Lipid peroxidation, antioxidant status and survival in institutionalised elderly: a five-year longitudinal study

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. alpha-tocopherol, beta-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), beta-carotene and alpha-tocopherol were positively associated with survival. alpha-tocopherol and MDA were revealed as independent predictors in a joint survival model, being the group with low MDA and high alpha-tocopherol that with the lowest mortality. In conclusion, a higher risk of death was associated with increased lipid peroxidation and lower antioxidant defenses.     

Effect of α-Tocopherol, β-Carotene, Monogalactosyldiglyceride and Phosphatidylcholine on Light-Induced Degradation of Chlorophyll a in Acetone

The effect of α-tocopherol, β-carotene, monogalactosyldi-glyceride and phosphatidylcholine on red light induced degradation of chlorophyll a was studied in acetone at 4°C. Monogalaclosyldi-glyceride was ineffective up to a molar ratio of monogalactosyldi glyceride to chlorophyll of 1:10. α-Tocopherol, β-carotene and phosphatidylcholine inhibited chlorophyll degradation. Maximal protection by α tocopherol and β-carotene was similar (76%) but on a molar basis a tocopherol was less effective. Protection by phosphatidylcholine was less than by a tocopherol and α-carotene but the lipid was effective at a lower ratio of chlorophyll to protectant. Inhibition by phosphatidylcholine was independent of the degree of unsaturation of the fatty acids. Effects of β-carotene and α-tocopherol were additive at suboptimal concentrations, but addition did not increase the maximal protection of 76% by these substances alone. Phosphatidylcholine increased the effectiveness of α-tocopherol and β-carotene independent of their concentrations. It is suggested that interactions between lipids participate in the mechanism protecting chlorophyll a against photooxidation in the chloroplast membrane.     

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}.     

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.     

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.     

Critical difference applied to exercise-induced oxidative stress: the dilemma of distinguishing biological from statistical change

Even though intense exercise has traditionally been associated with a statistically significant accumulation of blood-borne biomarkers of free radical-mediated lipid peroxidation, it remains to be determined if the oxidative stress response is biologically significant. To examine biological significance, we calculated the critical difference of selected biomarkers of oxidants-antioxidants in the peripheral circulation of ten male subjects aged 24±3 years. Venous blood was drawn in the resting supine position every hour over an 8-h period (Study 1). As proof-of-concept, supine venous blood was also obtained at rest and following maximal cycling exercise in a separate group of 13 males, mean age 22±3 years (Study 2). The critical difference of electron paramagnetic resonance spin-trapped alkoxyl free radicals, lipid hydroperoxides, malondialdehyde, ascorbic acid, retinol, lycopene, α-tocopherol, β-carotene and α-carotene was calculated as 121%, 28%, 50%, 9%, 29%, 106%, 13%, 28% and 107%, respectively (Study 1). Maximal exercise was associated with a statistically significant (P<0.05 vs. rest) reduction in α-tocopherol and retinol, and a corresponding rise in alkoxyl free radicals and lipid hydroperoxides (Study 2). However, these changes were all within the critical difference percentage value. In conclusion, these findings highlight the importance of distinguishing biological from statistical significance when assessing the physiological and clinical impact of exercise-induced oxidative stress.     

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.     

Evaluation of Serum and Tissue Levels of α-Tocopherol

This study was designed to test the effect of supplementation of several antioxidants, including α-tocopherol, on the clinical reduction of premalignant oral lesions. Samples of oral mucosa and serum were taken from baseline to 9 months of supplementation from patients with premalignant oral lesions and analyzed for α-tocopherol by HPLC. Statistical increases in both serum and tissue α-tocopherol were found after supplementation. There was no statistical relationship between α-tocopherol and β-carotene levels.     

Cyclosporine A induced changes to plasma and erythrocyte antioxidant defences

Abstract

Organ transplant recipients develop pronounced cardiovascular disease, and decreased antioxidant capacity in plasma and erythrocytes is associated with the pathogenesis of this disease. These experiments tested the hypothesis that the immunosuppressant cyclosporine A (CsA) alters erythrocyte redox balance and reduces plasma antioxidant capacity. Female Sprague-Dawley rats were randomly assigned to a control or CsA treated group. Treatment animals received 25 mg/kg/day of CsA via intraperitoneal injection for 18 days. Control rats were injected with the same volume of the vehicle. Three hours after the final CsA injection, rats were exsanguinated and plasma analysed for total antioxidant status (TAS), α-tocopherol, malondialdehyde (MDA), and creatinine. Erythrocytes were analysed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities, α-tocopherol, and MDA. CsA administration resulted in a significant (P < 0.05) decrease in plasma TAS and significant increases (P < 0.05) in plasma creatinine and MDA. Erythrocyte CAT was significantly (P < 0.05) increased in CsA treated rats compared to controls. There were no significant differences (P > 0.05) in erythrocyte SOD, GPX, G6PD, α-tocopherol or MDA between groups. In summary, CsA alters erythrocyte antioxidant defence and decreases plasma total antioxidant capacity.     

