Obituary: Alex Sevanian

Oxidative stress has been related to ageing and risk of death. To determine whether oxidative status was associated with all-cause risk of death we carried out a prospective study in 154 non-smoking Spanish elderly without major illness. Baseline glutathione peroxidase (GPx) and superoxide dismutase (SOD) were analysed in plasma and erythrocytes. α-tocopherol, β-carotene, lycopene and retinol were determined in serum samples and malondialdehyde (MDA), as a lipid peroxidation marker, in plasma. Mean survival time was 4.3 years. A total of 31 death cases (20.1%) occurred during the follow-up. Plasma-MDA predicted mortality independently of all other variables, while erythrocyte-SOD (e-SOD), β-carotene and α-tocopherol were positively associated with survival. α-tocopherol and MDA were revealed as independent predictors in a joint survival model, being the group with low MDA and high α-tocopherol that with the lowest mortality. In conclusion, a higher risk of death was associated with increased lipid peroxidation and lower antioxidant defenses.     

Antioxidant status and oxidative stress at rest and in response to acute exercise in judokas and sedentary men

It is well recognized that acute strenuous exercise is accompanied by an increase in free-radical production and subsequent oxidative stress, in addition to changes in blood antioxidant status. Chronic exercise provides protection against exercise-induced oxidative stress by upregulating endogenous antioxidant defense systems. Little is known regarding the protective effect afforded by judo exercise. Therefore, we determined antioxidant and oxidative stress biomarkers at rest and in response to acute exercise in 10 competitive judokas and 10 sedentary subjects after mixed exercise (anaerobic followed by aerobic). The subjects performed a Wingate test, followed by 30 minutes of aerobic exercise performed at 60% of maximal aerobic power. Blood samples were taken, by an intravenous catheter, at rest (R), immediately after the physical exercise (P0), and at 5 (P5), 10 (P10), and 20 (P20) minutes postexercise. The measured parameters included the activity of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and glutathione reductase, in addition to α-tocopherol, and total antioxidant status. Malondialdehyde was measured as a representation of lipid peroxidation. At rest, the judokas had higher values for all antioxidant and oxidative stress markers as compared to the sedentary subjects (p < 0.05). Plasma concentrations of all parameters except for α-tocopherol increased significantly above resting values for both the judokas and sedentary subjects (p < 0.05) and remained elevated at 20 minutes postexercise. A significant postexercise decrease was observed for α-tocopherol (p < 0.05) at P20 for judokas and at P5 for sedentary subjects. These data indicate that competitive judo athletes have higher endogenous antioxidant protection compared to sedentary subjects. However, both groups of subjects experience an increase in exercise-induced oxidative stress that is not different.     

Antioxidant effect of FeAOX-6 on free radical species produced during iron catalyzed breakdown of tert-butyl hydroperoxide

There is a body of evidences demonstrating, in biological systems, a cooperative interaction between tocopherols and carotenoids. FeAOX-6 is a novel antioxidant that combines the chroman head of α-tocopherol and a fragment of the isoprenyl chain of lycopene. We have tested its antioxidant effect on different radical species generated in a chemical system, where peroxyl, alkoxyl and methyl radicals are generated by the ferrous ion-mediated decomposition of tert-butyl hydroperoxide. We found that FeAOX-6 has the same effectiveness of α-tocopherol in quenching peroxyl radical with no contribution by lycopene. The antioxidant activity of FeAOX-6 on alkoxyl and methyl radicals is comparable to that of the equimolar mixture of the parent compounds. Lycopene is able to quench alkoxyl radical, while it has no effect on peroxyl radical, showing a different antioxidant activity compared to other carotenoids, such as β-carotene and lutein.     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediatedperoxidation of phosphatidyl choline liposomes

Abstract

The antioxidant efficacy of α-carotene and comparison with β-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2′-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS)at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of α- and β-carotenes, and the chain breaking antioxidant α-tocopherol were also included in the study.AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples.In this system, α-carotene retarded PCOOH formation better than β-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than α- and β-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, α-, β-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85%and 82%, respectively, while α-tocopherol elicited 90%reduction.AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P <0.001). α-Carotene significantly suppressed the TBARS formation by 78% whilst β-carotene, lutein, zeaxanthin and α-tocopherol elicited 60%, 91%and 80% reductions, respectively. Increasing the concentration of the carotenoid >1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack.Our findings suggest that α-carotene is a better antioxidant than is β-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids _{in vivo}.     

