Age-related Oxidative Modifications of Proteins and Lipids in Rat Brain

Oxidants have been shown to play a major role in ageing and ageing-related neurodegenerative diseases. In the present study, we investigated the effect of ageing on oxidative damage to lipids and proteins in brain homogenate, mitochondria and synaptosomes of adult (6-month-old), old (15-month-old), and senescent (26-month-old) Wistar rats. There was a significant increase in thiobarbituric acid-reactive substances and conjugated dienes in homogenates, which indicate increased lipid peroxidation (LPO). Oxidative modifications of homogenate proteins were demonstrated by a loss of sulfhydryl content, accumulation of dityrosines and formation of protein conjugates with LPO-end products. Increase in protein conjugates with LPO-end products and a decrease in SH groups were observed also in mitochondria and synaptosomes, but dityrosine content was elevated only in synaptosomes. Protein surface hydrophobicity, measured by fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS), was increased only in homogenate. These results suggest that besides mitochondria and synaptosomes other cellular compartments are oxidatively modified during brain ageing.     

Lipid peroxidation during ischemia depends on ischemia time in warm ischemia and reperfusion of rat liver

Prolonged hepatic warm ischemia has been incriminated in oxidative stress after reperfusion. However, the magnitude of oxidative stress during ischemia has been controversial. The aims of the present study were to elucidate whether lipid peroxidation progressed during ischemia and to clarify whether oxidative stress during ischemia aggravated the oxidative damage after reperfusion. Rats were subjected to 30 to 120 min of 70% warm ischemia alone or followed by reperfusion for 60 min. Lipid peroxidation (LPO) was evaluated by amounts of phosphatidylcholine hydroperoxide (PC-OOH) and phosphatidylethanolamine hydroperoxide (PE-OOH) as primary LPO products. Total amounts of malondialdehyde and 4-hydroxy-2-nonenal (MDA + 4-HNE), degraded from hydroperoxides, were also determined. PC-OOH and PE-OOH significantly increased at 60 and 120 min ischemia with concomitant increase of oxidized glutathione. These hydroperoxides did not increase at 60 min reperfusion after 60 min ischemia, whereas they did increase at 60 min reperfusion after 120 min ischemia with deactivation of phospholipid hydroperoxide glutathione peroxidase and superoxide dismutase. The amount of MDA + 4-HNE exhibited similar changes, but the velocity of production dropped with ischemic time longer than 60 min. In conclusion, oxidative stress progressed during ischemia and triggered the oxidative injury after reperfusion. Secondary LPO products are less sensitive, especially during ischemia, which may cause possible underestimation and discrepancy.     

Oxygen-dependent reperfusion injury in the isolated rat lung.

To further define the relationship between oxygen dependence of lung injury during ischemia and ischemia-reperfusion, we used the isolated, perfused, and ventilated rat lung model, so that oxygenation and perfusion could be separated. During ischemia, lungs were ventilated with various oxygen concentrations and then ventilated with 95% oxygen during the 60-min reperfusion period. Other lungs were ventilated with 0% oxygen (nitrogen) during ischemia, and the reperfusion phase oxygen concentration was varied. Tissue and perfusate lipid peroxidation products (thiobarbituric acid-reactive substances and conjugated dienes), dry-to-wet weight ratio, and lactate dehydrogenase were measured as indexes of lung damage. In addition, electron microscopy of some lungs was performed. Results demonstrate an oxygen dependence of lipid peroxidation in both the ischemic and reperfusion phases, but lipid peroxidation is severalfold greater in the reperfusion than in the ischemic phase. Products of lipid peroxidation closely correlate with indexes of lung injury (dry-to-wet weight ratio, lactate dehydrogenase, and electron microscopy).     

Fluorescence imaging of lipid peroxidation in isolated rat lungs during nonhypoxic lung ischemia

We have shown previously that ischemia results in reactive oxygen species production by lung endothelium that occurs within 3-5 s after flow cessation and is followed by lipid peroxidation at 15-30 min as determined by assay of thiobarbituric acid-reactive substances, conjugated dienes, and protein carbonyls in lung homogenate. The present study evaluated membrane lipid peroxidation in isolated, ventilated rat lungs using a fluorescence imaging method that permits continuous observation of pulmonary subpleural microvascular endothelial cells in situ. Diphenyl-1-pyrenylphosphine (DPPP), a fluorescent probe which localizes in the plasma membrane and shows increased fluorescence emission after its oxidation by lipid hydroperoxides, was used for detection of membrane lipid peroxidation. Compared to continuously perfused control lungs, endothelial cell DPPP fluorescence increased significantly within 1 min of ischemia (i.e., flow cessation); these changes were prevented by pretreatment with 0.5 mM alpha-tocopherol succinate (vitamin E) added to the perfusate. Increased DPPP fluorescence was confirmed by spectrofluorometry of lipid extracts of lung homogenates. These data indicate that DPPP can be used for the real-time detection of lipid peroxidation in an intact organ. Ischemia results in peroxidation of the pulmonary microvascular endothelial cell membrane and this insult can be detected as early as 1 min after the onset of ischemia compatible with a radical-mediated process.     

