Kinetic evaluation of lipophilic inhibitors of lipid peroxidation in DLPC liposomes

The authors have developed a kinetic method that allows one to obtain relative reactivity constants for lipophilic antioxidants in free radical systems. Two experimental model systems were developed: (a) a methanolic solution using AMVN as the free radical initiator and linoleic acid as the substrate, and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine and AAPH as the substrate and the initiator, respectively. The use of these two systems allows researchers not only to determine the intrinsic reactivity of a potential antioxidant, but also to evaluate its potency in a membranous system where the contribution of the physical properties of the antioxidant to the inhibition of lipid peroxidation is important. These results show that all antioxidants tested acted in these systems as free radical scavengers, and they validate the synergism between intrinsic scavenging ability and membrane affinity and/or membrane-modifying physical properties in the inhibition of lipid peroxidation.     

Plasma copper and lipid peroxidation in cigarette smokers

Plasma levels of copper and lipid peroxidation were evaluated in 14 smokers as compared to 14 nonsmokers. Plasma copper concentrations were higher in smokers than in nonsmokers (122.5 ± 19.15 vs. 101.5 ± 16.2 μg/dl, P < .01). Plasma lipoperoxidation, evaluated as fluorescent damage products of lipid peroxidation (FDPL), also was higher in smokers than in nonsmokers (20.35 ± 2.6 vs. 17.1 ± 2.95 units of relative fluorescence/ml, P < .01). A significant and positive correlation between the number of cigarettes smoked, expressed as pack years, and the levels of either FDPL (r = .61, P < .025) or copper (r = .55, P < .05) was found. Moreover, a significant and positive relationship between copper and FDPL values was observed in smokers (r = .64; P < .025), but not in nonsmokers. These data indicate that cigarette smoke-related plasma oxidant load may be partly due to enhanced levels of the prooxidant metal copper, potentially suggesting the supplementation of specific antioxidants (e.g., zinc) to counteract cigarette smoke-induced oxidative stress in smokers.     

Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression

Ultraviolet A (UVA) irradiation causes human skin aging and skin cancer at least partially through the activation of matrix metalloproteinases (MMPs). MMP-1, the interstitial collagenase, is responsible for the degradation of collagen and is involved in tumor progression in human skin. The present study uses human skin fibroblast cells (FEK4) to investigate the involvement of lipid peroxidation and the role of peroxides as possible mediators in MMP-1 activation by UVA. Preincubation with the antioxidants butylated hydroxytoluene and Trolox reduced UVA-dependent MMP-1 upregulation, suggesting that peroxidation of membrane lipids is involved. Blocking the iron-driven generation of lipid peroxides and hydroxyl radicals by different iron chelators led to a decrease in UVA-induced MMP-1 mRNA accumulation. Moreover, modulation of glutathione peroxidase activity by use of the specific inhibitor mercaptosuccinate (MS) or by the depletion of glutathione (using buthionine-S, R-sulfoximine, BSO), enhanced the UVA-dependent MMP-1 response. Finally, UVA irradiation generated a significant increase in intracellular peroxide levels which is augmented by pretreatment of the cells with BSO or MS. Our results demonstrate that lipid peroxidation and the production of peroxides are important events in the signalling pathway of MMP-1 activation by UVA.     

Endogenous ubiquinol prevents protein modification accompanying lipid peroxidation in beef heart submitochondrial particles

This article is a study of the relationship between lipid peroxidation and protein modification in beef heart submitochondrial particles, and the protective effect of endogenous ubiquinol (reduced coenzyme Q) against these effects. ADP-Fe^3· and ascorbate were used to initiate lipid peroxidation and protein modification, which were monitored by measuring TBARS and protein carbonylation, respectively. Endogenous ubiquinone was reduced by the addition of succinate and antimycin. The parameters investigated included extraction and reincorporation of ubiquinone, and comparison of the effect of ubiquinol with those of various antioxidant compounds and enzymes, as well as the iron chelator EDTA. Under all conditions employed there was a close correlation between lipid peroxidation and protein carbonylation, and the inhibition of these effects by endogenous ubiquinol. SDS-PAGE analysis revealed a differential effect on individual protein components and its prevention by ubiquinol. Conceivable mechanisms behind the observed oxidative modifications of membrane phospholipids and proteins and of the role of ubiquinol in preventing these effects are considered.     

