Oxidative Modification Of Lipids Research Articles

Ultrasound extraction and thin layer chromatography–flame ionization detection analysis of the lipid fraction in marine mucilage samples

This paper reports an analytical procedure based on ultrasound to extract lipids in marine mucilage samples. The experimental conditions of the ultrasound procedure (solvent and time) were identified by a FT-IR study performed on different standard samples of lipids and of a standard humic sample, before and after the sonication treatment. This study showed that diethyl ether was a more suitable solvent than methanol for the ultrasonic extraction of lipids from environmental samples because it allowed to minimize the possible oxidative modifications of lipids due to the acoustic cavitation phenomena. The optimized conditions were applied to the extraction of total lipid amount in marine mucilage samples and TLC–flame ionization detection analysis was used to identify the relevant lipid sub-fractions present in samples.

Lipophilic Antioxidants and Iron Status in ESRD Patients on Hemodialysis

Cardiovascular disease remains the major cause of mortality in hemodialysis patients. Abnormal oxidative stress and impaired antioxidant defense may contribute to accelerated atherogenesis associated with uremia. As oxidative modification of lipids appears to be a prerequisite for the development of atherosclerotic lesions, lipophilic antioxidants may be protective. The aim of this study was to determine the plasma levels of lipophilic antioxidants in 82 hemodialysis patients and 30 controls and to investigate the influence of body iron status on the levels of lipophilic antioxidants. The patients were categorized into 3 groups according to their serum ferritin levels. We found that the plasma levels of lycophene, δ-tocopherol, γ-tocopherol and retinol of hemodialysis patients were lower than those of controls. On the other hand, both absolute and lipid-normalized plasma lycophene levels were significantly reduced in those patients in the groups with higher ferritin levels as compared to those with lower ferritin levels. In addition, our study showed that the lipid-normalized plasma levels of β-carotene and α-carotene of hemodialysis patients with higher ferritin levels were lower than those of the patients with lower levels. These data suggest that the plasma levels of lipophilic antioxidants are altered in end-stage renal disease on hemodialysis and may be considered as markers of oxidative stress in these patients. Most importantly, elevated serum ferritin levels may affect the levels of these lipophilic antioxidants.

C-reactive protein, oxidative stress, homocysteine, and troponin as inflammatory and metabolic predictors of atherosclerosis in ESRD.

Mortality in patients with end-stage renal disease remains high, with cardiovascular disease accounting for half of these deaths. Novel risk factors such as inflammation, oxidative stress, hyperhomocysteinemia, and high troponin levels are associated with cardiovascular risk in the general population. While there are substantial epidemiologic data confirming that these novel risk factors are associated with cardiovascular risk in end-stage renal disease patients, a causal relationship has not been established. Inflammation is readily identified by the presence of high levels of C-reactive protein, while studies of oxidative stress are hampered by the lack of a standardized test. The cause of both is unknown. Hyperhomocysteinemia results from decreased remethylation to methionine, although vitamin supplementation only partially corrects the defect, suggesting that uremic inhibition of the enzymatic process may be important. The most promising strategies for correcting oxidative stress and hyperhomocysteinemia are vitamin E and folinic acid therapy, respectively. Troponin I appears to be a more specific marker of myocardial injury than Troponin T, but troponin T retains its ability to predict cardiovascular mortality as well as all-cause mortality. Sorting out the role of each of these risk factors may be difficult since the factors may influence each other, may increase oxidative stress, and may mediate atherosclerosis through oxidative modification of lipids.

Synthetically prepared Amadori-glycated phosphatidylethanolamine can trigger lipid peroxidation via free radical reactions

This study for the first time confirmed the peroxidative role of the Amadori product derived from the glycation of phosphatidylethanolamine (PE), namely Amadori–PE. The product was synthesized from the reaction of dioleoyl PE with D-glucose, and then purified by a solid-phase extraction procedure, which was a key step in the next HPLC technique for the isolation of essentially pure Amadori–PE. When the synthetically prepared Amadori–PE was incubated with linoleic acid in the presence of Fe 3+ in micellar system, a remarkable formation of thiobarbituric acid reactive substances was observed together with increases in lipid hydroperoxides. In addition, the lipid peroxidation caused by Amadori–PE was effectively inhibited by superoxide dismutase, mannitol, catalase and metal chelator. These results indicated that Amadori–PE triggers oxidative modification of lipids via the generation of superoxide, and implied the involvement of ‘lipid glycation’ along with membrane lipid peroxidation in the pathogenesis of diabetes and aging.

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1–6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4–6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (14C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (∼ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.

Oxidative modification of lipids and proteins in aniline-induced splenic toxicity.