Oxidative damage following cerebral ischemia depends on reperfusion - a biochemical study in rat

The extent of brain injury during reperfusion appears to depend on the experimental pattern of ischemia/reperfusion. The goals of this study were: first, to identify the rate of free radicals generation and the antioxidant activity during ischemia and reperfusion by means of biochemical measurement of lipid peroxidation (LPO) and both enzymatic (superoxid dismutase - SOD, catalase - CAT, glutathion peroxidase - GPx) and non-enzymatic antioxidants activity (glutathione - GSH); and second, to try to find out how the pattern of reperfusion may influence the balance between free radical production and clearance. Wistar male rats were subject of four-vessel occlusion model (Pulsinelly & Brierley) cerebral blood flow being controlled by means of two atraumatic arterial microclamps placed on carotid arteries. The level of free radicals and the antioxidant activity were measured in ischemic rat brain tissue homogenate using spectrophotometrical techniques. All groups subjected to ischemia shown an increase of LPO and a reduction of the activity of enzymatic antioxidative systems (CAT, GPx, SOD) and non-enzymatic systems (GSH). For both groups subjected to ischemia and reperfusion, results shown an important increase of LPO but less significant than the levels found in the group with ischemia only. Statistically relevant differences (p<0.01) between continuous reperfusion and fragmented reperfusion were observed concerning the LPO, CAT, SOD and GSH levels, oxidative aggresion during fragmented reperfusion being more important.     

Role of tumor necrosis factor alpha and sphingomyelin cycle activation in the induction of apoptosis by ischemia/reperfusion of the liver

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF- secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF- detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF- is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.     

Potentiation of lipid peroxides by ischemia in rat brain

Post-ischemic changes in energy metabolites and natural antioxidant compounds have been measured in rat brain in vitro concurrent with two different assays for peroxidized lipids. No exogenous free radical initiators were employed. In vitro oxygenation of minced brain preparations for periods of 10 minutes to 4 hours, following 5 minutes of preparatory ischemia, yielded increased levels of lipid conjugated dienes and TBA-reactive material, in contrast to anaerobically incubated preparations. However, either aerobic or anaerobic incubation of brain minces facilitated increased ratios of lactate: pyruvate and glutathione (oxidized): glutathione (reduced), as well as increased total ubiquinone content and loss of -tocopherol. Observation of lipid radical formation in vivo was then attempted using rats given embolic stroke in one hemisphere and left in the post-ischemic condition for times up to 24 hours. Conjugated dienes were found in lipids extracted from the ipsilateral hemisphere but not from the contralateral hemisphere. These observations of conjugated dienes in vivo (formed presumably during post-ischemic reperfusion) and in vitro (facilitated by oxygenation of brain minces), indicate that lipid radical intermediates and associated chain peroxidation processes are potentiated by ischemia and occur during tissue reoxygenation.     

The characteristics of the radiation-initiated peroxidation of the phosphatidylcholine making up liposomes containing phospholipids which are susceptible to free-radical fragmentation

The regularities of accumulation of conjugated dienes and thiobarbituric acid (TBA)-reactive substances under gamma-irradiation of liposomes from rat liver phosphatidylcholine (PC) and its mixtures with the resistant to lipid peroxidation saturated phospholipids and bovine brain sphingomyelin (SM) were studied. It was established that the incorporation of negatively charged dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylethanol (DPPET) into lipid bilayer resulted in the increase of primary and secondary products of LPO, whereas neutral dipalmitoylphosphatidylcholine (DPPC) and SM involving in the phospholipid mixtures inhibited the peroxidation of PC. For anionic phospholipids, DPPG had more profound activating action on LPO, amongst the neutral phospholipids SM was more potent inhibitor of the reaction. Unlike DPPET and DPPC, DPPG and SM were subjected to free radical fragmentation on gamma-radiation. It is suggested that the intermediates and products of free radical fragmentation may modulate the progress of LPO.     