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.     

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.     

Elicitors,SA and MJ enhance carotenoids and tocopherol biosynthesis and expression of antioxidant related genes in Moringa oleifera Lam. leaves

Moringa oleifera Lam. leaves are rich source of carotenoids (provitamin A) and α-tocopherol (vitamin E), and there is a scope for their further enhancement, through elicitor mediation, thereby a great potential for addressing these vitamins deficiency. In the present study, we report the efficacy of foliar administration of biotic elicitors, carboxy-methyl chitosan and chitosan, and signaling molecules, methyl jasmonate (MJ) and salicylic acid (SA) for enhancement of major carotenoids and α-tocopherol. Highest α-tocopherol content of 49.7 mg/100 g FW was recorded upon foliar application of 0.1 mM SA after 24 h of treatment, which represented a 187.5 % increase in comparison to the untreated control. Similarly, a maximum of 52.6 mg/100 g FW lutein, and 21.8 mg/100 g FW β-carotene content were observed in leaves after 24 h of treatment with MJ, which represented a 54.0 and 20.3 % increase in comparison to the untreated control, respectively. Among the major genes of carotenoid biosynthetic pathway, the expression of lycopene β-cyclase (LCY-β) was maximum influenced after treatment with elicitors and signaling molecules, compared to phytoene synthase and phytoene desaturase, suggesting the LCY-β-mediated enhancement in the production of β-carotene in elicitor treated M. oleifera leaves. Enhanced production of α-tocopherol under respective elicitor treatment was further supported by 2.0–2.7 fold up-regulation of γ-tocopherol methyl transferase, compared to untreated control. This is the first report on elicitor-mediated enhanced production of tocopherol and carotenoids in foliage of economically important food plant.     

Sea buckthorn (Hippophae rhamnoides L.) vegetative parts as an unconventional source of lipophilic antioxidants

The profile of lipophilic antioxidants in different vegetative parts (leaves, shoots, buds and berries) was studied in sea buckthorn (Hippophae rhamnoides L.) male and female plants collected in the end of spring. Five lipophilic compounds, i.e. three tocopherol homologues (α, β and γ), plastochromanol-8 and β-carotene, were identified in each vegetative part of male and female sea buckthorn plants at the following concentrations: 7.25–35.41, 0.21–2.43, 0.41–1.51, 0.19–1.79 and 4.43–24.57 mg/100 g dry weight basis. Additionally, significant amounts of α-tocotrienol (1.99 mg/100 g dry weight basis) were detected in buds. The α-tocopherol and β-carotene were predominant lipophilic antioxidants in each vegetative part, accounting for 78.3–97.0% of identified compounds. The greatest amounts of lipophilic antioxidants were found in leaves, especially of female plants. Nevertheless, apart from leaves, also shoots of plants of both sexes seem to be a good source of α-tocopherol and β-carotene.     

Tocopherols and carotenes are differently distributed in subfractions of high-density lipoprotein

An apolipoprotein (apo) E-rich and an apo E-poor fraction of high-density lipoprotein (HDL) were isolated from four healthy men by heparin-Sepharose affinity chromatography. On a cholesterol basis, the apo E-poor HDL fraction contained a third more α- and γ-tocopherol and about a third less α- and β-carotene than the apo E-rich HDL fraction. Plasma concentrations of HDL cholesterol were highly correlated with the contribution of the apo E-rich HDL subfraction to total HDL α-tocopherol (r = − 0.990, P < 0.001).     

Oral a-tocopherol Supplementation Inhibits Lipid Oxidation in Established Human Atherosclerotic Lesions

Background: Much experimental evidence suggests that lipid oxidation is important in atherogenesis and in epidemiological studies dietary antioxidants appear protective against cardiovascular events. However, most large clinical trials failed to demonstrate benefit of oral antioxidant vitamin supplementation in high-risk subjects. This paradox questions whether ingestion of antioxidant vitamins significantly affects lipid oxidation within established atherosclerotic lesions. Methods and results: This placebo-controlled, double blind study of 104 carotid endarterectomy patients determined the effects of short-term α-tocopherol supplementation (500 IU/day) on lipid oxidation in plasma and advanced atherosclerotic lesions. In the 53 patients who received α-tocopherol there was a significant increase in plasma α-tocopherol concentrations (from 32.66±13.11 at baseline to 38.31±13.87 (mean±SD) μmol/l, p&lt;0.01), a 40% increase (compared with placebo patients) in circulating LDL-associated α-tocopherol (p&lt;0.0001), and their LDL was less susceptible to ex vivo oxidation than that of the placebo group (lag phase 115.3±28.2 and 104.4±15.7 min respectively, p&lt;0.02). Although the mean cholesterol-standardised α-tocopherol concentration within lesions did not increase, α-tocopherol concentrations in lesions correlated significantly with those in plasma, suggesting that plasma α-tocopherol levels can influence lesion levels. There was a significant inverse correlation in lesions between cholesterol-standardised levels of α-tocopherol and 7β-hydroxycholesterol, a free radical oxidation product of cholesterol. Conclusions: These results suggest that within plasma and lesions α-tocopherol can act as an antioxidant. They may also explain why studies using &lt;500 IU α-tocopherol/day failed to demonstrate benefit of antioxidant therapy. Better understanding of the pharmacodynamics of oral antioxidants is required to guide future clinical trials.     

