Antioxidant activity of the pyridoindole stobadine in liposomal and microsomal lipid peroxidation.

Stobadine, a pyridoindole derivative, is an efficient inhibitor of lipid peroxidation in phosphatidylcholine liposomes and in rat liver microsomes treated with iron/ADP/NADPH as pro-oxidant. Accumulation of thiobarbituric acid-reactive substances (TBARS) or low-level chemiluminescence were taken as a measure of lipid peroxidation and 5 microM stobadine doubled the duration of the lag phase preceding the onset of rapidly increasing chemiluminescence. Inhibition of lipid peroxidation was not observed with tocopherol-deficient microsomes, suggesting that the antioxidant effect of stobadine depends on vitamin E in the membrane. The cis(-) isomer was most effective, with the cis(+) and trans(rac) as well as dehydro- or acetyl derivatives being less active. In liposomes, the presence of reductant (NADPH or ascorbate) protects from the loss of stobadine.     

Phospholipid Hydroperoxides and Lipid Peroxidation in Liver and Plasma of ODS Rats Supplemented with α-Tocopherol and Ascorbic Acid

Forty-five mutant male ODs rats, unable to synthesize ascorbic acid, were fed nine diets containing 5, 50 or 250 mg of vitamin E/kg diet and 150,300 or 900 mg of vitamin C/kg diet for 21 days. The concentrations of vitamins C and E increased in liver and plasma in relation to the level of these vitamins in the diet. Vitamin C dietary supplementation increased the plasma vitamin E content at low levels of vitamin E intake, supporting the concept of an in vivo synergism between both antioxidant vitamins. Vitamin C, at the dietary levels studied, did not affect the lipid peroxidation. Vitamin E decreased liver and plasma endogenous levels of thiobarbituric acid-reactive substances and liver sensitivity to non-enzymatic lipid peroxidation. This was confirmed by a highly specific assay of lipid hydroperoxides using high performance liquid chromatography with chemiluminescence detection. The hepatic concentration of both phosphatidylcholine and phosphatidylethanolamine hydroperoxides decreased as the vitamin E content of the diet increased. The results show for the first time the capacity of vitamin E to protect against peroxidation of major phospho-lipids in vivo under basal unstressed conditions.     

UVA-induced lipid peroxidation in cultured human fibroblasts

The UVA irradiation of cultured human fibroblasts leads to the formation and to the release of thiobarbituric acid-reactive substances in the supernatant. The major thiobarbituric acid-reactive substance is identified by fluorescence spectroscopy and HPLC, as malondialdehyde or malondialdehyde-forming substances under the thiobarbituric acid assay conditions. Malondialdehyde formation strongly suggests a UVA-induced lipid peroxidation. Lipid peroxidation is also supported by the inhibitory effect of D,L-alpha-tocopherol, the well-known chain breaking antioxidant, by the additional malondialdehyde formation in the dark after the photooxidative stress and by membrane damage revealed by lactate dehydrogenase leakage.     

Effect of ethanol and the catalase inhibitor aminotriazole on lipid peroxidation in the rat myocardium

The influence of chronic alcohol consumption and catalase inhibitor aminotriazole administration on the level of nonenzymatic lipid peroxidation has been studied in the rat myocardium. It was demonstrated that combined as well as separate treatment with ethanol or aminotriazole elevated the levels of chemiluminescence and enhanced the rate of accumulation of thiobarbituric acid-reactive products in the nuclei-free and total particulate fraction of the rat heart homogenate. The most pronounced effect was noted during combined application of ethanol and aminotriazole. The induction of chemiluminescence by ethanol was prevented by addition of natural (vitamin E, reduced glutathione) or artificial (dibunol) antioxidants into the incubation media. A putative role of the myocardial catalase-containing micro-peroxisomes in stimulation of the intracellular lipid peroxidation is discussed.     

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.     

Changes in membrane constituents and chemiluminescence in vitamin E-deficient red blood cells induced by the xanthine oxidase reaction

The oxidation of vitamin E-deficient rat red blood cells (RBCs) induced by the hypoxanthine-xanthine oxidase (HX-XOD) system has been performed in an aqueous suspension. The generation of chemiluminescence and the accumulation of thiobarbituric acid-reactive substances (TBARS) were observed initially and were followed by hemolysis. Interestingly, the total counts of chemiluminescence were closely related to the amount of TBARS. The predominant change of membrane proteins induced by the reaction was the depletion of spectrin bands in gel electrophoresis. When RBC ghosts were oxidized with HX-XOD, the sulfhydryl (SH) groups of membrane proteins decreased at an early stage of the incubation, which was coincident with the above protein alteration. Membrane alpha-tocopherol suppressed not only the formation of TBARS but also chemiluminescence and hemolysis; nevertheless, it did not inhibit the protein damage and the loss of SH groups. Moreover, it was concluded that the chemiluminescence observed during the oxidation of RBC membranes was associated mainly with the peroxidation of lipids and only to a minor extent with the oxidation of proteins.     