Exercise,free radicals,and lipid peroxidation in type 1 diabetes mellitus

Indirect biochemical techniques have solely been used to ascertain whether type 1 diabetes mellitus patients are more susceptible to resting and exercise-induced oxidative stress. To date there is no direct evidence to support the contention that type 1 diabetic patients have increased levels of free radical species. Thus, the aim of this study was to use electron spin resonance (ESR) spectroscopy in conjunction with alpha-phenyl-tert-butylnitrone (PBN) spin trapping to measure pre- and postexercise free radical concentration in the venous blood of young male patients with type 1 diabetes mellitus (HbA(1c) = 8.2 +/- 1%, n = 12) and healthy matched controls (HbA(1c) = 5.5 +/- 0.2%, n = 13). Supporting measures of lipid peroxidation (malondialdehyde and lipid hydroperoxides), ambient blood glucose and selected antioxidants were also measured. The diabetic patients presented with a comparatively greater concentration of free radicals as measured by ESR and lipid hydroperoxides (LH) compared to the healthy group (p <.05, pooled rest and exercise data), although there was no difference in malondialdehyde (MDA) concentration. alpha-Tocopherol was comparatively lower in the healthy group (p <.05, pooled rest and exercise data vs. diabetic group) due to a selective decrease during physical exercise (p <.05 vs. rest). The hyperfine coupling constants recorded from the ESR spectra (a(Nitrogen) = 1.37 mT and abeta(Hydrogen) = 0.17 mT) are suggestive of either oxygen or carbon-centered species and are consistent with literature values. We suggest that the greater concentration of oxidants seen in the diabetic group may be due to increased glucose autoxidation as a function of this pathology and/or a lower exercise-induced oxidation rate of the major lipid soluble antioxidant alpha-tocopherol. We suggest that the ESR-detected radicals are secondary species derived from decomposition of LH because these are the major initial reaction products of free radical attack on cell membranes.     

Electron paramagnetic spectroscopic evidence of exercise-induced free radical accumulation in human skeletal muscle

The present study determined if acute exercise increased free radical formation in human skeletal muscle. Vastus lateralis biopsies were obtained in a randomized balanced order from six males at rest and following single-leg knee extensor exercise performed for 2 min at 50% of maximal work rate (WR(MAX)) and 3 min at 100% WR(MAX). EPR spectroscopy revealed an exercise-induced increase in mitochondrial ubisemiquinone (UQ*-) [0.167 +/- 0.055 vs. rest: 0.106 +/- 0.047 arbitrary units (AU)/g total protein (TP), P < 0.05] and alpha-phenyl-tert-butylnitrone-adducts (112 +/- 41 vs. rest: 29 +/- 9 AU/mg tissue mass, P < 0.05). Intramuscular lipid hydroperoxides also increased (0.320 +/- 0.263 vs. rest: 0.148 +/- 0.071 nmol/mg TP, P < 0.05) despite an uptake of alpha-tocopherol, alpha-carotene and beta-carotene. There were no relationships between mitochondrial volume density and any biomarkers of oxidative stress. These findings provide the first direct evidence for intramuscular free radical accumulation and lipid peroxidation following acute exercise in humans.     

Detection of radicals produced by reaction of hydroperoxides with rat liver microsomal fractions.

EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals produced on reaction of a number of primary, secondary and lipid hydroperoxides with rat liver microsomal fractions in both the presence and absence of reducing equivalents. Two major mechanisms of radical generation have been elucidated. In the absence of NADPH or NADH, oxidative degradation of the hydroperoxide occurs to give initially a peroxyl radical which in the majority of cases can be detected as a spin adduct to DMPO; these radicals can undergo further reactions which result in the generation of alkoxyl and carbon-centered radicals. In the presence of NADPH (and to a lesser extent NADH) alkoxyl radicals are generated directly via reductive cleavage of the hydroperoxide. These alkoxyl radicals undergo further fragmentation and rearrangement reactions to give carbon-centered species which can be identified by trapping with DBNBS. The type of transformation that occurs is highly dependent on the structure of the alkoxyl radical with species arising from beta-scission, 1,2-hydrogen shifts and ring closure reactions being identified; these processes are in accord with previous chemical studies and are characteristic of alkoxyl radicals present in free solution. Studies using specific enzyme inhibitors and metal-ion chelators suggest that most of the radical generation occurs via a catalytic process involving haem proteins and in particular cytochrome P-450. An unusual species (an acyl radical) is observed with lipid hydroperoxides; this is believed to arise via a cage reaction after beta-scission of an initial alkoxyl radical.     

ESR study of a biological assay on whole blood: antioxidant efficiency of various vitamins

This study deals with the activity of various vitamins against the radical-mediated oxidative damage in human whole blood. We have used a biological method that allows both the evaluation of plasma and that of red blood cell resistance against the free radicals induced by 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH). Spin trapping measures using mainly 5-(diethoxyphosphoryl)-5-methyl-1-pyrolline N-oxide nitrone (DEPMPO) were carried out under several conditions to identify the free radicals implicated in this test. Only the oxygenated-centred radical generated from AAPH was found highly reactive to initiate red blood cell lysis. With DEPMPO only alkoxyl radicals were observed and no evidence was found for alkylperoxyl radicals. The antioxidant activity of several lipid- and water-soluble vitamins has been assessed by the biological assay and through two chemical methods. We have noticed high antioxidant activities for tocopherols (in the order δ>γ>α) in the biological test but not through chemical methods. At 1 μM, the δ-tocopherol efficiency in inhibiting radical-induced red blood cell hemolysis was three times as high as the α-tocopherol efficiency. For β-carotene no significant activity even in whole blood was shown. Highly surprising antioxidant activities were observed for acid folic and pyridoxine, compared to ascorbic acid. At 10 μM, the effectiveness of folic acid was almost three times as high as vitamin C. The biological test seems clinically more relevant than most other common assays because it can detect several classes of antioxidants.     

Light-Induced Lipid Peroxidation in Isolated Chloroplasts and Role of α-Tocopherol

Lipid peroxidation in isolated chloroplasts illuminated by visible light and the role of α-tocopherol in chloroplasts were studied. The TBA reactants and fluorescent products derived from lipid peroxidation were formed by illumination. Peroxidation was inhibited by free radical scavengers and ¹O₂ quenchers. Hydroxy methyl octadecanoates, which were the reduced and hydrogenated products of lipid hydroperoxides, were detected. Among them, 10-and 15-hydroxy methyl octadecanoates were generated from ¹O₂ oxidation. On the other hand, lipid hydroperoxides did not accumulate in this peroxidation process. The amount of α-tocopherol in the chloroplasts decreased with lipid peroxidation, and α-tocopheryl quinone was produced. The results indicate that α-tocopherol acts as a free radical scavenger for photo-oxidation of chloroplasts.     

Isocratic rapid liquid chromatographic method for simultaneous determination of carotenoids,retinol, and tocopherols in human serum

An improved isocratic and rapid HPLC method was developed for the measurement of carotenoids, retinol and tocopherols in human serum. Vitamins were extracted with hexane. Mobile phase consisted of a mixture acetonitrile:methylene chloride:methanol with 20 mM ammonium acetate. This method used a small bead size (3 μm) Spherisorb ODS2 column with titane frits. Diode array and fluorescence detectors were used respectively for the detection of carotenoids and retinol/tocopherols. Chromatographic separation was complete in 13 min for β-cryptoxanthin, cis–trans-lycopene, α-carotene, β-carotene, cis-β-carotene, retinol, δ-tocopherol, γ-tocopherol and α-tocopherol. Echinenone and tocol were employed as internal standards for diode array and fluorescence detection. Accuracy was validated using standard reference material (SRM) 968C. Intra-assay and inter-assay precision were respectively 0.2–7.3% and 3.6–12.6%. Sensitivity was verified using the ICH recommendations and the limit of detection (LOD) obtained was sufficient for routine clinical application.     

