Increase In Lipid Hydroperoxides Research Articles

Susceptibility of VLDL to oxidation in patients on regular haemodialysis

Oxidation of VLDL in vitro increases macrophage uptake and promotes foam cell formation, and the dyslipidaemia of chronic renal failure is characterised by an increase in VLDL. However, little information is available with regard to the susceptibility of VLDL to oxidation in patients at increased risk of atherosclerosis. We have therefore assessed the composition and susceptibility to oxidation of VLDL from haemodialysis patients and control subjects. VLDL from haemodialysis patients contained increased lipid hydroperoxides (81.6±12.6 versus 16.1±3.4 nmol/mg protein, P<0.001) and malondialdehyde (35.9±7.3 versus 16.0±4.1 nmol/mg protein, P<0.05). Susceptibility to oxidation was increased as shown by an increased rate of propagation of the copper induced lipid peroxidation chain-reaction (11.6±1.5×10 −5 versus 7.6±1.1×10 −5abs. U/min, P<0.05) and a greater increase in conjugated diene formation during peroxidation (0.47±0.04 versus 0.25±0.03 abs. U, P<0.001). Increased VLDL peroxidation in dialysis patients may contribute to the increased risk of cardiovascular disease observed in this group of patients.

Peroxidative changes associated with chilling injury in soybean seeds during imbibition

Summary The relationship between lipid peroxidation and fatty acid unsaturation was investigated during imbibitional chilling injury in soybean seeds. Transient changes in lipid hydroperoxide levels were observed in both chilled and non-chilled seeds and axes. However in axes the increase in lipid hydroperoxides after 12 hours of imbibition at chilling temperatures was associated with small but non-significant declines in the levels of linoleic acid, and linolenic acids (9 % and 8 % respectively) of the total lipid fraction. Increases in lipid hydroperoxides during the chilling of whole seeds and embryonic axes preceded ion leakage by some 40 hours. In an attempt to separate temperature effects from the imbibition process, seeds and axes were imbibed for 12 hours at 5 °C or 25 °C before transfer to 25 °C or 5 °C, respectively. Seeds showed no significant changes in fatty acid levels, although axes showed marked changes in the levels of linoleic acid. The capacity of soybean seeds to control lipid peroxidation may be an important component in chilling injury and lead to a loss of unsaturated fatty acids.

Increase of Lipid Hydroperoxides in Tissues of Vitamin E-Deficient Rats

The level of lipid hydroperoxides was determined by a newly developed method in rat tissues of vitamin E deficiency, which was a good in viuo model of enhanced radical reactions. In the heart, lung and kidney, the level of lipid hydroperoxides increased significantly as early as 4 weeks after feeding on a tocopherol-deficient diet compared with that of the control group. After 8 weeks of the deficiency, similar results were obtained. These results indicate that the lipid hydroperoxide is available as an extremely sensitive indicator of lipid peroxidation in these organs, because it takes several months to detect manifestations of the vitamin deficiency based on conventional indices.

Effects of UV-Sensitization of Hematoporphyrin on Lipid Hydroperoxides in Erythrocytes and on Their Hemolysis.

We have already reported the effect of ultraviolet-A (UVA)-sensitization of hematoporphyrin (HP) on the production of lipid hydroperoxides in erythrocytes and on their hemolysis. In this report, we investigated these effects under ultraviolet-B (UVB) irradiation and compared the results with those under UVA irradiation. It was found that an increase in lipid hydroperoxide preceded hemolysis, and that the UVB irradiation resulted in an increase in phosphatidylethanolamine hydroperoxide (PEOOH) and 2-thiobarbituric acid reactive substances (TBA-RS) at an earlier stage in comparison with UVA irradiation. Under either UVA or UVB irradiation, hemolysis was inhibited by anti-oxidants such as sodium azide and ascorbic acid (singlet oxygen scavengers), but not by mannitol, sorbitol (hydroxyl radical scavengers) or superoxide disumutase (SOD) (superoxide radical scavenger). These results suggest that singlet oxygen (1O2) produced by UV irradiation peroxidized the lipids of erythrocyte membranes, and therefore, the hemolysis of erythrocytes occured when the amount of hydroperoxides increased to a constant level, but auto-oxidation of lipids did not affect the hemolysis.