Melatonin reduces Fenton reaction-induced lipid peroxidation in porcine thyroid tissue

Free radicals and reactive oxygen species (ROS) participate in physiological and pathological processes in the thyroid gland. Bivalent iron cation (ferrous, Fe(2+)), which initiates the Fenton reaction (Fe(2+) + H2O2 --> Fe(3+) + *OH + OH(-)) is frequently used to experimentally induce oxidative damage, including that caused by lipid peroxidation. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of carcinogenesis. In turn, melatonin is a well-known antioxidant and free radical scavenger. The aim of the study was to estimate the effect of melatonin on basal and iron-induced lipid peroxidation in homogenates of the porcine thyroid gland. In order to determine the effect of melatonin on the auto-oxidation of lipids, thyroid homogenates were incubated in the presence of that indoleamine in concentrations of 0.0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, or 5.0 mM. To study melatonin effects on iron-induced lipid peroxidation, the homogenates were incubated in the presence of FeSO(4) (40 microM) plus H2O2 (0.5 mM), and, additionally, in the presence of melatonin in the same concentrations as above. The degree of lipid peroxidation was expressed as the concentration of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. Melatonin, in a concentration-dependent manner, decreased lipid peroxidation induced by Fenton reaction, without affecting the basal MDA + 4-HDA levels. In conclusion, melatonin protects against iron + H2O2-induced peroxidation of lipids in the porcine thyroid. Thus, the indoleamine would be expected to prevent pathological processes related to oxidative damage in the thyroid, cancer initiation included.     

Melatonin reduces lipid and protein oxidative damage in synaptosomes due to aluminium

Prolonged exposure to excessive aluminium (Al) concentrations is involved in the ethiopathology of certain dementias and neurological disorders. Melatonin is a well-known antioxidant that efficiently reduces lipid peroxidation due to oxidative stress. Herein, we investigated in synaptosomal membranes the effect of melatonin in preventing Al promotion of lipid and protein oxidation when the metal was combined with FeCl₃ and ascorbic acid. Lipid peroxidation was estimated by quantifying malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations in the membrane suspension and protein carbonyls were measured in the synaptosomes as an index of oxidative damage. Under our experimental conditions, the addition of Al (0.0001–1 mmol/L) enhanced MDA+4-HDA formation in the synaptosomes. In addition, Al (1 mmol/L) raised protein carbonyl contents. Melatonin reduced, in a concentration-dependent manner, lipid and protein oxidation due to Al, FeCl₃ and ascorbic acid in the synaptosomal membranes. These results show that melatonin confers protection against Al-induced oxidative damage in synaptosomes and suggest that this indoleamine may be considered as a neuroprotective agent in Al toxicity because of its antioxidant activity.     

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.     

Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release

Cardiolipin oxidation is emerging as an important factor in mitochondrial dysfunction as well as in the initial phase of the apoptotic process. We have previously shown that exogenously added peroxidized cardiolipin sensitizes mitochondria to Ca²⁺-induced mitochondrial permeability transition (MPT) pore opening and promotes the release of cytochrome c. In this work, the effects of intramitochondrial cardiolipin peroxidation on Ca²⁺-induced MPT and on the cytochrome c release from mitochondria were studied. The effects of melatonin, a compound known to protect the mitochondria from oxidative damage, on both of these processes were also tested. tert-Butylhydroperoxide (t-BuOOH), a lipid-soluble peroxide that promotes lipid peroxidation, was used to induce intramitochondrial cardiolipin peroxidation. Exposure of heart mitochondria to t-BuOOH resulted in the oxidation of cardiolipin, associated with an increased sensitivity of mitochondria to Ca²⁺-induced MPT and with the release of cytochrome c from the mitochondria. All these processes were inhibited by micromolar concentrations of melatonin. It is proposed that melatonin inhibits cardiolipin peroxidation in mitochondria, and this effect seems to be responsible for the protection afforded by this agent against the MPT induction and cytochrome c release. Thus, manipulating the oxidation sensitivity of cardiolipin with melatonin may help to control MPT and cytochrome c release, events associated with cell death, and thus, be used for treatment of those disorders characterized by mitochondrial cardiolipin oxidation and Ca²⁺ overload.     

Lipid peroxidation products in human subretinal fluid

The concentrations of thiobarbituric acid reacting substances (TBARS) and proteins in the subretinal fluid (SF) of patients undergoing retinal detachment surgery have been determined. We have tried to establish the correlations between these biochemical and other clinical features of these patients: evolution time of the retinal detachment, age, degree of myopia, and macular affection. Caucasian patients, 19 men and 19 women (57.42 ±12.85 average age, interval 20–80) were randomly selected for this study. SF samples were obtained by puncture after scleral indentation. TBARS and protein concentrations were determined by the corresponding colorimetric assays. A linear correlation exists between TBARS and protein contents in these samples. No correlation could be established between evolution time of the retinal detachment and TBARS content in SF. TBARS in SF increases with increasing age in nonmyopic patients. In the samples of myopic patients the correlation was established between TBARS content and degree of myopia. The group of patients with more than 10 dioptres show a significant higher TBARS concentration in SF than any of the other groups studied. It can be concluded that lipid peroxidation products in SF originate, at least partially, from rod outer segments, and that lipid peroxidation is a process that might play a role in the pathogenesis of retinal detachment, specially in myopic patients.     

NADPH- and iron-dependent lipid peroxidation inhibit aromatase activity in human placental microsomes

During pregnancy placenta is the most significant source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides and other ROS is often linked to pre-eclampsia. It is already proved that placental endoplasmic reticulum may be an important place of lipid peroxides and superoxide radical production. In the present study we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) inhibit placental aromatase--a key enzyme of estrogen biosynthesis in human placenta. We showed that significant inhibition of this enzyme is caused by small lipid peroxidation (TBARS (thiobarbituric acid-reactive substances)<4nmol/mg microsomal protein (m.p.)). More intensive lipid peroxidation (TBARS>9nmol/mg microsomal protein) diminishes aromatase activity to value being less than 5% of initial value. NADPH- and iron-dependent lipid peroxidation also causes disappearance of cytochrome P450 parallel to observed aromatase activity inhibition. EDTA, alpha-tocopherol, MgCl(2) and superoxide dismutase (SOD) prevent aromatase activity inhibition and cytochrome P450(AROM) degradation. Mannitol and catalase have not effect on TBARS synthesis, aromatase activity and cytochrome P450 degradation. In view of the above we postulate that the inhibition of aromatase activity observed is mainly a consequence of cytochrome P450(AROM) degradation induced by lipid radicals. The role of hydroxyl radical in cytochrome P450 degradation is negligible in our experimental conditions. The results presented here also suggest that the inhibition of aromatase activity can also take place in placenta at in vivo conditions.     

Melatonin, lipid peroxidation, and age in heterophils from the ring dove (Streptopelia risoria)

Numerous recent studies have shown the ability of physiological as well as all pharmacological concentrations of melatonin to prevent oxidative stress. We have found that incubating avian heterophils from young birds with a pharmacological concentration of 100 μM (23 × 10⁶ pg/ml) melatonin reduced superoxide anion levels by modulating the activity of superoxide dismutase while also enhancing phagocytosis. There was also a decline in lipid peroxidation levels with both physiological and pharmacological concentrations of this indolamine.

