Lipid Peroxidation in the Cortex and Medulla of Rabbit Kidneys Subjected to Cold Ischaemia and the Value of Protective Agents

The storage of rabbit kidneys for 24hr at 0 C in isotonic saline resulted in significantly increased rates of lipid peroxidation, as measured by the formation of thiobarbituric acid-reactive material and Schiff bases during in vitro incubation of homogenates prepared from the cortex and medulla. In addition, the content of thiobarbituric acid-reactive material in the medulla was also significantly elevated as a result of cold storage for 24 hr.

The effects of antioxidants (vitamin E), iron-chelation (desferoxamine) and inhibitors of arachidonic acid oxidation (indomethacin and dazmegrell on the rate of lipid peroxidation in homogenates prepared from ischaemic kidneys were studied. This demonstrated that lipid peroxidation in the cortex was predominantly non-specific and iron-catalysed whereas in the medulla approximately 50% of the TBA-reactive material was formed enzymically from arachidonic acid by cyclooxygenase.     

Protection against oxidative damage in cold-stored rabbit kidneys by desferrioxamine and indomethacin

The storage of rabbit kidneys in hypertonic citrate solution at 0 degree C for 48-72 hr of cold ischemia resulted in oxidative damage to membranes as measured by the in vitro formation of two markers of lipid peroxidation (Schiff's base and thiobarbituric acid (TBA)-reactive material). This damage was further increased when the organs were autografted and reperfused for 60 min. The intravenous (iv) administration of desferrioxamine (a powerful iron-chelating agent) prior to the removal of the kidneys reduced the production of Schiff's bases and TBA-reactive material to low levels in the cortex of stored kidneys and decreased these measures of lipid peroxidation in the medulla by approximately 50%. Intravenous administration of indomethacin (a cyclooxygenase inhibitor) had no effect on the rate of lipid peroxidation in the renal cortex, but significantly reduced the formation of TBA-reactive material and Schiff's bases in the medulla of kidneys following storage for 72 hr. The existence of two separate pathways of lipid peroxidation (one iron-catalyzed and the other cyclooxygenase-catalyzed) in the medulla of stored kidneys was further confirmed when administration of desferrioxamine and indomethacin together resulted in significantly greater protection against lipid peroxidation than when these compounds were administered singly. The value of this combination of agents for protecting kidneys against the damage due to cold ischemia followed by reperfusion was further suggested by a trend toward improved long-term survival of the animals following replantation of the stored kidneys.     

Phospholipid Degradation and Cellular Edema Induced by Free Radicals in Brain Cortical Slices

Abstract: Cellular edema and increased lactate production were induced in rat brain cortical slices by xanthine oxidase and xanthine, in the presence of ferric ions. Lipid peroxidation, as measured by thiobarbituric acid-reactive malon-dialdehyde, was increased 174%. Among the various subcellular fractions of brain cortex, xanthine oxidase-stimulated lipid peroxidation was highest in myelin, mitochondria, and synaptosomes, followed by microsomes and nuclei. Antioxidants, catalase, chlorpromazine, and butylated hydroxytoluene inhibited lipid peroxidation in both homogenates and synaptosomes, indicating H₂O₂ and radicals were involved. Further, several free fatty acids, especially oleic acid (18:1), arachidonic acid (20:4), and docosahexaenoic acid (22:6) were released from the phospholipid pool concomitant with the degradation of membrane phospholipids in xanthine oxidase-treated synaptosomes. These data suggest that Upases are activated by free radicals and lipid peroxides in the pathogenesis of cellular swelling.     

Glutathione peroxidase and iron-thiol dependent lipid peroxidation

Role of glutathione peroxidase in iron-thiol-mediated lipid peroxidation was examined. The enzyme was unable to prevent peroxidation of extracted rat liver microsomal lipids. In contrast, when arachidonic acid was the substrate, glutathione peroxidase did decrease the formation of thiobarbituric acid-reactive material. Superoxide dismutase produced a consistent but partial inhibition of peroxidation and catalase was without effect. Our results suggest that iron-thiol-dependent lipid peroxidation cannot be completely blocked by protective enzymes that are effective in other systems.     