Plasma tocopherol,retinol, and carotenoid concentrations in free-ranging Humboldt penguins (Spheniscus humboldti) in Chile

Plasma retinol and α-tocopherol concentrations were measured in heparinized blood samples collected from 51 free-ranging adult Humboldt penguins (Sphenicus humboldti) residing at two colonies off the Chilean coast. Thirty samples were collected in April 1992 from penguins inhabiting the Ex-islote de los Pájaros Niños in Algarrobo, Chile. In September 1992, 21 samples were collected from birds inhabiting Isla de Cachagua, Chile. Samples were assayed for retinol, retinyl palmitate, α-tocopherol, γ-tocopherol, lutein, β-cryptoxanthin, lycopene, α-carotene, and β-carotene. Retinol, α-tocopherol, and lutein were detected in all samples, while lycopene and γ-tocopherol were not detected in any. A significantly higher percentage of samples had detectable levels of retinyl palmitate and α-carotene in April (P < 0.001): for β-cryptoxanthin the percentage was higher in September (P < 0.001). Plasma concentrations of α-tocopherol and lutein were higher in September. Alpha-tocopherol concentrations were 1,877.1 ± 99.0 (SEM) μg/dl in April compared to 2.289 ± 122.3 μg/dl in September (P < 0.05); lutein concentrations were 4.16 ± 0.43 μg/dl in April vs. 10.68 ± 1.02 μg/dl in September (P < 0.001). Retinol concentrations were not significantly different (117 ± 8.0 μg/dl in April vs. 105.3 ± 7.6 μg/dl in September). Both physiologic changes associated with season, and the change in locale may have contributed to the differences seen in the assay means and the number of samples with detectable levels. © 1996 Wiley-Liss, Inc.     

β-Carotene as a high-potency antioxidant to prevent the formation of phospholipid hydroperoxides in red blood cells of mice

In order to investigate the antioxidant effect of β-carotene in vivo, phospholipid hydroperoxides and β-carotene isomers in red blood cells (RBC), plasma and tissue organelles were quantitatively measured after the oral administration of β-carotene (94.8% all-trans-β-carotene) to mice. Three groups of 24 mice each were fed for 1 week on a semisynthetic diet supplemented with either 0.6% or 3.0% β-carotene/diet or maintained on a control (β-carotene-unsupplemented) diet. The RBC phospholipid hydroperoxides showed a significant decrease followed by an increase of β-carotene intakes; i.e., 201, 16 and 4 pmol of phosphatidylcholine hydroperoxide/ml packed RBC, and 108, 22 and 8 pmol of phosphatidylethanolamine hydroperoxide/ml packed RBC, in the mice given the control diet, 0.6% carotene diet and 3.0% carotene diet, respectively. The RBC β-carotene increased from 14 to 43 pmol/ml packed RBC as followed by the increase of β-carotene intakes. Such a potent antioxidant effect of β-carotene as observed in RBC was not confirmed in the plasma, liver or lungs, although their β-carotene contents increased. The β-carotene ingestion increased the all-trans-β-carotene d and retinol contents in RBC, plasma, liver and lungs, but the α-tocopherol content decreased. In the β-carotene-supplemented (6 g and 30 g/kg diet) mice, cis-β-carotene content was relatively higher in the RBC (25–35% of total β-carotene) than that in plasma, liver and lungs. The present findings indicate that not only does β-carotene act as a potent antioxidant in vivo but also its antioxidant effect is very specific in the RBC phospholipid bilayers rather than in the plasma and other tissue organelles.     

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated with Cu(2+) ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu(2+) to Cu(+) and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu(+) oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides.     