Antioxidant capacity of allopurinol in biological systems

The antioxidant capacity of allopurinol was investigated in three biological systems by measurements of visible chemiluminescence, oxygen uptake and production of thiobarbituric acid reactive substances (TBARS). The addition of allopurinol to rat brain homogenates undergoing autoxidation and erythrocyte ghost membranes supplemented with 2,2'-azo-bis-(2-amidinopropane), in concentrations up to 2 mM, has a negligible effect on lipid peroxidation development. In erythrocyte ghost membranes exposed to gamma irradiation (9.5 Gy/min), allopurinol inhibits the radiation-induced lipid peroxidation with a Q(1/2) of 2.0 mM. It is suggested that allopurinol may have an alternative antioxidant pathway of action in biological systems, probably through a scavenging action upon hydroxyl radicals.     

Glycine Provokes Lipid Oxidative Damage and Reduces the Antioxidant Defenses in Brain Cortex of Young Rats

Patients affected by nonketotic hyperglycinemia (NKH) usually present severe neurological symptoms and suffer from acute episodes of intractable seizures with leukoencephalopathy. Although excitotoxicity seems to be involved in the brain damage of NKH, the mechanisms underlying the neuropathology of this disease are not fully established. The objective of the present study was to investigate the in vitro effects of glycine (GLY), that accumulate at high concentrations in the brain of patients affected by this disorder, on important parameters of oxidative stress, such as lipid peroxidation (thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence) and the most important non-enzymatic antioxidant defense reduced glutathione (GSH) in cerebral cortex from 30-day-old rats. GLY significantly increased TBA-RS and chemiluminescence values, indicating that this metabolite provokes lipid oxidative damage. Furthermore, the addition of high doses of the antioxidants melatonin, trolox (soluble vitamin E) and GSH fully prevented GLY-induced increase of lipid peroxidation, indicating that free radicals were involved in this effect. GLY also decreased GSH brain concentrations, which was totally blocked by melatonin treatment. Finally, GLY significantly reduced sulfhydryl group content from a commercial GSH solution, but did not oxidize reduced cytochrome C. Our data indicate that oxidative stress elicited in vitro by GLY may possibly contribute at least in part to the pathophysiology of the neurological dysfunction in NKH.     

Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O₂ consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radical. This protein may be an important defense against free radical injury to the microsomal membrane.     

Prooxidant action of aluminum Ion – Stimulation of iron-mediated lipid peroxidation by aluminum

Prooxidant nature of aluminum ion was analyzed in relation to iron coordination. Aluminum ion effectively enhanced the formation of thiobarbituric acid-reactive substances as a marker of lipid peroxidation of microsomes from rat liver under the acidic conditions, and this metal further attenuated the antioxidant action of flavonoids such as quercetin and baicalein under neutral conditions. Autooxidation of ferrous ion was markedly inhibited by aluminum ion. Aluminum can act as a prooxidant by stabilizing reduced iron the initiating species for lipid peroxidation, and by inhibiting the antioxidant action of flavonoid.     

Ascorbate-dependent recycling of the vitamin E homologue Trolox by dihydrolipoate and glutathione in murine skin homogenates

In the redox antioxidant network, dihydrolipoate can synergistically enhance the ascorbate-dependent recycling of vitamin E. Since the major endogenous thiol antioxidant in biological systems is glutathione (GSH) it was of interest to compare the effects of dihydrolipoate with GSH on ascorbate-dependent recycling of the water-soluble homologue of vitamin E, Trolox, by electron spin resonance (ESR). Trolox phenoxyl radicals were generated by a horseradish peroxidase (HRP)-hydrogen peroxide (H2O2) oxidation system. In the presence of dihydrolipoate, Trolox radicals were suppressed until both dihydrolipoate and endogenous levels of ascorbate in skin homogenates were consumed. Similar experiments made in the presence of GSH revealed that Trolox radicals reappeared immediately after ascorbate was depleted and that GSH was not able to drive the ascorbate-dependent Trolox recycling reaction. However, at higher concentrations GSH was able to increase ascorbate-mediated Trolox regeneration from the Trolox radical. ESR and spectrophotometric measurements demonstrated the ability of dihydrolipoate or GSH to react with dehydroascorbate, the two-electron oxidation product of ascorbate in this system. Dihydrolipoate regenerated greater amounts of ascorbate at a much faster rate than equivalent concentrations of GSH. Thus the marked difference between the rate and efficiency of ascorbate generation by dihydrolipoate as compared with GSH appears to account for the different kinetics by which these thiol antioxidants influence ascorbate-dependent Trolox recycling.     