Reduced susceptibility to lipid peroxidation in cold ischemic rabbit kidneys after addition of desferrioxamine, mannitol, or uric acid to the flush solution

Rabbit kidneys were stored for 24 hr at 0 degree C after single passage arterial flush with 30 ml of cold isotonic 0.9% sodium chloride (saline) solution alone or saline to which was added 12, 30, or 60 mM desferrioxamine, 1 or 3 mM uric acid, or 100 mM mannitol. They were then subjected to in vitro biochemical assay for evidence of free radical damage immediately after storage. Results were compared to those obtained with fresh, unstored kidneys. Levels of Schiff base fluorescence, diene conjugates, and thiobarbituric acid-reactive material were each significantly elevated in kidneys stored for 24 hr after flush with saline alone. These levels were in turn each significantly reduced by the addition of 60 mM desferrioxamine, 3 mM uric acid, and 100 mM mannitol to the flush solution. Likewise, glutathione redox activity fell in those flushed with saline alone, presumably in line with increased lipid peroxidation, but was restored to control levels by inclusion of the three scavenging agents.     

Diabetic heart and kidney exhibit increased resistance to lipid peroxidation

Alloxan-diabetic rats and age-matched controls were killed after 6 weeks of diabetes; heart and kidneys were removed and assayed for thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides, lipid phosphorus, total fatty acid composition and glutathione. Tissue homogenates from a second group of diabetic and control rats were incubated in oxygen-saturated buffer with and without the free radical generating system Fe2+/ascorbate (0.1/1.0 mM) and were assayed for lipid peroxidation. Diabetic hearts contained markedly lower levels of TBARS and lipid hydroperoxides (40% and 18%, respectively) than control hearts, whereas differences in TBARS were less pronounced in kidneys (9%). Incubation of homogenates of both organs in the presence or absence of Fe2+/ascorbate for up to 2 h yielded significantly lower levels of TBARS and lipid hydroperoxides with diabetic tissue. Diabetic hearts and kidneys contained higher levels of glutathione (28% and 13% over controls) and both diabetic tissues showed much higher linoleate/arachidonate ratios than did the controls (9.86 vs. 2.56 for heart, 2.01 vs. 0.86 for kidney). We conclude that diabetic tissues develop enhanced defense systems against oxidative stress and we assume tha the lower levels of arachidonate contribute to their resistance to lipid peroxidation as well.     

Peroxidative Changes in Brain Cortical Fatty Acids and Phospholipids, as Characterized During Fe2+- and Ascorbic Acid-Stimulated Lipid Peroxidation In Vitro

Abstract: The occurrence of peroxidative damage, as distinguished from anaerobic damage, to brain fatty acids and phospholipids was characterized in vitro. Fe²⁺ and ascorbic acid were used to stimulate peroxidation in cortical homogenates from rat brain incubated with or without oxygen. Lipid peroxidation was established in samples incubated with oxygen by increased diene conjugation, accumulation of thiobarbituric acid-reactive material (TBAR) and of lipid-soluble fluorescent products. No peroxidation occurred in samples incubated in the absence of oxygen (100% N₂). Lipid peroxidation was characterized by a selective loss of arachidonic acid and docosahexaenoic acid and by degradation of ethanolamine phosphoglyceride, while choline phosphoglyceride did not change. During the course of peroxidation there were parallel increases in products of lipid peroxidation concomitant with the decrease in polyenoic fatty acids. The maximal changes in diene conjugation and TBAR occurred earlier than the maximal changes in fluorescent material and fatty acids. It is concluded that measurements of changes in brain fatty acid and phospholipid composition may be a useful tool to establishment of whether peroxidative damage is important in vivo in situations with a critically reduced oxygen supply. Estimation of lipid-soluble fluorescence in vivo may also be useful, since it is considered to reflect the accumulation of stable end products of peroxidation.     