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.     

Time course of lipid peroxidation during incomplete ischaemia followed by reperfusion in rat brain

The time course of lipid peroxidation was studied in the rat brain cortex after ischaemia and reperfusion. The ischaemia was induced by 4-hour occlusion of both common carotid arteries and was followed by reperfusion of different duration (10, 30 or 60 min). The extent of lipid peroxidation was determined by measurement of conjugated dienes (CD) and TBA reactive products. Maximal values of CD and TBA reactive products were found after 10- and 30-minute reperfusion. This indicated the most suitable time interval for studying the effect of antioxidants and oxygen radical scavengers in this model of brain ischaemia.     

Reaction of xanthine oxidase-derived oxidants with lipid and protein of human plasma

Xanthine oxidase and purines have recently been detected in the circulation during acute viral infection and following hepatotoxicity and shock. Reactions of xanthine oxidase-generated oxidants with human plasma or bovine serum albumin (BSA) and egg phosphatidylcholine (PC) liposomes have been studied by measuring protein sulfhydryl oxidation and two markers of free radical-mediated lipid peroxidation, thiobarbituric acid reactive substances (TBARS) and conjugated dienes. Plasma incubated with 5 mU/ml xanthine oxidase (XO) and 0.5 mM hypoxanthine (Hx) for 2 h at 37 degrees C had 25-53% oxidation of sulfhydryl groups, with greater than 80% of the oxidation occurring during the first 20 min of the reaction. Concentrations of BSA similar to those present in serum, when exposed to XO/Hx-mediated oxidative stress, showed an even greater decrease in sulfhydryl concentration than that of plasma. No significant increase in plasma TBARS and conjugated dienes was observed during the 2-h incubation period in the presence of XO. Egg PC liposomes, suspended to a plasma phospholipid-equivalent concentration, showed a minor increase in TBARS and conjugated dienes under similar XO/Hx incubation conditions. In the presence of 0.23 mM BSA, lipid peroxidation was completely inhibited. A similar inhibition of lipid peroxidation was induced by cysteine but not by uric acid. Electrophoretic and arsenite-mediated sulfur reduction analysis revealed that BSA was oxidized beyond the disulfide form, with sulfenic acid formed during the initial period of oxidation. Protein sulfhydryls served as sacrificial antioxidants, preventing plasma lipid peroxidation, as well as being targets for oxidative damage. Plasma protein thiol oxidation was determined to be a more sensitive and specific indication of oxidant stress to the vascular compartment than assessment of lipid oxidation byproducts.     

Cocaine hepatotoxicity during protein undernutrition of retrovirally infected mice.

Effects of cocaine administration on lipid peroxidation and liver damage in immunocompromised mice fed different levels of dietary proteins were investigated. Indices of lipid peroxidation and serum aminotransferases as evidence of free radical attack and liver damage were compared in mice fed a low protein (4%) or regular protein diet (20% protein) for 3 weeks and then infected with murine leukemia virus and given daily intraperitoneal injections of increasing progressive doses of 5-45 mg.kg-1.day-1 of cocaine for 11 weeks. Cocaine administration significantly increased hepatic triglycerides, serum aminotransaminases, conjugated dienes, lipid fluorescence, and malondialdehyde levels. These changes were exacerbated by retroviral infection and also by protein undernutrition. Retroviral infection additively increased indices of cocaine-induced lipid peroxidation and hepatic damage. Significant increases in indices of lipid peroxidation and greater liver injury were also detected in similarly treated mice that received the low protein diet compared with well-nourished mice. These results show that immunocompromised mice fed low levels of dietary protein form significantly increased immunogenic lipid peroxidation adducts during cocaine treatment.     

Activation of lipid peroxidation and changes in vitamin E contents in the lungs under oxidative stress]

The lipid peroxidation (LPO) of the lung tissue and the bronchoalveolar lavage in rats under the influence of immobilization has been investigated. The effects accompanying the development of oxidative stress in animals--an increase in the content of conjugated dienes and fluorescent LPO products in biological objects and a strong decrease in the content of vitamin E in the lung tissue were registered.     

Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium

Oxygen free radical injury has been postulated to occur during myocardial ischemia. We have used Electron Spin Resonance and Spin Trapping techniques to directly demonstrate the production of carbon-centered (R.) and oxygen-centered lipid radical (RO.) in ischemic canine heart. In addition, venous effluent from the ischemic region showed that conjugated dienes (lipid peroxidation products) increased with ischemic duration. Our results suggest that the formation of the oxygen-centered and carbon-centered lipid radical species during ischemia are a consequence of oxy-radical peroxidation of myocardial membrane lipids.     

