Aging increases Nepsilon-(carboxymethyl)lysine and caloric restriction decreases Nepsilon-(carboxyethyl)lysine and Nepsilon-(malondialdehyde)lysine in rat heart mitochondrial proteins

The present investigation studies the effect of aging, short-term and long-term caloric restriction on four different markers of oxidative, glycoxidative or lipoxidative damage to heart mitochondrial proteins: protein carbonyls (measured by ELISA); N epsilon -(carboxyethyl)lysine (CEL), N epsilon -(carboxymethyl)lysine (CML), and N epsilon -(malondialdehyde)lysine (MDA-lys) measured by gas chromatography/mass spectrometry. Aging increased the steady state level of CML in rat heart mitochondria without changing the levels of the other three markers of protein damage. Short-term caloric restriction (six weeks) did not change any of the parameters measured. However, long-term (one year) caloric restriction decreased CEL and MDA-lys in heart mitochondria and did not change protein carbonyls and CML levels. The decrease in MDA-lys was not due to changes in the sensitivity of mitochondrial lipids to peroxidation since the measurements of the fatty acid composition showed that the total number of fatty acid double bonds was not changed by caloric restriction. The decrease in CEL and MDA-lys in caloric restriction agrees with the previously and consistently described finding that caloric restriction agrees with the previously and consistently described finding that caloric restriction lowers the rate of generation of reactive oxygen species (ROS) in rodent heart mitochondria, although in the case of CEL a caloric restriction-induced lowering of glycaemia can also be involved. The CEL and MDA-lys results support the notion that caloric restriction decreases oxidative stress-derived damage to heart mitochondrial proteins.     

Increase in oxidative damage to lipids and proteins in skeletal muscle of uremic patients

Muscle weakness and reduced exercise capacity are frequent complaints of patients with chronic uremia. Several lines of evidence have suggested that chronic uremia result in a state of increased oxidative stress. Reactive oxygen species (ROS) and free radicals are capable of damaging lipids and proteins but it remains unclear whether oxidative damage plays a role in the skeletal myopathy commonly seen in chronic uremia. In this cross-sectional study, we compared the levels of oxidative damage to proteins and lipids of skeletal muscle from 40 chronic uremic patients and 20 age- and sex-matched healthy subjects. Protein carbonyls were determined by a spectrophotometric method to assess the oxidative damage to proteins. Our results showed that the mean content of protein carbonyls in skeletal muscles was significantly elevated in the hemodialysis patients ( 3.78 ±0.14 nmol of 2,4-dinitrophenyl-hydrazone per mg of protein) as compared to healthy controls (2.97 ±0.28 nmol per mg of protein, p =0.017 vs normal controls). In addition, we found that the mean malondialdehyde (MDA) level was also significantly increased in the uremic patients compared to healthy controls. Further analysis revealed that there was an age-dependent increase in both oxidative damages in these patients. Regression analysis between plasma protein carbonyl and MDA levels showed a significant correlation between these two parameters ( r =0.43, p =0.002). The finding of increased oxidative damage to protein and lipids provide support that oxidative damage may play a role in the pathogenesis of skeletal myopathy in chronic uremic patients on hemodialysis.     

Species differences in membrane susceptibility to lipid peroxidation

The susceptibility of liver microsomes to lipid peroxidation was evaluated in seven species: rat, rabbit, trout, mouse, pig, cow, and horse. Lipid peroxidation was measured as thiobarbituric acid reactive substances formed in the presence of either FeCl₃-ADP/ascorbate or FeCl₂/H₂O₂ initiating systems. For rat, rabbit, and trout microsomes, the order of susceptibility to peroxidation was rat > rabbit >> trout. The lack of peroxidation in trout microsomes could be explained by high microsomal vitamin E levels. Membrane fatty acid levels differed between species. Docosahexaenoic acid predominated in the trout, arachidonic acid in the rat, and linoleic acid in the rabbit. The contribution of individual fatty acids to lipid peroxidation reflected the degree of unsaturation with docosahexaenoic > arachidonic >>> linoleic. For all species except trout, the predicted susceptibility to peroxidation, based on the response of individual fatty acids, agreed well with directly measured microsomal peroxidation. With the exception of the trout, vitamin E content ranged from 0.083–0.311 nmol/mg microsomal protein between species, and low levels did not influence susceptibility to peroxidation. Trout microsomes peroxidized only after vitamin E depletion by prolonged incubation. The data indicate that below a vitamin E threshold, species differences in membrane susceptibility to peroxidation can be reasonably predicted based only on content of individual peroxidizable fatty acids.     