In the present work, we evaluated malonaldehyde (MDA) levels as an indicator of lipid peroxidation (both basal and antigen-induced) in young and old animals (ring doves) at different times of day (16:00 and 00:00) and with two incubation times (15 and 60 min). The lipid peroxidation was also measured in heterophils from old animals, incubated with the physiological concentrations of melatonin measured in young animals (50 and 300 pg/ml, diurnal and nocturnal, respectively). The results, expressed as nmol MDA/mg protein, show that MDA levels were higher in heterophils of old animals than in the young birds in all the experimental groups studied at both 16:00 and 00:00 (00:00 is the time at which the lowest peroxidation levels were obtained). Incubation with melatonin was found to reduce MDA levels, with the maximum reduction being after the 60 min incubation time and the nocturnal melatonin concentration. At both concentrations (diurnal and nocturnal), melatonin also counteracted the enhancement of MDA levels caused by latex beads, with the effect being greater at the longer incubation time. In conclusion, the results are further evidence of the antioxidant effect of melatonin even at physiological concentrations, and suggest its utility as a therapeutic agent in some pathological processes associated with age.     

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with α-linolenic acid

Lipid hydroperoxide (LOOH)–dependent lipid peroxidation was induced in α-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 μM. The effects of structurally related flavonoids at concentrations from 10 to 500 μM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid–metal complexes.     

Protective effects of lazaroid U74389F (16-desmethyl tirilazad) on endrin-induced lipid peroxidation and DNA damage in brain and liver and regional distribution of catalase activity in rat brain

Endrin, a poly-halogenated cyclic hydrocarbon, induces hepatic lipid peroxidation, modulates calcium homeostasis, decreases membrane fluidity, and increases nuclear DNA damage. Little information is available on the neurotoxicity of endrin. The effects of endrin on lipid peroxidation, DNA damage, and regional distribution of catalase activity were assessed in rat brain and liver 24 h following an acute oral dose of 4.5 mg endrin/kg. Lipid peroxidation associated with whole brain mitochondria increased 2.4-fold, whereas microsomal lipid peroxidation increased 2.8-fold following endrin administration. Lipid peroxidation also increased 2.0-fold both in hepatic mitochondria and microsomes. Catalase activity decreased 24% in the hypothalamus, 23% in the cortex, 38% in the cerebellum, and 11% in the brain stem in response to endrin. A 4.3-fold increase in brain nuclear DNA-single strand breaks (SSB) was observed in endrin-treated rats. Pretreatment of rats intraperitoneally with the lazaroid U74389F (16-desmethyl tirilazad) (10 mg/kg in two doses) attenuated the biochemical consequences of endrin-induced oxidative stress. The administration of U74389F in citrate buffer (pH 3.8) provided better protection than administering the lazaroid in corn oil, decreasing endrin-induced lipid peroxidation by 50–80% and DNA-SSB by approximately 72% in liver and 85% in brain, while ameliorating the suppressed catalase activity. The data suggest an involvement of an oxidative stress in the neurotoxicity and hepatotoxicity induced by endrin, which can be attenuated by the lazaroid U74389F.     

Pantothenic acid and its derivatives protect ehrlich ascites tumor cells against lipid peroxidation