Protective effect of a new anti-oxidant on the rat brain exposed to ischemia-reperfusion injury: inhibition of free radical formation and lipid peroxidation.

A new oligomeric derivative was synthesized from prostaglandin B2 and ascorbic acid, and its effect on rat brain ischemia-reperfusion injury was studied. Brain ischemia was produced in the rat by the combination of bilateral common carotid artery occlusion and hemorrhagic hypotension (30 mmHg, 20 min). The cerebral cortex was homogenized in the presence of the spin trap agent, N-tert-butyl-alpha-phenyl-nitrone (PBN). Spin-adducts were detected using an electron spin resonance spectrometer (EPR). Lipid peroxidation was estimated from the amounts of both thiobarbituric acid reactive substances (TBAR) and conjugated diene. In control experiments, reperfusion induced a burst of free radical formation which peaked at 5 min reperfusion time (238 +/- 41%). Lipid peroxidation increased significantly after 20 min of reperfusion (TBAR, 161 +/- 50%; conjugated diene, 160 +/- 29%). When the oligomeric derivative was administered (9 mg/kg i.p. 30 min before ischemic insult), it significantly reduced both spin adduct formation (103 +/- 13%) and lipid peroxidation (TBAR, 109 +/- 14%; conjugated diene, 97 +/- 33%).     

Role of oxygen in oxidation of lipid and protein during ischemia/reperfusion in isolated perfused rat lung.

Considerable evidence has accumulated that oxygen free radicals play a major role in ischemic injury, particularly when followed by reperfusion. Few reports have demonstrated the occurrence of oxidative damage during the ischemic period, itself. Our laboratory has demonstrated that events occurring during an ischemic period with adequate oxygen supply can mimic the "oxygen paradox," using lipid peroxidation as an index of oxidative stress and lung edema as an index of tissue injury. The present study compares lipid peroxidation and oxidation of soluble (100,000g supernatant) protein during ischemia and reperfusion in isolated rat lung model perfused with artificial medium and ventilated with varying alveolar oxygen tension. Protein oxidation was determined by a modified dinitrophenylhydrazine (DNPH) method using Sephadex G-25 column chromatography to isolate the DNPH bound proteins. Global ischemia was produced by discontinuing perfusion while ventilation continued with gas mixtures containing 5% CO2 and a fixed oxygen concentration between 0 and 95%. After 1 h ischemia in the isolated rat lung ventilated with 20% oxygen, protein carbonyls and thiobarbituric acid reactive substances (TBARS) increased significantly compared with controls. These changes were more pronounced after 60 min of reperfusion with 95% oxygen in the ventilation gas. With 0% oxygen (95% nitrogen and 5% CO2) content of the ventilating gas during ischemia, TBARS and protein carbonyls remained at the control level. The wet/dry weight ratio showed changes parallel to the indices of tissue oxidation. The presence of 5,8,11,14-eicosatetraynoic, an inhibitor of cyclooxygenase and lipoxygenase pathways, in the perfusate had no effect on the generation of protein carbonyls although inhibition of lipid peroxidation was demonstrated. This implies that the oxidation of soluble protein is not mediated by the eicosanoid metabolic cascade. These data indicate that oxidative processes occur during ischemia and are dependent on the alveolar oxygen concentration. Oxidation of soluble protein can be used as an index of oxidative damage during lung ischemia and reperfusion.     

Comparative evaluation of manganese peroxidase- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids by different methods

The peroxidation of C18 unsaturated fatty acids by fungal manganese peroxidase (MnP)/Mn(II) and by chelated Mn(III) was studied with application of three different methods: by monitoring oxygen consumption, by measuring conjugated dienes and by thiobarbituric acid-reactive substances (TBARS) formation. All tested polyunsaturated fatty acids (PUFAs) were oxidized by MnP in the presence of Mn(II) ions but the rate of their oxidation was not directly related to degree of their unsaturation. As it has been shown by monitoring oxygen consumption and conjugated dienes formation the linoleic acid was the most easily oxidizable fatty acid for MnP/Mn(II) and chelated Mn(III). However, when the lipid peroxidation (LPO) activity was monitored by TBARS formation the linolenic acid gave the highest results. High accumulation of TBARS was also recorded during peroxidation of linoleic acid initiated by MnP/Mn(II). Action of Mn(III)-tartrate on the PUFAs mimics action of MnP in the presence of Mn(II) indicating that Mn(III) ions are involved in LPO initiation. Although in our experiments Mn(III) tartrate gave faster than MnP/Mn(II) initial oxidation of the unsaturated fatty acids with consumption of O₂ and formation of conjugated dienes the process was not productive and did not support further development of LPO. The higher effectiveness of MnP/Mn(II)-initiated LPO system depends on the turnover of manganese provided by MnP. It is proposed that the oxygen consumption assay is the best express method for evaluation of MnP- and Mn(III)-initiated peroxidation of C18 unsaturated fatty acids.     