Identification of the several new radicals formed in the reaction mixture of oxidized linoleic acid with ferrous ions using HPLC-ESR and HPLC-ESR-MS

ESR, HPLC-ESR and HPLC-ESR-MS analyses were performed for the reaction mixtures of oxidized linoleic acid with ferrous ions combined use of spin trapping technique. More than 14 peaks were detected on the HPLC-ESR elution profile. In addition to 7-carboxyheptyl and pentyl radicals, several new radicals such as 7-carboxy1dihydroxyheptyl, 1,5-dihydroxypentyl, 8-carboxy-1-hydroxyoctyl, 7-carboxy-1-hydroxyheptyl, 1-hydroxypentyl and 1-hydroxyhexyl were identified using HPLC-ESR and HPLC-ESR-MS.     

Angeli's salt induces neurotoxicity in dopaminergic neurons in vivo and in vitro

In this study, we investigated the hypothesis that the pro-oxidative properties of Angeli's salt (AS), a nitroxyl anion (HNO/NO -) releasing compound, cause neurotoxicity in dopaminergic neurons. The pro-oxidative properties were demonstrated in vitro by measuring hydroxylation products of salicylate and peroxidation of lipids under various redox conditions. AS (0-1000 μM) released high amounts of hydroxylating species in a concentration dependent manner. AS also increased lipid peroxidation in brain homogenates at concentrations below 100 μM, while inhibiting it at 1000 μM concentration. The AS induced pro-oxidative effects were completely suppressed by copper (II), which converts nitroxyl anion to nitric oxide, as well as by a potent nitroxyl anion scavenger glutathione. Neurotoxicity towards dopaminergic neurons was tested in rat nigrostriatal dopaminergic system in vivo and by using primary mesencephalic dopaminergic neuronal cultures in vitro . Intranigral infusion of AS (0-400 nmol) caused neurotoxicity reflected as a dose dependent decrease of striatal dopamine seven days after treatment. The effect of the 100 nmol dose was more pronounced when measured 50 days after the infusion. Neurotoxicity was also confirmed as a decrease of tyrosine hydroxylase positive neurons in the substantia nigra. Neither sulphononoate, a close structural analog of AS, nor sodiumnitrite caused changes in striatal dopamine, thus reflecting lack of neurotoxicity. In primary dopaminergic neuronal cultures AS reduced [ 3 H] dopamine uptake with concentrations over 200 μM confirming neurotoxicity. In line with the quite low efficacy to increase lipid peroxidation in vitro , infusion of AS into substantia nigra did not cause increased formation of fluorescent products of lipid peroxidation. These results support the hypothesis that AS derived species oxidize critical thiol groups, rather than membrane lipids, potentially leading to protein oxidation/dysfunction and demonstrated neurotoxicity. These findings may have pathophysiological relevance in case of excess formation of nitroxyl anion.     

Effect of docosahexaenoic acid and ascorbate on peroxidation of retinal membranes of ODS rats

Mutant male osteogenic disorder Shionogi (ODS) rats, unable to synthesize ascorbic acid, were fed diets containing a high content of docosahexaenoic acid (DHA) and different amounts of ascorbic acid, to study the effect of DHA on peroxidative susceptibility of the retina and possible antioxidant action of ascorbic acid. ODS rats were fed from 7 weeks of age with diets containing high DHA (6.4% of total energy). A control group received a diet high in linoleic acid. The diets also contained varying amounts of ascorbic acid. Fatty acid compositions and phospholipid hydroperoxides in rod outer segment (ROS) membranes, and retinal ascorbic acid were analyzed. DHA in ROS membranes was significantly increased in rats fed high DHA, compared with the linoleic acid diet. Levels of phospholipid hydroperoxides in the DHA-fed rats were significantly higher than the linoleic acid-fed rats. Ascorbic acid supplementation did not suppress the phospholipid hydroperoxide levels after a high DHA diet, even when the supplement increased the content of retinal ascorbic acid. In conclusion, high DHA feeding induced a marked increase of phospholipid hydroperoxides in ROS membranes of ODS rats. Supplementation of ascorbic acid did not reverse this increase.     

Laser Mediated Production of Reactive Oxygen and Nitrogen Species; Implications for Therapy

Laser therapy has gained wide acceptance applications to many medical disciplines. The side effect-effects from laser therapy involve the potential for interaction with cellular and extracellular matrix molecules to generate reactive oxygen species and reactive nitrogen species which in turn can initiate lipid peroxidation, protein damage or DNA modification. These issues are addressed in this short overview in the context of experimental models of laser-induced thrombosis.     

