Measurement of lipid peroxidation

Lipid peroxidation results in the formation of conjugated dienes, lipid hydroperoxides and degradation products such as alkanes, aldehydes and isoprostanes. The approach to the quantitative assessment of lipid peroxidation depends on whether the samples involve complex biological material obtained in vivo, or whether the samples involve relatively simple mixtures obtained in vitro. Samples obtained in vivo contain a large number of products which themselves may undergo metabolism. The measurement of conjugated diene formation is generally applied as a dynamic quantitation e.g. during the oxidation of LDL, and is not generally applied to samples obtained in vivo. Lipid hydroperoxides readily decompose, but can be measured directly and indirectly by a variety of techniques. The measurement of MDA by the TBAR assay is non-specific, and is generally poor when applied to biological samples. More recent assays based on the measurement of MDA or HNE-lysine adducts are likely to be more applicable to biological samples, since adducts of these reactive aldehydes are relatively stable. The discovery of the isoprostanes as lipid peroxidation products which can be measured by gas chromatography mass spectrometry or immunoassay has opened a new avenue by which to quantify lipid peroxidation in vivo, and will be discussed in detail.     

Nitric oxide and lipid peroxidation

Nitric oxide (NO) is a free radical produced enzymatically in biological systems from the guanidino group of L-arginine. Its large spectrum of biological effects is achieved through chemical interactions with different targets including oxygen (O2), superoxide (O2o-) and other oxygen reactive species (ROS), transition metals and thiols. Superoxide anions and other ROS have been reported to react with NO to produce peroxynitrite anions that can decompose to form nitrogen dioxide (NO2) and hydroxyl radial (OHo). Thus, NO has been reported to have a dual effect on lipid peroxidation (prooxidant via the peroxynitrite or antioxydant via the chelation of ROS). In the present study we have investigated in different models the in vitro and in vivo action of NO on lipid peroxidation. Copper-induced LDL oxidation were used as an in vitro model. Human LDL (100 micrograms ApoB/ml) were incubated in oxygene-saturated PBS buffer in presence or absence of Cu2+ (2.5 microM) with increasing concentrations of NO donnors (sodium nitroprussiate or nitroso-glutathione). LDL oxidation was monitored continuously for conjugated diene formation (234 nm) and 4-hydroxynonenal (HNE) accumulation. Exogenous NO prevents in a dose dependent manner the progress of copper-induced oxidation. Ischaemia-reperfusion injury (I/R), characterized by an overproduction of ROS, is used as an in vivo model. Anaesthetized rats were submitted to 1 hour renal ischaemia following by 2 hours of reperfusion. Sham-operated rats (SOP) were used as control. Lipid peroxidation was evaluated by measuring the HNE accumulated in rats kidneys in presence or absence of L-arginine or D-arginine infusion. L-arginine, but not D-arginine, enhances HNE accumulation in I/R but not in SOP (< 0.050 pmol/g tissue in SOP versus 0.6 nmol/g tissue in I/R), showing that, in this experimental conditions, NO produced from L-arginine, enhances the toxicity of ROS. This study shows that the pro- or antioxydant effects of NO are different in vivo and in vitro and could be driven by environmental conditions such as pH, relative concentrations of NO and ROS, ferryl species.     

Marchantinquinone, isolated from Reboulia hemisphaerica, as inhibitor of lipid peroxidation and as free radical scavenger

Recent years have witnessed a renewed interest in plants as pharmaceuticals in the Western world. This interest is channeled into the discovery of new biologically-active molecules by the pharmaceutical industry and into the adoption of crude extracts of plants for self-medication by the general public. In both of these areas some attention is being paid to the investigation and use of ethnopharmacology, the traditional use of plants for medicinal purposes by particular cultural groups. Ethnopharmacologic leads have resulted in the introduction of new single molecule drugs but have a greater role to play if crude extracts are accepted for clinical use in the West. The problems confronting such usage are discussed. Considerable benefits for developing countries are possible when the local medicinal plants are subjected to scientific methods of validation of traditional use and quality control. This approach has met with success in some parts of the world but is not always appreciated by national governments and international agencies. Related areas of concern such as conservation of ecology and culture must be integrated with any such program. Plants used in traditional medicine therefore have an important role to play in the maintenance of health in all parts of the world and in the introduction of new treatments.     