Fish oil diet affects on oxidative senescence of red blood cells linked to degeneration of spleen cells in mice

The effect of dietary polyunsaturated fatty acids and α-tocopherol supplementation on erythrocyte lipid peroxidation and immunocompetent cells in mice was studied comparatively using seven dietary oils (15% oil/diet, w/w) including fish oil rich in eicosapentaenoic acid (EPA, 20:5, n–3) and docosahexaenoic acid (DHA, 22:6, n–3). A 43% increase in spleen weight, about twice as many spleen cells and no change in the subpopulations of spleen cells, as well as a significant depression of mitogen-induced blastogenesis of both T and B cells in the spleen were observed in mice fed fish oil for 30 days in comparison with soybean oil diet-fed mice. In the fish oil diet-fed mice, membranous lipid hydroperoxide (hydroperoxides of phosphatidylcholine and phosphatidylethanolamine) accumulation as a marker of oxidative senescence in red blood cells (RBC) was 2.7–3.5 times higher than that in mice fed soybean oil, although there was no difference in the plasma phosphatidylcholine hydroperoxide concentration. In spite of the supplementation of α-tocopherol to up to 10 times the level in the basal diet, the degeneration of spleen cells and the stimulated oxidative senescence of RBC found by the fish oil feeding could not be prevented. The results suggest that oral intake of excess polyunsaturated fatty acids, i.e. EPA and DHA, in a fish oil diet can lead to acceleration of membrane lipid peroxidation resulting in RBC senescence linked to the lowering of immune response of spleen cells, and that supplementation of α-tocopherol as antioxidant does not always effectively prevent such oxidative degeneration as observed in spleen cells and RBC in vivo.     

α-Tocopherol increases caspase-3 up-regulation and apoptosis by β-carotene cleavage products in human neutrophils

It has been found that β-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as α-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that α-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by α-tocopherol. Both α-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of α-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of α-tocopherol and β-ionone, a cyclohexenyl degradation product of β-carotene with shorter aliphatic chain.     

Free radical formation from organic hydroperoxides in isolated human polymorphonuclear neutrophils.

We have demonstrated with electron paramagnetic resonance (EPR) that organic hydroperoxides are decomposed to free radicals by both human polymorphonuclear leukocytes (PMNs) and purified myeloperoxidase. When tert-butyl hydroperoxide was incubated with either PMNs or purified myeloperoxidase, peroxyl, alkoxyl, and alkyl radicals were trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the case of ethyl hydroperoxide, DMPO radical adducts of peroxyl and alkyl (identified as alpha-hydroxyethyl when trapped by tert-nitrosobutane) radicals were detected. Radical adduct formation was inhibited when azide was added to the incubation mixture. Myeloperoxidase-deficient PMNs produced DMPO radical adduct intensities at only about 20-30% of that of normal PMNs. Our studies suggest that myeloperoxidase in PMNs is primarily responsible for the decomposition of organic hydroperoxides to free radicals. The finding of the free radical formation derived from organic hydroperoxides by PMNs may be related to the cytotoxicity of this class of compounds.     

Rapid quantitative determination of fat-soluble vitamins and coenzyme Q-10 in human serum by reversed phase ultra-high pressure liquid chromatography with UV detection

We are presenting the first ultra-high pressure LC (UHPLC) method for rapid quantitative measurement of vitamin A, E (α- and γ-tocopherol), β-carotene and CoQ₁₀ from human serum. The chromatography was performed on Shield RP₁₈ UHPLC column with UV detection. The method was validated based on linearity, accuracy, matrix effects study, precision and stability. The calibration was linear over the following range: 0.09–10.0 for retinol and γ-tocopherol, 0.05–5 for β-carotene, 0.9–100 for α-tocopherol and 0.14–15 mg/L for CoQ₁₀. The limit of detection and quantitation for retinol, γ-tocopherol, β-carotene, α-tocopherol and CoQ₁₀ were as follows 0.07/0.024, 0.018/0.06, 0.004/0.12, 0.078/0.261, 0.008/0.028 mg/L. The recoveries were above 85%. The inter- and intra-assay precision was below 10%. Reference intervals were established for children and adults. Because of its low cost, extremely short analysis time (2 min) and excellent chromatographic reproducibility this UHPLC method can easily be adopted for high-throughput clinical and pharmacokinetics studies.