Oxidation of ferrous iron during peroxidation of lipid substrates

Oxidation of Fe2+ in solution was dependent upon medium composition and the presence of lipid. The complete oxidation of Fe2+ in 0.9% saline was markedly accelerated in the presence of phosphate or EDTA and the ferrous oxidation product formed was readily recoverable as Fe2+ by ascorbate reduction. In contrast, in the presence of either brain synaptosomal membranes, phospholipid liposomes, fatty acid micelles or H2O2, less than 50% of the Fe2+ oxidized during an incubation could be recovered as Fe2+ via reduction with ascorbate. In the presence of unsaturated lipid, oxidation of Fe2+ was associated with peroxidation of lipid, as assessed by the uptake of O2 and formation of thiobarbituric acid-reactive products during incubations. Although relatively little Fe2+ oxidation or lipid peroxidation occurred in saline with synaptosomes or linoleic acid micelles during an incubation with Fe2+ alone, significant Fe2+ oxidation and lipid peroxidation occurred in incubations containing a 1:1 ratio of Fe2+ and Fe3+. Extensive Fe2+ oxidation and lipid peroxidation also occurred with Fe2+ alone in saline incubations with either linolenic or arachidonic acid acid micelles or liposomes prepared from dilinoleoylphosphatidylcholine. While a 1:1 ratio of Fe2+ and Fe3+ enhanced thiobarbituric acid-reactive product formation in incubations containing linolenic or arachidonic micelles, it reduced the rate of O2 consumption as compared with Fe2+ alone. The results demonstrate that oxidation of Fe2+ in incubations containing lipid substrates is linked to and accelerated by peroxidation of those substrates. Furthermore, the results suggest that oxidation of Fe2+ in the presence of lipid or H2O2 creates forms of iron which differ from those formed during simple Fe2+ autoxidation.     

Criteria for determination of lipid peroxidation in tissues: estimation in liver of mice intoxicated with carbon tetrachloride

The profiles of lipid peroxidation products in liver homogenates or microsomal membranes prepared from CCl4-intoxicated mice were determined by several commonly employed methods. The level of conjugated dienes peaked within 30 min and then decreased, suggesting the transitory nature of lipid peroxides in vivo. Values for thiobarbituric acid positive material peaked 30 min after CCl4 treatment, diminished thereafter for a time, and gradually rose to a new maximum at 24 h; the first peak appears to represent lipid peroxides and the second represents further degradation products including malondialdehyde. Fluorescence intensity (excitation, 360 nm; emission, 430 nm) was closely correlated with the second peak. Our findings support the involvement of lipid peroxidation in CCl4-induced hepatotoxicity in mice and emphasize the necessity for several analytical indices of lipid peroxidation performed at different time intervals.     

Regional lipid peroxidation in rat brain in vitro: Possible role of endogenous iron

Lipoperoxidative capacity of various brain areas of aging rats was examined in vitro using the thiobarbituric acid test. Significant regional differences in the generation of lipid peroxides were found in freshly prepared homogenates from different areas of brain incubated under air. Incubation under oxygen resulted in marked stimulation of lipid peroxidation, with highest increases in hypothalamus (144%). Addition of exogenous Fe²⁺ and ascorbic acid resulted in stimulation of lipid peroxidation ranging from 10-fold in cortex to 20-fold in hypothalamus homogenates during incubation in air. A linear relationship was found between endogenous iron content in brain regions and their ability to produce lipid peroxides in vitro under oxygen for all areas except striatum. Several iron chelating agents effectively inhibited lipid peroxidation under hyperbaric oxygen whereas oxygenfree radical scavengers, as well as catalase and superoxide dismutase were not effective. It is concluded that regional differences in lipoperoxidative capacity of brain areas in vitro are in part governed by local endogenous iron content and may indicate regional susceptibility to oxidative damage.     