Enhanced lipid peroxidation in hepatic cirrhosis

> Objective: This paper describes the deliberations of an interdisciplinary group of clinical and basic scientists who met at the National Institute of Child Health and Human Development to discuss the potential role of fetal behavior in assessing fetal well being and predicting neonatal outcome. The conference focused on three aspects of fetal behavior: 1) habituation; 2) state transitions; and 3) movement. Methods: The participants consisted of 25 leaders in the fields of obstetrics, perinatal medicine, neonatology, developmental psychobiology, developmental neuroscience, developmental psychology, ethology, and mathematics. The meeting was divided into three parts. In each of these a plenary speaker (a recognized expert in his field) began the session with an overview of the scientific theme. Two respondents, with research expertise in fetal research (animal models or human fetuses) followed with remarks on the plenary talk and comments based on their own studies. At the conclusion of these comments, the participants met in small groups to discuss the plenary proceedings and their implications for assessing human fetal well being and predicting outcome. At the conclusion of the small group deliberations all of the participants reconvened in a plenary session. During this part of the meeting a rapporteur from each small group summarized their discussions. Results and Conclusions: 1) Fetal habituation: there was a general consensus that research on this aspect of fetal behavior may have a high payoff for assessing human fetal well being and predicting neonatal outcome. 2) Behavioral state transitions: participants agreed that transitions afford investigators with an indication of when (timing) and how (models) behavior changes within and between developmental periods. Knowledge of transitions during development allows for tracking of behaviors that may be necessary for the fetus to adapt to its in utero environment or prepare for its postnatal life. 3) Chaos theory and fetal movement: participants concluded that non-linear dynamics systems analysis models could be useful to analyze "noise" within a measurement system; better define time scales; and increase resolution and thereby better identify "signals."     

Involvement of lipid peroxidation in platelet signalling

A well-known signalling pathway in blood platelets consists in the release of arachidonic acid (AA) from membrane phospholipids and its specific oxygenation into bioactive derivatives. In particular, cyclic prostaglandin endoperoxides and thromboxane A2 are potent inducers of platelet functions and are produced in greater amounts when the level of lipid hydroperoxides is higher than normal, as 'physiological concentrations' of such peroxides activate the cyclooxygenation of AA. In this context, a lower activity of platelet glutathione peroxidase (GPx), the key-enzyme for the degradation of lipid hydroperoxides, has been reported in aging, which will ensure a longer life span to those peroxides. Accordingly, the biosynthesis of pro-aggregatory prostanoids is elevated in platelets from the elderly. On the other hand, fatty acids from marine origin have been recognized as inhibitors of platelet functions, and they may alter the redox status of cells. They may for instance increase the platelet GPx activity, an effect that can be prevented by antioxidants. Overall, these data point out the relevance of the redox status in platelet functions.     

Inhibition of lipid peroxidation mediated by indolizines

Esters, ethers, carbonates and carbamates of 1-indolizinols and azaindolizinols exhibit a profound inhibition of lipid peroxidation in vitro. The antioxidants were prepared by cyclization of pyridines and diazines with diphenylcyclopropenone followed by introduction of the O-substituent.     

Low temperature phosphorescence of lipid peroxidation products

The phosphorescence exitation and emission spectra and the phosphorescence lifetimes of polymerized malonic aldehyde, Shiff bases, linoleic acid, phosphatidylcholine, phosphatidylethanolamine, cardiolipine, total lipid fraction from human erythrocyte membranes were measured at 77 K. The nature of chromophores of lipid peroxidation products capable of phosphorescence was discussed.     