Our earlier studies with aniline suggested the involvement of oxidative stress as an early toxic event in the spleen. In order to understand the status and consequences of the damaging oxidative reactions, especially during the progression of characteristic splenic lesions, time-dependent subchronic studies were conducted in rats. Male Sprague-Dawley rats were treated with 65 mg/kg/day aniline in the drinking water, while control rats received drinking water only. The animals were euthanized after 1, 2, or 3 months of aniline exposure. Total iron content was remarkably greater in the aniline-treated rats than in age-matched controls. There were time-dependent increases in splenic lipid peroxidation of aniline-treated rats. Malondialdehyde-protein adducts were quantitated by a competitive ELISA and showed greater concentrations in the spleens of aniline-treated rats, further substantiating our lipid peroxidation results. Protein oxidation in the spleens of aniline-treated rats was also greater, with a maximum increase of approximately 76% at 3 months. Western blot analysis for oxidized proteins showed two distinct protein bands at approximately 114 kD and approximately 69 kD in both post-nuclear and mitochondrial fractions of the spleens. Furthermore, densitometric analysis of the blot showed increased band intensities of the oxidized proteins in both these spleen fractions from aniline-treated rats, suggesting the susceptibility of these proteins to aniline-induced oxidative stress. The most prominent morphological changes in the spleens of aniline-treated rats included thickening of the capsule, and capsular cells with nuclear prominence and hyperchromia indicative of capsular hyperplasia. These capsular changes and fibrosis of capsule, splenic trabeculae, and red pulp were noted at all three time points after aniline exposure. Our studies thus suggest that aniline-induced oxidative stress in the spleen is an ongoing event that leads to oxidative modifications of biomolecules. Such oxidative modifications, directly or indirectly, could contribute to the splenic toxicity leading to deleterious consequences, including capsular hyperplasia and fibrosis, as observed in this study, and possibly tumorigenesis in chronic aniline exposure conditions.

Autoantibodies against Oxidatively Modified Low-Density Lipoprotein in Patients with Behçet’s Disease

Background: Change of lipids and lipoprotein metabolism and an imbalance of the oxidant-antioxidant system related to the disease activity have been reported in Behçet’s disease. Therefore, there is a tendency of oxidative modification of lipids and lipoproteins in patients with the disease. Objective: To investigate serum autoantibodies against oxidatively modified low-density lipoprotein (oxLDL) as a marker for the degree of in vivo oxidation of lipoproteins in Behçet’s disease. Methods: Serum autoantibodies against oxLDL, total cholesterol, triacylglycerol, HDL cholesterol, LDL cholesterol, apolipoprotein (Apo) AI, Apo B, α₁-antitrypsin, α₂-macroglobulin and erythrocyte sedimentation rate were determined in 37 patients and 30 sex- and age-matched healthy volunteers. Autoantibodies against oxLDL were measured by a commercial enzyme-linked immunosorbent assay. Results: Serum autoantibody levels against oxLDL were significantly higher in patients than in controls (425 ± 365 and 187 ± 132 mU/ml, respectively; p < 0.05). The levels of autoantibodies against oxLDL in the patients were found to correlate with total cholesterol, LDL cholesterol, HDL cholesterol and α₁-antitrypsin levels (r = 0.38, p < 0.05; r = 0.42, p < 0.05; r = –0.38, p < 0.05; r = 0.42, p < 0.05, respectively). Conclusion: It has been shown in previous studies that high autoantibody titers against oxLDL may be important in diseases with atherosclerosis as seen in systemic lupus erythematosus and rheumatoid arthritis. High autoantibody titers against oxLDL are not specific for Behçet’s disease but probably important for pathologic processes in the disease. We suggest that increased levels of autoantibodies against oxLDL may be a factor responsible for endothelial dysfunction and development of vascular pathology in Behçet’s disease.

γ-glutamyltransferase dependent generation of reactive oxygen species from a glutathione/transferrin system

In the presence of molecular oxygen and iron or copper ions, a number of antioxidants paradoxically generate reactive oxygen species (ROS) leading to free radical damage of nucleic acids and oxidative modification of lipids and proteins. The present work demonstrates that the combination of three components, which are often considered as part of an antioxidant protection system, can generate ROS. Purified human γ-glutamyltransferase (GGT) in the presence of 2 mM glutathione (GSH) and 80 μM transferrin, as an iron source, at pH 7.4 generates ROS, as measured by chemiluminescence of luminol. Initiated by the addition of purified GGT, generation of ROS reached a maximal rate in the first 6 min. Intensity of the chemiluminescence was only slightly enhanced by addition of 200 μM hydrogen peroxide. Generation of ROS was also investigated in transfected V79 cells expressing human GGT. In comparison with GGT negative V79 cells, only recombinant cells expressing a high level of GGT on the cell membrane were able to generate ROS. Generation of ROS in these cells reached a maximum within 2 min and was enhanced by 200 μM hydrogen peroxide. We further confirmed the hypothesis that cysteinylglycine (CysGly), a product of GGT/GSH reaction, identified by high-performance liquid chromatography, but not GSH, was responsible for ROS formation initiated by the reductive release of iron from transferrin. These data clearly indicate that under physiological conditions, GGT is directly involved in ROS generation.