Activation of peroxisomal acyl-CoA oxidase and lipid peroxidation in the rat myocardium as affected by prolonged administration of ethanol

The influence of chronic alcoholic intoxication on the activity of peroxisomal acyl-CoA oxidase and antioxidative defensive enzymes (catalase, glutathione reductase, glutathione-S-transferase, superoxide dismutase, glucose-6-phosphate dehydrogenase) was studied in the rat myocardium. The parameters of lipid peroxidation in cardiomyocytes (the level of spontaneous chemiluminescence, accumulation of thiobarbituric acid-reactive material), as well as reduced glutathione content were also examined. The data obtained suggest that ethanol-induced activation of lipid peroxidation in the myocardium may be due to the elevation of hydrogen peroxide-generating activity of peroxisomes.     

Fluorescence emitted from microsomal membranes by lipid peroxidation

The fluorescence emitted from rat liver microsomal membranes which had undergone enzymatic and nonenzymatic lipid peroxidation was detected directly. This fluorescence produced in peroxidized membranes increased progressively with peroxidation reaction time, and the fluorescent substances produced were retained in the membranes without being released into the aqueous phase. Extracts of the peroxidized membranes with organic solvents (chloroform/methanol) emitted fluorescence which was also dependent on the peroxidation reaction time. The generation profiles of fluorescence emitted from both the peroxidized membranes and their extracted membrane lipids differed essentially from that of thiobarbituric acid-reactive substances which reached a plateau at a relatively early stage of peroxidation reaction. These results indicate that lipid peroxidation induces stepwise chemical and physical changes in membranes and that the fluorescence from peroxidized membranes will be useful in studying such changes occurring in biological membranes.     

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.     

Stimulation of lipid peroxidation by methyl mercury in rats

As an index lipid peroxidation, thiobarbituric acid (TBA)-reactive substances in the liver, kidney, and serum, and hydrocarbons (ethane and pentane) in the exhalation of rats injected subcutaneously with 10 mg/kg/day of methylmercuric chloride (MMC) were determined. Formation of TBA-reactive substances in the liver and kidney of rats was significantly increased 4 and 2 days after initial injection of MMC, respectively. The result for serum was similar to that for the kidney. The maximum ethane production in the exhaled gases was observed 4 days after initial injection of MMC, and thereafter decreased slowly. Pentane production was significantly increased 5 days after initial injection of MMC, and thereafter continued to increase. Glutathione peroxidase activity and amount of vitamin C in the liver were depleted 4 days after initial injection of MMC; vitamin E was not depleted. In the kidney, significant decreases of glutathione peroxidase activity and vitamin C content were also seen 4 days after initial injection of MMC, but vitamin E content was unaltered.Thus, a clear increase of lipid peroxidation as determined by measurement of TBA-reactive substances in tissues and of hydrocarbons in the exhaled gases of rats after MMC treatment was demonstrated, though there was a lag phase of several days before the increase of lipid peroxidation. It is suggested that the significant increase of lipid peroxide formation may be a result of depletion of defending factors against lipid peroxidation.     

Dietary vitamin E supplementation affects tissue lipid peroxidation of hybrid tilapia,Oreochromis niloticus x O. aureus

A feeding trial was conducted to evaluate the effects of dietary vitamin E contents on the growth, ascorbate induced iron-catalyzed lipid peroxidation in post-mortem muscle and liver tissue, and Raman spectral changes in lens of juvenile hybrid tilapia (Oreochromis niloticus x O. aureus). Experimental fish were fed practical diets supplemented with 0, 50, 100, 200, 450 and 700 mg alpha-tocopheryl acetate/kg diet for 14 weeks. There was no significant difference in weight gain, feed conversion ratio and protein efficiency ratio among fish fed test diets (P>0.05). Protein content of fish fed diet containing the lowest vitamin E level was the lowest (P<0.05) among all groups. No difference was found in other body constituents among test fish (P>0.05). The thiobarbituric acid-reactive substances produced by iron-catalyzed lipid peroxidation in muscle and liver tissue of fish fed the diet without alpha-tocopheryl acetate supplementation were significantly (P<0.05) greater than those from fish fed diets containing higher levels of alpha-tocopheryl acetate. Dietary vitamin E supplementation increased the antioxidant capability of tilapia tissues against lipid peroxidation. Further, dietary vitamin E supplementation also influenced the lens cortical membrane structure of tilapia.     