The lipid peroxidation inhibitor indenoindole H290/51 protects myocardium at risk of injury induced by ischemia-reperfusion

OBJECTIVE: Elevated blood ammonia is an important pathogenic factor of hepatic encephalopathy. Although colonic bacteria are considered the main source of ammonia, the stomach in subjects with urease-producing Helicobacter pylori (H. pylori) is an alternative site. The objective of this study was to determine whether H. pylori is associated with this complication. METHODS: After assessing liver function and portal hypertension, 55 cirrhotics were evaluated for encephalopathy and H. pylori infection. Response to 2 weeks of amoxicillin (2 g/day) and omeprazole (40 mg/day) was then assessed in 17 (13 H. pylori-positive, four H. pylori-negative) encephalopathic subjects. RESULTS: H. pylori infection was more common (67 % vs 33%, p = 0.004) among encephalopathic patients. Additional factors associated with encephalopathy included older age (60.1 +/- 1.5 vs 49.8 +/- 2.4 yr, p = 0.001), lower albumin (3.17 +/- 0.08 vs 3.69 +/- 0.12 g/dl, p = 0.001), higher total bilirubin (2.24 +/- 0.20 vs 1.53 +/- 0.23 mg/dl, p = 0.034), greater ascites score (0.8 +/- 0.1 vs 0.3 +/- 0.1, p = 0.01), greater diuretic score (1.1 +/- 0.1 vs 0.3 +/- 0.1, p = 0.002), and greater modified Child score (6.7 +/- 0.3 vs 5.1 +/- 0.3, p = 0.001). When adjusted for severity of cirrhosis and age, H. pylori continued to demonstrate a statistical association (p = 0.039). After anti-H. pylori therapy, symptomatology in infected encephalopathic patients appeared to improve, whereas noninfected subjects were unaffected. CONCLUSION: In cirrhotic patients, H. pylori infection is associated with hepatic encephalopathy, especially in younger patients with decompensated liver disease.     

Postischemic application of lipid peroxidation inhibitor U-101033E reduces neuronal damage after global cerebral ischemia in rats

BACKGROUND AND PURPOSE: The lipid peroxidation inhibitor U-101033E was examined for effects on cerebral blood flow (CBF), cortical tissue hemoglobin oxygen saturation (HbSo2), and neuronal damage. METHODS: Fifteen minutes of global cerebral ischemia was induced by two-vessel occlusion and hypobaric hypotension. Wistar rats (n = 25) were randomized to receive vehicle (n = 9) or 40 mg/kg U-101033E (n = 9) intraperitoneally during 2 hours of reperfusion. A sham group (n = 7) had neither ischemia nor therapy. Histology was evaluated 7 days after ischemia. RESULTS: During late hyperperfusion (at 17 minutes), vehicle-treated animals had a higher (P = 0.044) cortical tissue HbSo2 (72.0 +/- 1.4%) than did U-101033E-treated animals (65.8 +/- 2.5%). Neuronal counts in the superficial cortex layer found after 7 days correlated negatively with rCBF (r = -0.76; P < 0.001) or cortical tissue HbSo2 (r = -0.56; P = 0.028) assessed during the late hyperperfusion phase. U-101033E reduced neuronal damage in hippocampal CA1 from 64.3 +/- 9.2% to 31.2 +/- 8.4% (P = 0.020), as well as in the superficial cortical layer from 53.5 +/- 14.6% to 12.8 +/- 11.7% (P = 0.046). While animals in the vehicle group had reduced counts in all four examined cortex layers (P < 0.05 versus sham group), there was significant cortical neuron loss in the U-101033E group in only one of four areas. U-101033E had no effect on resting CBF or CO2 reactivity. CONCLUSIONS: Postischemic application of U-101033E protects hippocampal CA1 and cortical neurons after 15 minutes of global cerebral ischemia. The results indicate that free radical-induced lipid peroxidation contributes to reperfusion injury, a process that can be inhibited by antioxidants such as U-101033E.     