Linoleic acid activates nuclear transcription factor-κB (NF-κB) and induces NF-κB-dependent transcription in cultured endothelial cells

High dietary intakes of unsaturated fats may be atherogenic by disrupting normal functions of the vascular endothelium, due in part to the ability of linoleic acid (18:2n-6) to contribute to an increase in cellular oxidative stress and related injurious events. Exposing endothelial cells to 90 micromol linoleic acid/L for 6 h resulted in a significant increase in lipid hydroperoxides that coincided wih an increase in intracellular calcium concentrations. Treatment with this fatty acid caused an initial decrease in glutathione concentrations, which was followed by an increase at later time points. Most importantly, a significant activation of the oxidative stress-sensitive nuclear transcription factor-kappa B (NF-kappa B) was achieved after a 6-h exposure to 18:2n-6, which is the time point at which maximal depletion of cellular glutathione was observed. The fatty acid-mediated NF-kappa B activation was accompanied by induction of NF-kappa B-dependent transcription, as measured by chloramphenicol acetyltransferase (CAT) assay of an NF-kappa B-responsive promoter construct. Pretreatment of endothelial cells with vitamin E and N-acetyl cysteine inhibited the fatty acid-induced activation of NF-kappa B and formation of lipid hydroperoxides. These data suggest that oxidative stress-induced cellular changes are critical early events in fatty acid-mediated endothelial cell dysfunction.

Change in the Level of Vitamin C and Lipid Peroxidation in Tissues of the Inherently Scorbutic Rat during Ascorbate Deficiency

To investigate an accurate profile of vitamin C deficiency, ascorbate deficiency was caused in the inherently scorbutic rat [Osteogenic Disorder Shionogi (ODS)], and changes in the level of the vitamin in 12 tissues of the animals (plasma, liver, stomach, small and large intestines, lung, heart, kidney, adrenal gland, spleen, muscle, and brain) were followed based on the specific method (Kishida et al. Anal. Chem. 1992, 64, 1505-1507). The level of ascorbate in plasma decreased most rapidly, and the rate of decline of the vitamin was the slowest in the brain among the 12 tissues. Based on the kinetic profile of ascorbate decay, these tissues were classified into four groups. After 25 days of ascorbate deficiency, indicators of oxidative stress changed significantly compared with the control group. The indices included increased lipid hydroperoxide level determined by the specific method (Tokumaru et al. Anal. Chim. Acta 1995, 307, 97-102) in the brain, elevated thiobarbituric acid-reactive substances (TBARS) and glutathione peroxidase activity in the heart, and the fall of glutathione in plasma and the liver.

Role of glutathione redox cycle in TNF-α-mediated endothelial cell dysfunction

Modulation of the glutathione redox cycle may influence tumor necrosis factor-α (TNF)-mediated disturbances of endothelial integrity. To test this hypothesis, normal endothelial cells or cells with either increased or decreased glutathione levels were exposed to 100 ng (500 U) TNF/ml. Increased glutathione levels were achieved by exposure to 0.2 mM N-acetyl-L-cysteine (NAC) and decreased glutathione levels by exposure to 25 μM buthionine sulfoximine (BSO). Several components of the glutathione redox cycle as well as markers of endothelial integrity, such as cytoplasmic free calcium and transendothelial albumin transfer, were measured in the treated cells. Exposure to TNF for 3 and 6 h decreased total glutathione levels, which was followed by an increase at later time points. Moreover, treatment with TNF resulted in an increase in the ratio of oxidized to reduced glutathione, intracellular free calcium, albumin transfer across endothelial monolayers and lipid hydroperoxides. However, an increase in lipid hydroperoxides was seen only when endothelial cell cultures were supplemented with iron. BSO treatment increased susceptibility of endothelial cells to TNF-mediated metabolic disturbances. On the other hand, NAC partially protected against TNF-induced injury to endothelial monolayers. Our results demonstrate the important role of the glutathione redox cycle in TNF-mediated disturbances of the vascular endothelium and indicate that modulation of glutathione levels may potentiate the injurious effects of this inflammatory cytokine.