Autopsy Samples of Alzheimer''s Cortex Show Increased Peroxidation In Vitro

The formation of thiobarbituric acid-reactive products was measured as an index of peroxidation by oxygen free radicals in homogenates of frontal cortex and cerebellum from brains taken at autopsy and verified histologically as being Alzheimer's (n = 6) or normal (n = 6). Compared with controls, basal peroxidation is significantly higher in Alzheimer's cortex, and this difference is also evident in the presence of exogenous iron. Peroxidation in cerebellum and levels of total glutathione, RNA, and DNA in cortex and cerebellum do not differ significantly between Alzheimer's brain and controls. Iron-induced peroxidation in cortex is reduced by the lazaroid U-74500A, with calculated IC50 values that are significantly higher in Alzheimer's samples (10 microM) than in controls (2.5 microM). These observations suggest that cerebral cortex from Alzheimer's patients differs from controls with respect to in vitro peroxidation.     

Importance of two iron-reducing systems in lipid peroxidation of rat brain: implications for oxygen toxicity in the central nervous system

The mechanism of lipid peroxidation in the central nervous system has been studied using oxygen-flushed rat brain homogenates at pH 7.4. Brain lipid peroxidation was monitored by chemiluminescence and by determination of thiobarbituric acid-reactive substances. Less involvement of O2-., H2O2 and probably .OH in the initiation of lipid peroxidation was indicated. Deferroxamine was an extremely potent inhibitor of lipid peroxidation, suggesting that lipid peroxidation was catalyzed by endogenous iron. Brain tissues were shown to contain at least two iron-reducing systems for promotion of lipid peroxidation. One was ascorbate-dependent and the other NADPH-dependent. The former was much more potent than the latter with respect to iron-reducing activity.     

Inactivation of transforming activity of plasmid DNA by lipid peroxidation

DNA damage due to NADPH-dependent lipid peroxidation of liposomes was examined using liposomes prepared from lipids, NADPH-cytochrome P-450 reductase and cytochrome P-450 isolated from rat liver microsomes. Plasmid pBR322 DNA was incubated in the reaction mixture for liposomal lipid peroxidation and introduced to Escherichia coli CSR603 (uvrArecA). More of the transforming activity of the DNA was lost as the lipid peroxidation progressed, and this inactivation was dependent on the extent of lipid peroxidation. Single strand breaks occurred in the plasmid DNA. Hydroxyl radical scavengers could not prevent most of the strand breaks or the lipid peroxidation reaction. Chloroform extracts from the reaction mixture of peroxidized microsomes also inactivated the transforming activity of pBR322 DNA but did not cause strand breaks. The 105 000 X g supernatant of the reaction mixture, which contained more than 85% of the thiobarbituric acid-reactive substances, did not inactivate the plasmid DNA. The degradative products of [U-14C]arachidonic acid in the liposomes did not bind to DNA. These results led to the conclusion that at least two types of DNA damaging agent are produced during NADPH-dependent microsomal lipid peroxidation. One induces single strand breaks of DNA and another inactivates the plasmid-transforming activity without inducing strand breaks.     

Nephrotoxicity and its prevention by vitamin E in ferric nitrilotriacetate-promoted lipid peroxidation

Iron and aluminum complexes of nitrilotriacetic acid cause severe nephrotoxicity in Wistar rats. In addition, a high incidence of renal cell carcinoma is seen in ferric nitrilotriacetate-treated animals. The present study was performed to see if lipid peroxidation is involved in ferric nitrilotriacetate toxicity. Ferric nitrilotriacetate had more bleomycin-detectable 'free' iron than any ferric salt, while iron complexed with desferrioxamine or ferric chondroitin sulfate had none. The toxicity of ferric nitrilotriacetate in vivo was more pronounced in vitamin E-deficient rats. A thiobarbituric acid-reactive substance was present in the kidneys of vitamin E-deficient rats in amounts markedly elevated compared to vitamin E-sufficient, or vitamin E-supplemented rats. Non-complexed nitrilotriacetate or aluminum nitrilotriacetate did not produce any thiobarbituric acid-reactive substance in vitamin E-sufficient rats died by the 58th day of administration. We suggest that the iron-stimulated production of free radicals leading to lipid peroxidation is the major cause of ferric nitrilotriacetate-mediated renal toxicity. Vitamin E, a known scavenger of free radicals, is effective in protecting against this iron-induced toxicity.     