Diaphragmatic lipid peroxidation in chronically loaded rats

Since the functional outcome of effector T lymphocytes depends on a balance between activatory and inhibitory receptors, we studied the ability of CTLA-4 (CD152) to inhibit the cytolytic function of CTL. In 22 TCR alpha/beta+ CD3+ 8+ CTL clones, activation induced by anti-CD3, anti-CD28, or anti-CD2 mAb was inhibited by anti-CD152 mAb in a redirected killing assay. In eight clones inhibition was >40%, in 10 it ranged between 20-40%, and in four it was <20%. This suggests the existence of a clonal heterogeneity as well as for the ability of CTLA-4 to inhibit CD3/TCR-, CD28-, or CD2-mediated CTL activation. To support further this contention, we used an experimental model based upon Ag-specific CTL. Eight Ag-specific T cell clones that lyse autologous EBV-infected B lymphocytes, but are unable to lyse allogeneic EBV-infected B cell lines, were used in a cytolytic assay in which anti-CD152 mAb or soluble recombinant receptor (i.e., CTLA-4 Ig) were included. In this system, at variance from the redirected killing assay, cross-linking of surface molecules by mAb does not occur. Thus, addition of anti-CD152 mAb or of CTLA-4 Ig and anti-CD80/CD86 mAb to the assay should result in a blockade of receptor/ligand interactions. As a consequence, inhibition of a negative signal, such as that delivered via CD152, should enhance lysis. A >40% increment of target cell lysis was achieved in three of eight clones studied. Since it is not equally shared by all CTL clones, this feature also appears to be clonally distributed.     

Dehydroepiandrosterone-induced lipid peroxidation in rat liver mitochondria

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

The effect of exercise intensity on lipid peroxidation

This study characterizes exercise-induced lipid peroxidation during graded aerobic exercise in seven healthy men and women (36.4 +/- 3 yr). Levels of ethane and pentane in expired breath during cardiopulmonary exercise stress testing were measured at rest, lactic acidosis threshold (LAT), maximal exercise (VO2max), and recovery. Serum malonaldehyde (MDA) levels were measured at rest before exercise and 5 min after maximal exercise. Expired ethane and pentane flux levels were increased above resting levels at LAT, continued to rise at VO2max, then declined during recovery. Serum MDA levels were not significantly different before and after maximal exercise. Substantial exercise-induced lipid peroxidation (by expired ethane and pentane) apparently occurred in healthy individuals at LAT and continued to increase at VO2max, yet rapidly attenuated during post-exercise recovery. These findings indicate that in healthy individuals physical exercise induced lipid peroxidation transiently and that there was a removal of lipid peroxidation byproducts during recovery.     

Role of lipid peroxidation in dapsone-induced hemolytic anemia

Dapsone hydroxylamine (DDS-NOH) is a direct-acting hemolytic agent responsible for dapsone-induced hemolytic anemia in the rat. The hemolytic activity of DDS-NOH is associated with the formation of disulfide-linked hemoglobin adducts on membrane skeletal proteins. We have postulated that this membrane protein "damage" is a consequence of DDS-NOH-induced oxidative stress within the red cell and that it serves as the trigger for premature removal of injured but intact red cells from the circulation by splenic macrophages. Oxidative stress has also been associated with the induction of lipid peroxidation, and it is possible that direct damage to the lipoidal membrane may play a role in the premature sequestration of the damaged cells in the spleen. To investigate this possibility, rat and human red cells were incubated with hemolytic concentrations of DDS-NOH and examined for evidence of lipid peroxidation using two independent assays: thiobarbituric acid-reactive substances formation and cis-paranaric acid degradation. Phenylhydrazine, which is known to induce lipid peroxidation in red cells, was used as a positive control. The extent of thiobarbituric acid-reactive substances formation and cis-paranaric acid degradation in DDS-NOH-treated rat and human red cells was not significantly different from that in control cells. In contrast, thiobarbituric acid-reactive substances formation and cis-paranaric acid degradation were significantly increased in red cells treated with hemolytic concentrations of the positive control, phenylhydrazine. These data suggest that lipid peroxidation is not involved in the mechanism underlying dapsone-induced hemolytic anemia.     