Lung damage in paraquat poisoning and hyperbaric oxygen exposure: Superoxide-mediated inhibition of phospholipase a 2

Paraquat poisoning and hyperbaric oxygen exposure are well established models of oxidative stress in lung. The aim of this study was focused on the contribution of oxygen free radicals and other cytotoxic species, such as lipid hydroperoxides, to the overall toxicity. Adult Wistar rats were injected with paraquat (30 or 60 mg/kg b.w.) or exposed to hyperbaric oxygen (0.2 MPa), and several parameters of lung damage were measured. Both treatments resulted in increased spontaneous lung chemiluminescence, number of lung PMN, malondialdehyde content, lung edema, and pleural liquid. Of note, spontaneous lung chemiluminescence, used to monitor the steady-state concentration of oxygen free radicals in vivo, did not increase significantly after either treatment. The increase in spontaneous lung chemiluminescence started after PMN migration, being both maxima separated by a delay time of 4–6 h. After PMN migrated and became activated in the lung, the survival of the animals started to decline. Thus, PMN can be considered as additional sources of oxygen free radicals supported by the subsequent increase in chemiluminescence. Their role in lung damage was evidenced by the increase in lung edema, augmented pleural liquid, and decreased survival after PMN migration. Lipid hydroperoxide concentration in lung membranes was also increased after either treatment. This increased concentration may be a consequence of an increased rate of lipid peroxidation, initiated by oxidative stress on lipid membranes, or by an inhibition of their catabolism. Ester lipid hydroperoxides normally produced in membranes cannot be catabolized directly by the glutathione peroxidase-reductase system unless phospholipase A 2 catalyses the release of free lipid hydroperoxides. In both experimental models, phospholipase A 2 activity was decreased to almost negligible values. Betamethasone (1 mg/ml, IV) administered to the rats 3 h before paraquat injection accelerated the decrease in survival and phospholipase A 2 inactivation. Inactivation of phospholipase A 2, detected in paraquat or oxygen exposed rats, could be attributed to a O 2 ·−-driven Fenton reaction. However, phospholipase A 2 inactivation by betamethasone pretreatment may be attributed to the presence of lipocortin, a corticosteroid-inducible factor and inhibitor of phospholipase A 2. Besides the mechanism underlying the inactivation of phospholipase A 2, the increase in lipid hydroperoxides may indicate their role as long-lived cytotoxic species that contribute to the damage already inititated by oxidative stress. Indeed, lipid hydroperoxides are very well known modifiers of membrane physical properties.

Differential detection of plasma hydroperoxides in sepsis

To determine whether plasma lipid hydroperoxides may be a useful marker for sepsis. Exploratory, open-label study. Critical care unit at a university medical center. Twelve patients with sepsis syndrome requiring hemodynamic monitoring with pulmonary artery catheters. Seven patients were diagnosed with pulmonary infections and five patients had intra-abdominal infections. Fatty acid hydroperoxide was measured in the fresh arterial plasma (radial artery) and mixed venous plasma (pulmonary artery) from each patient. Hydroperoxide was determined using a sensitive assay based on activating the cyclooxygenase reaction of prostaglandin H synthase. The mean difference between the amount of fatty acid hydroperoxide measured in the plasma draining involved regions (arterial plasma for pulmonary sepsis, mixed venous plasma for intra-abdominal sepsis) compared with the paired, uninvolved regions was 0.45 +/- 0.14 microM (mean +/- SEM; p less than .005). Increased lipid hydroperoxides in blood-draining septic foci are markers of oxyradical release associated with severe infection, although they are not specific for infectious conditions, being released also from nonseptic regions of surgical trauma. Assays for hydroperoxides may be useful when relatively free of other tissue trauma.

Lipid peroxidation and altered vascular function in vitamin E-deficient rats.