Oxidative insult to human red blood cells induced by free radical initiator AAPH and its inhibition by a commercial antioxidant mixture.

This study was carried out to investigate sequel of oxidative insult to human erythrocytes induced by a water-soluble radical initiator, 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) and the effect of a commercially available mixed antioxidant (Blackmores, BioAce Excel), containing alpha-tocopherol, ascorbic acid, beta-carotene and some herbal extracts (containing grape seed catechins and milk thistle derived silybin), on lipid peroxidation, degradation of membrane proteins and haemolysis. We performed this study in order firstly to clarify aspects of the mechanism of AAPH induced free radical damage in human erythrocytes and secondly to establish in vitro conditions by which the efficacy of mixed antioxidant preparations may fairly and objectively be compared. In the process of oxidation initiated by peroxyl radical, a rapid loss of reduced glutathione occurred in the first 60 min. Formation of thiobarbitric acid-reactive substances indicative of lipid peroxidation increased subsequently and almost reached maximal levels at 180 min before significant apparent degradation of membrane proteins was detected. At this point, a significant haemolysis occurred. This sequence of events is consistent with the idea that haemolysis is a consequence of lipid peroxidation and the degradation of membrane proteins. The mixed commercial antioxidant, which suppressed lipid peroxidation and protected membrane proteins against degradation induced by peroxyl radicals, also effectively delayed AAPH induced haemolysis. The system we describe provides a sound objective basis for the in vitro comparison of the potential efficacy of the hundreds of antioxidant nutritional supplements currently available in the market place.     

Free radical-induced alterations of myocardial membrane proteins

Rat myocardial membranes exposed to the free radical-generating systems, Fe2+/ascorbate, Cu2+/t-butylhydro-peroxide, linoleic acid hydroperoxide, and soybean lipoxygenase (Type I) undergo lipid peroxidation. This is evidenced by the accumulation of thiobarbituric acid-reactive substances and the loss of both extractable phospholipids and their polyunsaturated acyl groups. Lipid peroxidation is accompanied by alterations of membrane proteins including the general loss of polypeptides and accumulation of high-molecular weight material. The most sensitive protein is a polypeptide with a molecular weight of 28 kDa. At low levels of oxidation, this protein moves incrementally to slightly higher apparent molecular weight. At higher oxidant levels or longer periods of oxidation, the protein disappears completely from the SDS-PAGE gel. The "28K reaction" occurs prior to the massive, oxidant-induced lipid alterations and may thus indicate specific adduct formation between this protein and certain peroxidized membrane phospholipids.     

The antioxidant action of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a chemiluminescence probe to detect superoxide anions

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O^⋅−₂, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2′-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe²⁺-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.     

Differential effects of 3 beta blockers on lipid peroxidation in hyperthyroid muscle.

To determine whether beta blockade protects against the acceleration of lipid peroxidation in hyperthyroid rat soleus (slow-oxidative) muscle, in vivo chronic (3 weeks) effects of 3 beta blockers with different ancillary properties on mitochondrial oxidative enzymes, antioxidant enzymes, and thiobarbituric acid-reactive substances were investigated. The rats were rendered hyperthyroid by the administration of thyroxine and treated simultaneously with either carteolol (a nonselective blocker with partial agonist activity; 30 mg/kg/day), atenolol (a beta 1-selective blocker; 50 mg/kg/day), or arotinolol (a nonselective blocker with weak alpha-blocking action; 50 mg/kg/day) over a 3 week period. Hyperthyroidism induced tachycardia, an increase in the mitochondrial oxidative enzymes, manganese (mitochondrial) superoxide dismutase and thiobarbituric acid-reactive substances, and a decrease in the other antioxidant enzymes. The tachycardia was alleviated completely by either atenolol or arotinolol, but partially by carteolol. Arotinolol, but neither carteolol nor atenolol, inhibited the increase in oxidative enzymes and thiobarbituric acid-reactive substances. The levels of antioxidant enzymes were minimally affected by the beta-blocker treatment. Beta 2-, and possibly alpha- as well, but not beta 1-, blockade suppressed mitochondrial hypermetabolism and protected against peroxidative injury in the hyperthyroid soleus muscle. Partial agonist activity was not beneficial.