Immunohistochemical localization of lipoperoxidation products in normal human placenta

4-Hydroxynonenal (4-HNE) is a major propagation product of lipid peroxidation that is supposed to be responsible for some of the effects associated with oxidative stress in tissues. We have investigated the possible occurrence and distribution of 4-HNE-immunoreactivity in human normal placenta using immunocytochemistry. Specific immunostaining was observed in cytotrophoblast cells, syncytiotrophoblast, some cells of the villous mesenchyme and some endothelial cells of first trimester and term placentae. The detection of 4-HNE-immunoreactivity in placenta raises the question whether lipoperoxidation products are produced locally in placental cells or represent exogenous products that derive from maternal blood flow. Since trophoblastic cells and villous macrophages are provided by a scavenger receptor, it is conceivable that these cells may play a protective role with regard to the diffusion of lipoperoxidation products from the mother to the embryo. However, since a significant degree of lipid oxidative modification does not take place in plasma, it is presumed that 4-HNE is a local product of placental metabolism. In line with this hypothesis, it is proposed that maternal low density lipoproteins, which are the major source of cholesterol for placental steroid synthesis, might be oxidized by villous cells during their traversal through the villous wall.

Regional lipid peroxidation and protein oxidation in rat brain after hyperbaric oxygen exposure

Reactive oxygen species may participate in development of neurological toxicity resulting from hyperbaric oxygen exposure. To explore the possibility that increased reactive O 2 metabolite generation may result in oxidative modification of lipids and proteins, rats were exposed to five atmospheres (gauge pressure) of O 2 until development of an electroencephalographic seizure. Lipid peroxidation (as thiobarbituric acid-reactive substances) and protein oxidation (as 2,4-dinitrophenyl-hydrazones) were measured in five brain regions. Oxidized and reduced glutathione were also determined because of their role in regulating lipid peroxidation. Lipid peroxidation was confined to the frontal cortex and hippocampus, while protein oxidation (in both cytoplasmic and membranous fractions) and increased oxidized glutathione was evident throughout the brain. These results support a role for formation of reactive O 2 metabolites from hyperbaric O 2 exposure and suggest that protein oxidation, especially in soluble proteins, may be one of the most sensitive measures.

Lipid advanced glycosylation: pathway for lipid oxidation in vivo.

To address potential mechanisms for oxidative modification of lipids in vivo, we investigated the possibility that phospholipids react directly with glucose to form advanced glycosylation end products (AGEs) that then initiate lipid oxidation. Phospholipid-linked AGEs formed readily in vitro, mimicking the absorbance, fluorescence, and immunochemical properties of AGEs that result from advanced glycosylation of proteins. Oxidation of unsaturated fatty acid residues, as assessed by reactive aldehyde formation, occurred at a rate that paralleled the rate of lipid advanced glycosylation. Aminoguanidine, an agent that prevents protein advanced glycosylation, inhibited both lipid advanced glycosylation and oxidative modification. Incubation of low density lipoprotein (LDL) with glucose produced AGE moieties that were attached to both the lipid and the apoprotein components. Oxidized LDL formed concomitantly with AGE-modified LDL. Of significance, AGE ELISA analysis of LDL specimens isolated from diabetic individuals revealed increased levels of both apoprotein- and lipid-linked AGEs when compared to specimens obtained from normal, nondiabetic controls. Circulating levels of oxidized LDL were elevated in diabetic patients and correlated significantly with lipid AGE levels. These data support the concept that AGE oxidation plays an important and perhaps primary role in initiating lipid oxidation in vivo.

Free radical generation by early glycation products: A mechanism for accelerated atherogenesis in diabetes

Non-enzymatic glycation of reactive amino groups in model proteins increased the rate of free radical production at physiologic pH by nearly fifty-fold over non-glycated protein. Superoxide generation was confirmed by electron paramagnetic resonance measurements with the spin-trap phenyl-t-butyl-nitrone. Both Schiff base and Amadori glycation products were found to generate free radicals in a ratio of 1:1.5. Free radicals generated by glycated protein increased peroxidation of membranes of linoleic/arachidonic acid vesicles nearly 2-fold over control, suggesting that the increased glycation of proteins in diabetes may accelerate vascular wall lipid oxidative modification.