     

The formation of carbon monoxide during peroxidation of microsomal lipids

The evolution of carbon monoxide during lipid peroxidation has been demonstrated in microsomal membranes. The formation of carbon monoxide was dependent on the peroxidation process, but independent of the initiators (NADPH-ADP x Fe(+3) or Ascorbate-Fe(+3)) used. Contrary to published results, the carbon monoxide does not result from heme catabolism. Carbon monoxide was generated during the peroxidation of isolated phospholipids, indicating that carbon monoxide may form directly during the peroxidative degradation of unsaturated fatty acids.     

Role of mitochondrial peroxidation of lipids in their hydrolysis by endogenous phospholipase A2

The analysis of lipids (18 compounds all in all) obtained from mitochondria and incubated for two hours was carried out. It has been shown that hydrolysis of individual phospholipids by endogenous phospholipase of these organelles depends on the intensity of lipid peroxidation (LPO). Thus, with the low level of this process the content of phosphatidylethanolamine, cardiolipin and phosphatidylcholine decreased by 25%, 33% and 18%, respectively of the initial level. However, with LPO activation, their content reduced by 63%, 19% and 4%.     

Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes

The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied. Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides. The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes. The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase. It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase. The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation. The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances. The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.     

Role of mitochondria in paricalcitol-mediated cytoprotection during obstructive nephropathy

Vitamin D slows the progression of chronic kidney disease. Furthermore, activators of vitamin D receptors (VDR) have suppressant effects on the renin-angiotensin system, as well as anti-inflammatory and antifibrotic actions. This study aimed to evaluate the cytoprotective effects of paricalcitol, a VDR activator, at the mitochondrial level using an obstructive nephropathy model [unilateral ureteral obstruction (UUO)]. Rats subjected to UUO and controls were treated daily with vehicle or paricalcitol. The control group underwent a sham surgery. The treatment was done for 15 days (30 ng/kg). The following were determined: biochemical parameters; fibrosis; apoptosis; mitochondrial morphology; VDR, AT(1) receptor, and NADPH oxidase 4 expression; and NADPH oxidase activity (in total and in mitochondrial fractions from the renal cortex). VDR activation prevented fibrosis (20 ± 5 vs. 60 ± 10%) and the number of TUNEL-positive apoptotic cells (10 ± 3 vs. 25 ± 4) in UUO. Biochemical, histological, and molecular studies suggest mitochondrial injury. Electron microscopy revealed in UUO electronically luminous material in the nucleus. Some mitochondria were increased in size and contained dilated crests and larger than normal spaces in their interiors. These changes were not present with paricalcitol treatment. Additionally, high AT(1)-receptor mRNA and NADPH activity was reverted in mitochondrial fractions from obstructed paricalcitol-treated animals (0.58 ± 0.06 vs. 0.95 ± 0.05 relative densitometry units and 9,000 ± 800 vs. 15,000 ± 1,000 relative fluorescence units·μg protein(-1)·min(-1), respectively). These changes were consistent with an improvement in VDR expression (0.75 ± 0.05 vs. 0.35 ± 0.04 relative densitometry units). These results suggest that paricalcitol confers a protective effect and reveal, as well, a possible AT(1) receptor-dependent protective effect that occurs at the mitochondrial level.     

Susceptibility of plasma lipids to peroxidation

Lipid peroxidation is an old and yet novel subject. It induces membrane disturbance and damage and its products are known to induce the generation of various cytokines and cell signaling. In the present work, the susceptibility and specificity of human plasma lipids to oxidation were studied, aiming specifically at elucidating the effects of oxidation milieu and oxidants. Cholesteryl esters (CEs) and phosphatidylcholines (PCs) were more readily oxidized in plasma than in organic solution under similar conditions. The susceptibilities of PC and free cholesterol (FC) relative to CE to free radical-mediated lipid peroxidation induced by peroxyl radicals and peroxynitrite were smaller in plasma than in organic solution. The higher rate of CE oxidation by free radicals than PC may be accounted for by the physical effects as well as higher content of polyunsaturated lipids in CE than PC. On the contrary, PC was more readily oxidized than CE by lipoxygenases. The lipid hydroperoxides were stable in organic solution but reduced to the corresponding hydroxides in plasma, the rate being much faster for PC hydroperoxides than for CE and FC hydroperoxides. It was confirmed that free radical-mediated oxidation gave both cis,trans and trans,trans, racemic, random hydroperoxides, while that by lipoxygenase gave only regio- and stereo-specific cis,trans-hydroperoxide.     

Dehydroepiandrosterone-induced lipid peroxidation in rat liver mitochondria

Administration of dehydroepiandrosterone (DHEA), a steroid hormone of the adrenal cortex which acts as a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in mitochondria isolated from the liver, kidney and heart, but not from the brain. The effect of DHEA on rat liver mitochondrial lipid peroxidation became discernible after feeding steroid-containing diet (0.6% w/w) for 3 days, and reached maximal levels between 1 and 2 weeks. DHEA in the concentration range 0.001–0.02% did not significantly increase lipid peroxidation compared to the control. Lipid peroxidation was significantly enhanced in animals given a diet containing ≥ 0.05% DHEA. The addition of DHEA in the concentration range 0.1–100 μM to mitochondria isolated from control rats had no effect on lipid peroxidation. It seems, therefore, that the steroid effect is mediated by an intracellular process. Our data indicate that induction of mitochondrial membrane lipid peroxidation is an early effect of DHEA administration at pharmacological doses.     

Calcium potentiates the peroxidation of erythrocyte membrane lipids

A clonal cell line from rat osteosarcoma was found to possess parathyroid hormone and isoproterenol sensitive adenylate cyclase. This study examines the relationship between the two hormones and triphosphoguanine nucleotide with respect to enzyme activation. Concentration-dependence curves, analyzed by computer-aided curve fitting, revealed: (1) in the presence of 5 microM GTP there were two apparent affinities for parathyroid hormone (Km 9 and 89 nM) and isoproterenol (Km 72 and 340 nM; (2) and two affinities for guanosine-5' (beta, gamma-imido)triphosphate (Km 0.25 and 1.3 microM); (3) hormones and guanine nucleotides reciprocally shifted each other's concentration dependence curve to the high affinity sites; (4) parathyroid hormone and isoproterenol interacting with high affinity sites competed for the same adenylate cyclase; (5) parathyroid hormone and isoproterenol, acting on low affinity sites had additive effects and also stimulated adenylate cyclase in the absence of added guanine nucleotides. The findings are consistent with (i) competition of parathyroid hormone and isoproterenol for the activation of the high (hormone) affinity complex containing: receptors, nucleotide subunit, triphosphoguanine nucleotide, catalytic unit (ii) the apparent presence of receptor-nucleotide sub-unit GDP-catalytic unit complexes with low hormone affinity which are stimulated by parathyroid hormone and isoproterenol separately.     

Role of the sympathetic nervous system during reparative regeneration and compensation of heart function in experimental myocardial infarct

Experiments on chemically sympathectomized rats have revealed a double effect of the sympathetic nervous system on compensation and adaptation of heart function and reparative regeneration in myocardial infarction. It has been established that if the sympathetic influences are excluded, the elements of the connective tissue are activated, which leads to myocardial infarction healing at a shorter period of time. At the same time it has been demonstrated that sympathectomy inhibits the development of the compensatory reactions and limits adaptation possibilities of the heart.