Preincubation of Ehrlich ascites tumor cells at 22 or 32°C, but not at 0°C, with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine reduced lipid peroxidation (measured by production of thiobarbituric acid-reactive compounds) induced by the Fenton reaction (Fe²⁺ + H₂O₂) and partly protected the plasma membrane against the leakiness to cytoplasmic proteins produced by the same reagent. Pantothenic acid and its derivatives did not inhibit (Fe²⁺ + H₂O₂)-induced peroxidation of phospholipid multilamellar vesicles, thus indicating that their effect on the cells was not due to the scavenging mechanism. Homopantothenic acid and its 4′-phosphate ester (which are not precursors of CoA) neither protected Ehrlich ascites tumor cells against lipid peroxidation nor prevented plasma membrane leakiness under the same conditions. Incubation of the cells with pantothenic acid, 4′-phosphopantothenic acid, pantothenol, or pantethine significantly increased the amount of cellular CoA and potentiated incorporation of added palmitate into phospholipids and cholesterol esters. It is concluded that pantothenic acid and its related compounds protect the plasma membrane of Ehrlich ascites tumor cells against the damage by oxygen free radicals due to increasing cellular level of CoA. The latter compound may act by diminishing propagation of lipid peroxidation and promoting repair mechanisms, mainly the synthesis of phospholipids.     

Hyaluronic acid and chondroitin-4-sulphate treatment reduces damage in carbon tetrachloride-induced acute rat liver injury

Oxidative stress is involved in the pathogenesis of chemically mediated liver injury. Since glycosaminoglycans possess antioxidant activity, the aim of this work was to assess the protective effects of hyaluronic acid and chondroitin-4-sulphate treatment in a model of carbon tetrachloride-induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of carbon tetrachloride (1 ml/kg in vegetal oil). Serum alanine aminotransferase and aspartate aminotransferase, hepatic malondialdehyde, plasma TNF-alpha, hepatic reduced glutathione and catalase, and myeloperoxidase, an index of polymorphonuclear infiltration in the jeopardised hepatic tissue, were evaluated 24 h after carbon tetrachloride administration. Carbon tetrachloride produced a marked increase in serum alanine aminotransferase and aspartate aminotransferase activities, primed lipid peroxidation, enhanced plasma TNF-alpha levels, induced a severe depletion of reduced glutathione and catalase, and promoted neutrophil accumulation. Intraperitoneal treatment of rats with hyaluronic acid (25 mg/kg) or chondroitin-4-sulphate (25 mg/kg) failed to exert any effect in the considered parameter, while the combination treatment with both glycosaminoglycans (12,5 + 12,5 mg/kg) decreased the serum levels of alanine aminotransferase and aspartate aminotransferase, inhibited lipid peroxidation by reducing hepatic malondialdehyde, reduced plasma TNF-alpha, restored the endogenous antioxidants, and finally decreased myeloperoxidase activity. These results suggest that hyaluronic acid and chondroitin-4-sulphate possess a different antioxidant mechanism and consequently the combined administration of both glycosaminoglycans exerts a synergistic effect with respect to the single treatment.     

Beta-glucan from Saccharomyces cerevisiae reduces plasma lipid peroxidation induced by haloperidol

Since oxidative stress observed in schizophrenia may be caused partially by the treatment of patients with various antipsychotics, the aim of the study was to establish the effects of beta-d-glucan, polysaccharide derived from the yeast cell walls of species such as Saccharomyces cerevisiae, and the antipsychotics (the first generation antipsychotic (FGA) - haloperidol and the second generation antipsychotic (SGA) - amisulpride) action on plasma lipid peroxidation in vitro. Lipid peroxidation in human plasma was measured by the level of thiobarbituric acid reactive species (TBARS). The samples of plasma from healthy subjects were incubated with haloperidol or amisulpride in the presence of beta-glucan (4 μg/ml). The action of beta-d-glucan was also compared with the properties of a well characterized commercial monomeric polyphenol - resveratrol (3,4′,5-trihydroxystilbene, the final concentration - 4 μg/ml). The two-way analysis variance showed that the differences in TBARS levels were depended on the type of tested drugs (p = 7.9 × 10⁻⁶). We observed a statistically increase of the level of biomarker of lipid peroxidation such as TBARS after 1 and 24 h incubation of plasma with haloperidol compared to the control samples (p < 0.01, p < 0.02, respectively). Amisulpride, contrary to haloperidol (after 1 and 24 h) did not cause plasma lipid peroxidation (p > 0.05). We showed that in the presence of beta-glucan, lipid peroxidation in plasma samples treated with haloperidol was significantly decreased. Moreover, we did not observe the synergistic action of beta-glucan and amisulpride on the inhibition of plasma lipid peroxidation. However, the beta-d-glucan was found to be more effective antioxidant, than the solution of pure resveratrol. The presented results indicate that beta-glucan seems to have distinctly protective effects against the impairment of plasma lipid molecules induced by haloperidol.     