Effect of ferritin-containing fractions with different iron loading on lipid peroxidation.

Ferritin-containing fractions with different degrees of iron loading were prepared. All ferritin fractions stimulated the peroxidation of bovine brain phospholipid liposomes, as measured by the formation of thiobarbituric acid-reactive material. This stimulation was increased in the presence of ascorbate. Iron salts of equivalent concentration to those of the ferritin fractions were more stimulatory to lipid peroxidation at the higher iron concentrations. None of the fractions inhibited ascorbate-dependent peroxidation in the presence of added iron salts.     

Protective effect of fosfomycin on gentamicin-induced lipid peroxidation of rat renal tissue

Fosfomycin is clinically recognized to reduce the aminoglycoside antibiotics-induced nephrotoxicity. However, little has been clarified why fosfomycin protects the kidney from the aminoglycosides-induced nephrotoxicity. Gentamicin, a typical aminoglycoside, is reported to cause lipid peroxidation. We focused on lipid peroxidation induced by gentamicin as a mechanism for the aminoglycosides-induced nephrotoxicity. The aim of this study is to investigate the effect of fosfomycin on the gentamicin-induced lipid peroxidation. In rat renal cortex mitochondria, fosfomycin was shown to depress the gentamicin-induced lipid peroxidation, which was evaluated by formation of thiobarbituric acid reactive substances (TBARS). Interestingly, this effect was observed in rat renal cortex mitochondria, but not in rat liver microsomes. However, fosfomycin did not affect lipid peroxidation of arachidonic acid caused by gentamicin with iron. Fosfomycin inhibited the gentamicin-induced iron release from rat renal cortex mitochondria. These results indicated that fosfomycin inhibited the gentamicin-induced lipid peroxidation by depressing the iron release from mitochondria. This may possibly be one mechanism for the protection of fosfomycin against the gentamicin-induced nephrotoxicity.     

Free fatty acids,lipid peroxidation,and lysosomal enzymes in experimental focal cerebral ischemia in primates: Loss of lysosomal latency by lipid peroxidation

Experimental focal cerebral ischemia was produced in monkeys (Macaca radiata) by occlusion of the right middle cerebral artery (MCA). The release of the lysosomal glycosidases, -d-hexosaminidase, -l-fucosidase and -d-mannosidase into the soluble fraction in the right basal ganglia of the experimental animals was measured at different periods from 30 min to 12 hr after occlusion and compared with the corresponding sham operated control animals. There was a significant increase in the released lysosomal enzymes in the MCA occluded animals at all periods and particularly at 4 hr after occlusion. The CSF from the experimental animals also showed elevated levels of hexosaminidase and fucosidase. The free fatty acids (FFA) measured in the basal ganglia at 30 min and 2 hr after occlusion showed a 100 fold increase in the experimental animals. The predominant fatty acid released was linoleic acid (18:2) followed by arachidonic acid (20:4). Lipid peroxidation in the basal ganglia measured by the thiobarbituric acid (TBA) reaction in the presence or absence of ascorbic acid also showed a significant increase in the experimental animals at all periods with a maximum at 30 min to 2 hr after occlusion. In order to assess whether lipid peroxidation causes damage to the lysosomes and release of the enzymes, a lysosome enriched P₂ fraction from the normal monkey basal ganglia was prepared and the effect of peroxidation studied. Maximum peroxidation in the P₂ fraction was observed in the presence of arachidonic acid, ascorbic acid and Fe²⁺. There was a good correlation between the extent of lipid peroxidation and the in vitro release of lysosomal hexosaminidase from the P₂ fraction. Anti-oxidants which strongly inhibited lipid peroxidation in the P₂ fraction prevented the release of hexosaminidase. The results suggested that in ischemia produced by MCA occlusion lipid peroxidation which damages the lysosomal membrane causes the release of lysosomal hydrolytic enzymes.Abbreviations used BHA butylated hydroxyanisole - BHT butylated hydroxytoluene - FFA free fatty acids - MCA middle cerebral artery - MDA malonaldehyde - PUFA polyunsaturated fatty acids - TBA thiobarbituric acid     