Female rats deprived of dietary vitamin E for 35 wk postweaning were analyzed for changes in vascular function. A functional state of vitamin E deficiency was indicated by a marked increase in spontaneous hemolysis of washed red cells by 22 wk of feeding. Elevated thiobarbituric acid-reactive material in aorta, liver, and plasma samples from vitamin-E deficient rats indicated increased lipid hydroperoxide formation. Systolic blood pressures and heart rates measured biweekly were unaltered by diet. Before being killed, the rats were catheterized and allowed to recover from anesthesia (methohexital sodium ip). The pressor response to graded doses of angiotensin II was significantly increased in the vitamin E-deficient group relative to its control. Isolated superior mesenteric artery segments from vitamin E-deficient rats demonstrated significantly decreased relaxation responses to acetylcholine. In contrast, artery contractile responses to 50 mM potassium and to graded doses of extracellular calcium did not differ, indicating that contractile capability was maintained. Surface blebbing of the femoral artery endothelium was observed by scanning electron microscopy. These data support a proposed link between lipid peroxidation and development of altered vascular function.

Decreased essential antioxidants and increased lipid hydroperoxides following high-dose radiochemotherapy.

The blood from 19 patients having bone marrow transplantation was examined for the essential antioxidants alpha-tocopherol and beta-carotene as well as lipid hydroperoxides before, at and after bone marrow transplantation (BMT). Conditioning therapy, preceding BMT in order to achieve marrow ablation and immunosuppression, consists of high-dose chemotherapy which is mostly combined with total body irradiation (TBI). In order to see a possible difference between patients with and without additional TBI, we divided the patients up into two groups; patients receiving TBI (RT+) and patients without TBI (RT-). All patients required total parenteral nutrition beginning one week prior to BMT. After conditioning therapy plasma levels of absolute and lipid-standardized alpha-tocopherol and beta-carotene decreased in both groups, presumably as a result of an enhanced breakdown of these antioxidants. The loss of these lipid-soluble antioxidants has to be considered as a possible cause for early post-transplant toxicity. Lipid hydroperoxides increase significantly in the group of patients with additional TBI, whereas the other group, receiving no additional TBI, showed no significant change. We suggest high-dose supplementation of essential antioxidants for patients undergoing BMT.

Effect of etoposide (VP16-213) on lipid peroxidation and antioxidant status in a high-dose radiochemotherapy regimen

A total of 13 patients receiving bone marrow transplants (BMT) for treatment of different haematological diseases were investigated. Conditioning therapy preceding BMT consisted of fractionated total-body irradiation (12 Gy) and high-dose chemotherapy with cyclophosphamide (2 +/- 60 mg/kg). Patients stratified to be at high risk for relapse (6/13) were additionally treated with etoposide (30 mg/kg). Plasma concentrations of absolute and lipid-standardized antioxidants (alpha-tocopherol and beta-carotene) decreased following conditioning therapy, presumably as the result of an enhanced breakdown of these antioxidants. Etoposide treatment did not amplify the loss of essential anti-oxidants but significantly increased lipid hydroperoxide concentrations in serum. We suggest that the abnormal generation of lipid hydroperoxides is the result of free radical formation.

Generation of toxic phospholipid(s) during oxyhemoglobin-induced peroxidation of phosphatidylcholines

When egg yolk diacylglycerophosphocholine (PC) liposomes were incubated with human oxyhemoglobin, peroxidation of liposomal lipid was induced, as monitored by an increase of thiobarbituric acid (TBA)-reactive substances, an increase of lipid hydroperoxides and the generation of chemiluminescence in the presence of luminol. During the reaction, cytotoxic substance(s), which induced shedding of acetylcholinesterase-enriched vesicles from human erythrocytes, were produced. Formation of TBA-reactive substances and lipid hydroperoxides preceded generation of chemiluminescence, conversion of oxyhemoglobin to methemoglobin and production of the toxic substances. Either Superoxide dismutase or catalase could suppress generation of chemiluminescence, but not other events. Methemoglobin or ferrous ion plus ascorbate could induce peroxidation of the liposomes without production of the cytotoxic substance(s). Synthetic PCs containing both saturated and polyunsaturated fatty acyl chains caused the production of cytotoxic products which induced shedding of vesicles from erythrocytes, whereas those containing only polyunsaturated fatty acyl chains did not, suggesting that the molecular species which can produce cytotoxic products may be phospholipids containing both saturated and polyunsaturated fatty acids. The mechanism of oxyhemoglobin-induced peroxidation of lipids will be also discussed.