The study of cytokine content and ganglioside metabolism in experimental brain edema

A blood cytokine profile and also the brain content of lipid peroxidation (LPO) products and gangliosides were investigated in rats with experimental brain edema. The development of brain edema was accompanied by the increase in pro-inflammatory and a decrease in anti-inflammatory cytokine content. In parallel, accumulation of LPO products (conjugated dienes, hydroperoxides, and malondialdehyde) was observed. The study of ganglioside content under conditions of experimental brain edema revealed a decrease of their hydrolytic degradation product, sphingosine.     

Lipid peroxidation as molecular mechanism of liver cell injury during reperfusion after ischemia

The pathophysiological importance of reactive oxygen species has been extensively documented in the pathogenesis of hepatic ischema-reperfusion injury. Kupffer cells and neutrophils were identified as the dominant sources of the postischemic oxidant stress. To test the hypothesis that a direct free radical-mediated injury mechanism (lipid peroxidation; LPO) may be involved in the pathogenesis, highly sensitive and specific parameters of LPO, i.e., hydroxy-eicosatetraenoic acids (HETES), and F₂-isoprostanes, were determined by gas chromatographic-mass spectrometric analysis in liver tissue and plasma during 45 min of hepatic ischemia and up to 24 h of reperfusion. A significant 60–250% increase of F₂-isoprostane levels in plasma was found at all times during reperfusion; the HETE content increased only significantly at 1 h of reperfusion and in severely necrotic liver tissue at 24 h with increases between 90–320%. On the other hand, in a model of LPO-induced liver injury (infusion of 0.8 μmol tert-butylhydroperoxide/min/g liver), the hepatic HETE content increased two to fourfold over baseline values at 45 min, i.e., before liver injury. A further increase to 12- to 30-fold of baseline was observed during moderate liver injury. Based on these quantitative comparisons of LPO and liver injury, it seems highly unlikely that LPO is the primary mechanism of parenchymal cell injury during reperfusion, although it cannot be excluded that LPO may be important as a damaging mechanism in a limited compartment of the liver, e.g., endothelial cells, close to the sources of reactive oxygen, e.g., Kupffer cells and neutrophils.     

Influence of different oxygen modes on the blood oxygen transport and prooxidant-antioxidant status during hepatic ischemia/reperfusion

Oxygen supply was corrected in rabbits during the hepatic ischemia/reperfusion by means of different breathing mixtures: hypoxic (14.8 % O(2)+85.2 % N(2)), hyperoxic (78 % O(2)+20.2 % N(2)+ 1.8 % CO(2)), or hypercapnic (5 % CO(2) in air). Hepatic ischemia was induced for 30 min by ligation of hepatic artery, reperfusion period lasted 120 min. Indices of blood oxygen transport (p50(act), pCO(2), pH, pO(2), etc.) and prooxidant-antioxidant balance (Schiff bases, conjugated dienes, catalase, retinol, alpha-tocopherol) were measured in the blood and liver. The severity of reperfusion damage was evaluated by the activities of alanine and aspartate aminotransferases (ALT, AST) in the blood. Hepatic ischemia/reperfusion resulted in higher p50(act) in hepatic venous and mixed venous blood in all experimental groups. The changes of p50(act) were most marked in the hypercapnic group and were the weakest in the hypoxic group. The rise in p50(act) was accompanied by higher levels of lipid peroxidation products, ALT and AST in blood and liver homogenates, and by a simultaneous fall of alpha-tocopherol and retinol concentrations, except in the hypoxic group. Catalase activity at the end of reperfusion increased under normoxia, decreased under hyperoxia or hypercapnia and did not change under hypoxia. The moderate hypoxia during reperfusion was accompanied by a better balance between the mechanisms of reactive oxygen species production and inactivation that may be observed by optimal changes in p50act and reduced the hepatic damage in this pathological condition.