Oxidative stress induction by cis-4-decenoic acid: Relevance for MCAD deficiency

Patients affected by medium-chain acyl-CoA dehydrogenase deficiency (MCADD) suffer from acute episodes of encephalopathy whose underlying mechanisms are poorly known. The present work investigated the in vitro effect of cis-4-decenoic acid (cDA), which accumulates in MCADD, on important parameters of oxidative stress in cerebral cortex of young rats. cDA markedly induced lipid peroxidation, as verified by the increased levels of spontaneous chemiluminescence and thiobarbituric acid-reactive substances. Furthermore, cDA significantly increased carbonyl formation and sulphydryl oxidation, which is indicative of protein oxidative damage, and promoted 2′,7′-dihydrodichlorofluorescein oxidation. It was also observed that the non-enzymatic tissue antioxidant defenses were decreased by cDA, whereas the antioxidant enzyme activities catalase, superoxide dismutase and glutathione peroxidase were not altered. Moreover, cDA-induced lipid peroxidation and GSH reduction was totally blocked by free radical scavengers, suggesting that reactive species were involved in these effects. The data indicate that oxidative stress is induced by cDA in rat brain in vitro and that oxidative damage might be involved in the pathophysiology of the encephalopathy in MCADD.     

Distribution of prostaglandins in rabbit kidney

Three prostaglandins (PGE(2), PGF(2alpha) and PGA(2)) are present in rabbit kidney medulla. An acidic lipid extract (0.165g) obtained from 2kg of frozen rabbit kidney cortex was separated by silicic acid chromatography to yield eluates containing fatty acids, possible non-polar prostaglandin metabolites, PGA, PGE and PGF compounds. Ultraviolet spectra of the eluates before and after treatment with sodium hydroxide did not yield chromophores typical of any known prostaglandins or related metabolites. By using more sensitive bioassay procedures (contraction of rabbit duodenum) weak activity equivalent to 60mug of PGE(2) and 10mug of PGF(2alpha) was detected in the PGE and PGF eluates respectively. Extraction and bioassay of fresh kidney cortex revealed no prostaglandin-like activity. Attempts to biosynthesize prostaglandins in fresh homogenates of rabbit kidney cortex from endogenous precursors and from added arachidonic acid were unsuccessful. When freshly prepared homogenates of rabbit kidney cortex were incubated with added PGE(1) no evidence of enzymic breakdown was obtained. It is concluded that rabbit kidney prostaglandins are present predominantly in the medulla and there are no cortical mechanisms for their biosynthesis or inactivation under normal conditions.     

Effects of deoxycholic acid and its epimers on lipid peroxidation in isolated rat hepatocytes

We studied the effects of deoxycholic acid and its three epimers with beta-hydroxyl groups (3alpha,12beta-, 3beta,12alpha-, and 3beta,12beta-dihydroxy-5beta-cholan-24-oic acids), which were hydrophilic and less cytotoxic, on lipid peroxidation to elucidate the relationship between structural features of bile acids and their effect on lipid peroxidation. Taurodeoxycholate markedly increased the production of thiobarbituric acid-reactive substances, end products of lipid peroxidation, in isolated rat hepatocytes, whereas epimers of taurodeoxycholate did not. Deoxycholic acid inhibited mitochondrial NADH dehydrogenase and NADH:ferricytochrome c oxidoreductase activities, leading to free radical generation, whereas epimers of deoxycholic acid had no effect on mitochondrial enzymes. These findings suggested that hydrophobic bile acids cause lipid peroxidation by impairment of mitochondrial function, leading to the generation of free radicals; and epimerization of alpha-hydroxyl groups in the steroid nucleus to beta-hydroxyl groups results in a decrease of the toxic effects of deoxycholic acid on lipid peroxidation.