Effect of lipid peroxidation on rhodopsin regeneration.

We have developed an in vitro system in which lipid peroxidation can be produced in a predictable fashion and have studied the effects of peroxidation on regenerability of rhodopsin in rod outer segments (ROS). ROS isolated by sucrose flotation from dark-adapted retinas of Rana pipiens were suspended in various concentrations of FeSO4 and 1 or 2 mM ascorbic acid for 10 minutes. An increase in lipid hydroperoxides (measured as conjugated dienes) and a decrease in 22:6 omega 3 were determined for each Fe+2 concentration and used as an index of peroxidation. Following incubation with and without FeSO4, ROS were pelleted and rhodopsin was regenerated with 15 micron 11-cis-retinal. The regenerability of rhodopsin in ROS in which significant lipid peroxidation had taken place was reduced by 40-50% compared to controls. DTPA (diethylenetriamine pentaacetic acid), EDTA, and EGTA protected against lipid peroxidation by Fe+2 and allowed almost complete regeneration of rhodopsin. Incubation with DTPA also prevented the destruction of vitamin E in ROS. Other compounds containing primary amine groups did not protect against peroxidation or loss of regenerability. These experiments indicate that lipid peroxidation is associated with a loss of regenerability of the visual pigment rhodopsin.

Copper increases superoxide dismutase activity in rat liver

Superoxide dismutase (SOD) activity in rat liver cytosol and submitochondrial fractions was characterized as enzymatic and nonenzymatic (due to the SOD-like activity of copper) by four approaches: (i) aerobic NBT 2+ (nitroblue tetrazolium) photoreduction in the absence of EDTA; (ii) aerobic NBT 2+ photoreduction in the presence of 10 −4 m EDTA; (iii) anaerobic NBT 2+ photoreduction; and (iv) o-dianisidine photooxidation. Under normal conditions nonenzymatic SOD activity has been observed only in the intermembrane space. The single subcutaneous injection of rats with CuSO 4 solution (5 mg Cu/kg body wt) led to (i) an elevation of the copper level in all submitochondrial fractions; (ii) an increase in enzymatic SOD activity in only cytosol and intermembrane spaces; (iii) the appearance of a new electrophoretic SOD activity band in the intermembrane space preparations; and (iv) the appearance of nonenzymatic SOD-like activity in the outer and inner mitochondrial membranes, and a twofold increase in lipid hydroperoxides. This suggests that the increased nonenzymatic copper in vivo has a prooxidant effect, and does not catalyze the dismutation of O 2 − as it has been shown in in vitro experiments [ E. M. Russanov S. G. Ljutakova, and S. I. Leutcher (1982) Arch. Biochem. Biophys. 215, 220–229 ]. The peculiarities of the SOD activity in the intermembrane space are explained by the lysosomal localization of the granular CuZnSOD.

Glutathione peroxidase activity and selenium concentration in the hearts of doxorubicin-treated rabbits

Doxorubicin-treated cardiotoxicity was studied in the rabbit. Rabbits given intravenous injections of 1.5 mg doxorubicin/kg body weight (3 times per week for 3 weeks) developed morphological and histological alterations in their hearts. In addition to these changes, both glutathione peroxidase activity and selenium concentration were significantly reduced in the hearts of the doxorubicin-treated rabbits. The decrease in glutathione peroxidase activity is probably related to the decrease in the concentration of selenium, since this enzyme requires selenium for activity and, furthermore, since in vitro studies failed to show any effect of doxorubicin on glutathione peroxidase. Although the mechanism responsible for the observed decrease in selenium is not known, an alteration in the selenium flux in the myocardial cell may account for the observation. The results of the present studies suggest that the observed increase in lipid hydroperoxides in the hearts of doxorubicin-treated animals may be the result of a decrease in glutathione peroxidase activity.