Laser mediated production of reactive oxygen and nitrogen species; implications for therapy

Laser therapy has gained wide acceptance applications to many medical disciplines. The side effect-effects from laser therapy involve the potential for interaction with cellular and extracellular matrix molecules to generate reactive oxygen species and reactive nitrogen species which in turn can initiate lipid peroxidation, protein damage or DNA modification. These issues are addressed in this short overview in the context of experimental models of laser-induced thrombosis.     

The isoprostanes: Novel prostaglandin-like products of the free radical-catalyzed peroxidation of arachidonic acid

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo from the free radical-catalyzed peroxidation of arachidonic acid. This review summarizes our current knowledge regarding these compounds. Novel aspects of the biochemistry and bioactivity of IsoPs are detailed and methods by which these compounds are analyzed are discussed. A considerable portion of this review deals with the utility of measuring IsoPs as markers of oxidant injury in human diseases particularly in association with risk factors that predispose to atherosclerosis, a condition in which excessive oxidative stress has been causally implicated.     

Lipid Peroxidation of Lung Surfactant due to Reactive Oxygen Species Released from Phagocytes Stimulated by Bacteria from Children with Cystic Fibrosis

We used Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia, live or heat-killed, isolated from the airways of children with Cystic Fibrosis, to stimulate human neutrophils (PMN) and rat alveolar macrophages (AM) to produce reactive oxygen metabolites in the presence or absence of Curosurf, a natural porcine lung surfactant. We determined: (1) the amount of lipid peroxidation (LPO) as assessed by the amounts of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE) using the LPO 586 test kit; (2) the production by AM of superoxide with the nitroblue tetrazolium test and (3) of nitric oxide (NO) with the Griess reaction. Stimulation of PMN or AM increases LPO of Curosurf and cell wall lipids. In both types of phagocytes, B. cepacia induced the highest LPO levels followed by P. aeruginosa and S. maltophilia. PMN, stimulated by live bacteria, induced higher LPO than those stimulated by heat-killed bacteria. B. cepacia stimulated AM to produce more superoxide and NO than did P. aeruginosa and S. maltophilia. The high phagocyte-stimulating ability of B. cepacia and its higher surfactant LPO than those of the other bacteria used in this in vitro study may play a role in vivo in the serious clinical condition known as the "Cepacia syndrome".     

Lipid peroxidation of lung surfactant due to reactive oxygen species released from phagocytes stimulated by bacteria from children with cystic fibrosis

We used Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia, live or heat-killed, isolated from the airways of children with Cystic Fibrosis, to stimulate human neutrophils (PMN) and rat alveolar macrophages (AM) to produce reactive oxygen metabolites in the presence or absence of Curosurf, a natural porcine lung surfactant. We determined: (1) the amount of lipid peroxidation (LPO) as assessed by the amounts of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE) using the LPO 586 test kit; (2) the production by AM of superoxide with the nitroblue tetrazolium test and (3) of nitric oxide (NO) with the Griess reaction. Stimulation of PMN or AM increases LPO of Curosurf and cell wall lipids. In both types of phagocytes, B. cepacia induced the highest LPO levels followed by P. aeruginosa and S. maltophilia. PMN, stimulated by live bacteria, induced higher LPO than those stimulated by heat-killed bacteria. B. cepacia stimulated AM to produce more superoxide and NO than did P. aeruginosa and S. maltophilia. The high phagocyte-stimulating ability of B. cepacia and its higher surfactant LPO than those of the other bacteria used in this in vitro study may play a role in vivo in the serious clinical condition known as the "Cepacia syndrome".     

Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate''s protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.     

Antioxidant Action of 2,2,4,6-Tetra-Substituted 2,3-Dihydro-5-hydroxybenzofuran Against Lipid Peroxidation: Effects of Substituents and Side Chain