The role of tocopherols in biomembrane lipid peroxidation

The rate constants of elementary reactions of oxidation inhibition by tocopherols of different structures (peroxy radical destruction by antioxidant molecules, interaction of inhibitor radicals with one another and with oxidation substrate) are discussed. In contrast with most synthetic phenol antioxidants, tocopherols exhibit a high affinity to peroxy radicals (the rate constants for their reactions with these radicals are one or two orders of magnitude greater than those for reactions involving most synthetic phenols: they possess a fairly high stability of phenoxyl radicals formed and activity in the reaction of oxidation-chain transfer). The activity of tocopherol radicals in chain transfer reactions depends on their structure and manifests itself in a marked dependence of the extent of inhibition on oxidation conditions and the antioxidant concentration, up to an opposite effect. This can be used to control the oxidation rate: the radicals formed from tocopherols can serve as a buffer maintaining the oxidation rate at a certain level in systems with different antioxidant concentrations (concentration control) and with substrates of various unsaturation extent (substrate control). Tocopherols exhibit a unique set of properties which allow them to control the rate of lipid peroxidation and the physico-chemical properties of biological membranes. This dual effect provides the feedback relations and thereby maintains homeostasis in the organism.     

Crosslinking of apolipoprotein E by products of lipid peroxidation

Apolipoprotein E (APOE) genotype and advancing aging are interacting ri sk factors in the expression of late onset and sporadic Alzheimer's Disease (AD). We tested the hypothesis that 2 products of lipid peroxidation, malondialdehyde (MDA) and 4 hydroxy-2-nonenal (HNE), covalently modify APOE and alter its metabolism. In vitro, both HNE and MDA crosslinked purified APOE3 and APOE4. HNE was a more potent crosslinker than MDA, and purified APO3 was more susceptible to crosslinking by HNE than was purified APOE4. In P19 neuroglial cultures, oxidative stress with lipid peroxidation led to increased intracellular accumulation of anti-HNE and anti-APOE immunoreactive proteins of approximately 50 kDa. Intercellular accumulation of the 50 kDa APOE-immunoreactive protein (APOE-50) was not prevented by cyclohexamide, suggesting formation by post-translational mechanisms. In CSF, a 50 kDa APOE-immunoreactive protein co-migrated with proteins most immunoreactive for HNE and MDA adducts, containing NaB3H4-reducible bonds. These proteins were in CSF from adult subjects (with or without dementia), and in AD patients homozygous for APOE3 or APOE4 alleles. These data suggest that HNE covalently crosslinks APOE in P19 neuroglial cultures to form a 50 kDa protein, and that similar modifications of APOE appear to occur in vivo.     

Ischemic tolerance and lipid peroxidation in the brain

Using a modified method of focal ischemic preconditioning (FIP) followed by permanent middle cerebral artery occlusion (MCAO), we studied the effects of ischemic tolerance on infarct volume. The FIP was achieved by obstructing the blood flow into the MCA using miniature clamps at two points simultaneously. The first point was proximal to its origin and the second was where it intersects the middle cerebral vein. In rats subjected to three short conditioning periods (3 x 3 min with 7 min reperfusion between occlusions), there were significant reductions in the infarct volume in the cerebral cortex induced by the subsequent MCAO in FIP-treated rats compared with sham-operated controls. This rapid FIP-induced ischemic tolerance to subsequent MCAO occurred within 1 day and lasted for 5 days. Tissue lipid peroxidation in the cortex was significantly reduced for up to 2 days after FIP. In contrast, lipid peroxidation increased rapidly after MCAO. A significant reduction in this increase was observed in FIP-treated rats, suggesting a correlation with the subsequent reduction in the infarct volume.     

Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation

The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-azobis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolox. When 5-/6-CF free in solution is incubated with red blood cells exposed to cumene hydroperoxide (CH), a similar loss of fluorescence occurs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluorescein (C11-fluor), a lipophilic fluorescein conjugate, has been incorporated into the membranes of RBC. Upon addition of CH, a decrease in fluorescence is fluorometrically observed that is proportional to the amount of hydroperoxide added and inhibited by preincubation with Trolox or vitamin E. Flow-cytometric studies are then performed to demonstrate that C11-fluor can monitor free radicals generated during lipid peroxidation on a cell-by-cell basis. When exposed to CH, a time-dependent shift of the flow-cytometric profile toward lower values is observed that is inhibited by Trolox or vitamin E. This approach in conjunction with multiparametric flow cytometry may allow examination of the biologic significance of lipid peroxidation by correlation to other cellular end points on single cells.