Increase in heart glutathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin e dietary supplementation in guinea pigs

Dietary treatment with three diets differing in vitamin E, Low E (15 mg of vitamin E/kg diet), Medium E (150 mg/kg), or High E (1,500 mg/kg), resulted in guinea pigs with low (but nondeficient), intermediate, or high heart a-tocopherol concentration. Neither the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and reductase, nor the nonenzymatic antioxidants, GSH, ascorbate, and uric acid were homeostatically depressed by increases in heart a-tocopherol. Protection from both enzymatic (NADPH dependent) and nonenzymatic (ascorbate-Fe²⁺) lipid peroxidation was strongly increased by vitamin E supplementation from Low to Medium E Whereas no additional gain was obtained from the Medium E to the High E group. The GSH/GSSG and GSH/total glutathione ratios increased as a function of the vitamin E dietary concentration closely resembling the shape of the dependence of heart a-tocopherol on dietary vitamin E. The results show the capacity of dietary vitamin E to increase the global antioxidant capacity of the heart and to improve the heart redox status in both the lipid and water-soluble compartments. This capacity occurred at levels six times higher than the minimum daily requirement of vitamin E, even in the presence of optimum dietary vitamin C concentrations and basal unstressed conditions. The need for vitamin E dietary supplementation seems specially important in this tissue due to the low constitutive levels of endogenous enzymatic and nonenzymatic antioxidants present of the mammalian heart in comparison with those of other internal organs.     

Isoprostanes: novel bioactive products of lipid peroxidation

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

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

Roles of catalase and cytochrome C in hydroperoxide-dependent lipid peroxidation and chemiluminescence in rat heart and kidney mitochondria

A recent report (Radi et al., J. Biol. Chem. 266:22028–22034, 1991) showed that rat heart mitochondria contain catalase. The protective role of mitochondrial catalase was tested by exposing heart or kidney mitochondria and mitoplasts to two oxidants (H₂O₂) or tert-butyl hydroperoxide, t-BOOH), estimating lipid peroxidation (as thiobarbituric acid-reactive substances, TBARS) and overall oxidative stress (as chemiluminescence). Additional controls included heart and kidney preparations from aminotriazole-treated (catalase-depleted) rats. Both oxidants increased TBARS in catalase-free preparations to similar extents over their respective controls (between 200 to 350%). In catalase-containing preparations, H₂O₂ lipid peroxidation increased by only 40 to 96% over controls. Similar qualitative results were obtained when measuring chemiluminescence. The catalytic role of cytochrome c in mitochondrial lipid peroxidation was investigated by exposing either control or cytochrome-c-depleted kidney mitoplasts (catalase free) to either H₂O₂ or t-BOOH. Hydrogen-peroxide-dependent mitochondrial lipid peroxidation varied with cytochrome c concentrations, remaining close to controls when cytochrome c concentration decreased by 66%, even though there was no catalase present. Tert-butyl hydroperoxide-dependent lipid peroxidation was less affected by cytochrome c remaining 2.3-fold above controls under the same conditions, suggesting that organic peroxides are more likely to remain in the less polar membrane environment being decomposed by heme or nonheme iron imbedded in the inner mitochondrial membrane. Chemiluminescence was less affected by cytochrome c depletion. Comparing control and cytochrome-c-deficient mitochondria, chemiluminescence was 1.7-fold and 2.8-fold higher when control preparations were challenged with t-BOOH or H₂O₂, respectively.