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di- tert -butylbenzofuran (BO-653) was designed, synthesized and has been evaluated as a novel antiatherogenic drug. In order to further understand the action of BO-653 and also radical-scavenging antioxidants in general, the dynamics of inhibition of oxidation by BO-653 were compared with those of the related compounds, 2,3-dihydro-5-hydroxy-2,2-dimethyl-4,6-di- tert -butylbenzofuran (BOB), 2,3-dihydro-5-hydroxy-2,2,4,6-tetramethylbenzofuran (BOM), &#102 -tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC), aiming specifically at elucidating the effects of substituents and side chain length of the phenolic antioxidants. These five antioxidants exerted substantially the same reactivities toward radicals and antioxidant capacities against lipid peroxidation in organic solution. When compared with di-methyl side chains, the di-pentyl side chains of BO-653 reduced its inter-membrane mobility but exerted less significant effect than the phytyl side chain of &#102 -tocopherol on the efficacy of radical scavenging within the membranes. Di- tert -butyl groups at both ortho-positions made BO-653 and BOB more lipophilic than di-methyl substituents and reduced markedly the reactivity toward Cu(II) and also the synergistic interaction with ascorbate. The results of the present study together with those of the previous work on the effect of substituents on the stabilities of aryloxyl radicals suggest that tert -butyl group is more favorable than methyl group as the substituent at the ortho-positions and that di-pentyl side chains may be superior to a phytyl side chain.     

The modulating effects of tumor necrosis factor alpha antibody on ricin-induced oxidative stress in mice

Previous studies in our laboratory have shown that the protein toxin ricin induces an oxidative stress in mice, resulting in increased urinary excretion of malondialdehyde (MDA), formaldehyde (FA), and acetone (ACON). Other toxicants have been shown to induce oxidative stress by macrophage activation with subsequent release of reactive oxygen species and tumor necrosis factor alpha (TNF-α). Therefore, the ability of TNF-α antibody to modulate ricin-induced urinary carbonyl excretion as well as hepatic lipid peroxidation, glutathione depletion, and DNA single-strand breaks was assessed. Ricin-induced urinary MDA, FA, and ACON were reduced significantly in mice receiving antibody (15,000 U/kg) 2 hours before treatment with ricin (5 μ/kg). At 48 hours following ricin treatment, MDA, FA, and ACON concentrations in the urine of TNF antibody-treated mice decreased 25.7, 53.2, and 64.5%, respectively, relative to ricin-treated mice receiving no antibody. In addition, anti-TNF-α (1500 U/kg) significantly decreased hepatic lipid peroxidation and DNA single-strand breaks, induced by 5 μg ricin/kg, by 49.3 and 44.2%, respectively. The results suggest that macrophage activation and subsequent release of TNF-α are involved in ricin toxicity.     

Serum and urine malondialdehyde levels in NIDDM patients with and without hyperlipidemia

Malondialdehyde (MDA), a marker of lipid peroxidation, was measured as thiobarbituric acid reactive substance (TBARS) in 78 noninsulin-dependent diabetic patients, 38 hyperlipidemic patients, and 28 healthy subjects. Diabetic patients were divided into groups and subgroups according to the existence of hyperlipidemia and other complications. Serum and urine MDA concentrations were significantly higher in diabetic and nondiabetic patient groups than in the control group. By contrast to urine MDA level, serum MDA level was significantly higher in hyperlipidemic diabetics than that of normolipidemic diabetics. Serum MDA levels in the hyperlipidemic diabetic group and urine MDA levels in both diabetic groups were significantly higher than those in hyperlipidemic nondiabetic group. In both diabetic groups, the existence of complications didn't affect serum and urine MDA levels. No correlation existed between serum and urine MDA levels in both patient groups and control subjects. This study confirmed the existence of lipid peroxidation disorders in diabetic patients.     

Structural Basis for Ligand Regulation of the Fatty Acid-binding Protein 5, Peroxisome Proliferator-activated Receptor β/δ (FABP5-PPARβ/δ) Signaling Pathway

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain “activating” fatty acids induce the protein''s cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5''s translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling.     

Effect of Reactive Oxygen Species on Myelin Membrane Proteins

Fresh myelin, isolated from brainstems of adult rats, was incubated in the presence of Cu2+ and H2O2. Electrophoretic analysis of the reisolated myelin membrane revealed a gradual loss of the protein moiety from the characteristic pattern and an increase in aggregated material appearing at the origin of the gel. The aggregation of proteins was time-dependent and was concomitant with the accumulation of lipid peroxidation products reactive with thiobarbituric acid. Furthermore, during the course of incubation, there was a gradual decrease in the amount of recovered light myelin and a quantitatively similar increase in heavier myelin subfractions. The aggregation of proteins seems not to be directly related to the buoyant densities of myelin fragments. The peroxidative damage to the myelin proteins may be an important contributor to pathochemistry of myelin sheath, in particular, and in general it implies the susceptibility of the protein moiety of cell membranes to oxygen-induced deterioration.