a-Tocopherol Content and a-Tocopherol Transfer Protein Expression in Leukocytes of Children with Acute Leukemia

α-Tocopherol (a form of vitamin E) is a fat-soluble vitamin that can prevent lipid peroxidation of cell membranes. This antioxidant activity of α-tocopherol can help to prevent cardiovascular disease, atherosclerosis and cancer. We investigated the α-tocopherol level and the expression of α-tocopherol transfer protein (α-TTP) in the leukocytes of children with leukemia. The plasma and erythrocyte α-tocopherol levels did not differ between children with leukemia and the control group. However, lymphocytes from children with leukemia had significantly lower α-tocopherol levels than lymphocytes from the controls (58.4±39.0 ng/mg protein versus 188.9±133.6, respectively; p&lt;0.05), despite the higher plasma α-tocopherol/cholesterol ratio in the leukemia group (5.83±1.64 μmol/mmol versus 4.34±0.96, respectively; p&lt;0.05). No significant differences in the plasma and leukocyte levels of isoprostanes (the oxidative metabolites of arachidonic acid) were seen between the leukemia patients and controls. The plasma level of acrolein, a marker of oxidative stress, was also similar in the two groups. Investigation of α-TTP expression by leukocytes using real-time PCR showed no difference between the two groups. These findings suggest that there may be comparable levels of lipid peroxidation in children with untreated leukemia and controls, despite the reduced α-tocopherol level in leukemic leukocytes.     

Retinol-deficient rats can convert a pharmacological dose of astaxanthin to retinol: antioxidant potential of astaxanthin, lutein, and β-carotene

Retinol (ROH) and provitamin-A carotenoids are recommended to treat ROH deficiency. Xanthophyll carotenoids, being potent antioxidants, can modulate health disorders. We hypothesize that nonprovitamin-A carotenoids may yield ROH and suppress lipid peroxidation under ROH deficiency. This study aimed to (i) study the possible bioconversion of astaxanthin and lutein to ROH similar to β-carotene and (ii) determine the antioxidant potential of these carotenoids with reference to Na(+)/K(+)-ATPase, antioxidant molecules, and lipid peroxidation (Lpx) induced by ROH deficiency in rats. ROH deficiency was induced in rats (n = 5 per group) by feeding a diet devoid of ROH. Retinol-deficient (RD) rats were gavaged with astaxanthin, lutein, β-carotene, or peanut oil alone (RD group) for 7 days. Results show that the RD group had lowered plasma ROH levels (0.3 μmol/L), whereas ROH rose in astaxanthin and β-carotene groups (4.9 and 5.7 μmol/L, respectively), which was supported by enhanced (69% and 70%) intestinal β-carotene 15,15'-monooxygenase activity. Astaxanthin, lutein, and β-carotene lowered Lpx by 45%, 41%, and 40% (plasma), respectively, and 59%, 64%, and 60% (liver), respectively, compared with the RD group. Lowered Na(+)/K(+)-ATPase and enhanced superoxide dismutase, catalase, and glutathione-S-transferase activities support the lowered Lpx. To conclude, this report confirms that astaxanthin is converted into β-carotene and ROH in ROH-deficient rats, and the antioxidant potential of carotenoids was in the order astaxanthin > lutein > β-carotene.     

Carotenoid-containing unilamellar liposomes loaded with glutathione: a model to study hydrophobic-hydrophilic antioxidant interaction

Unilamellar liposomes are used as a simple two-compartment model to study the interaction of antioxidants. The vesicle membrane can be loaded with lipophilic compounds such as carotenoids or tocopherols, and the aqueous core space with hydrophilic substances like glutathione (GSH) or ascorbate, mimicking the interphase between an aqueous compartment of a cell and its surrounding membrane.

Unilamellar liposomes were used to investigate the interaction of GSH with the carotenoids lutein, β-carotene and lycopene in preventing lipid peroxidation. Lipid peroxidation was initiated with 2,2′-azo-bis-[2,4-dimethylvaleronitrile] (AMVN). Malondialdehyde (MDA) formation was measured as an indicator of oxidation; additionally, the loss of GSH was followed. In liposomes without added antioxidant, MDA levels of 119 ± 6 nmol/mg phospholipid were detected after incubation with AMVN for 2 h at 37°C. Considerably lower levels of 57 ± 8 nmol MDA/mg phospholipid were found when the liposomal vesicles had been loaded with GSH. Upon incorporation of β-carotene, lycopene or lutein, the resistance of unilamellar liposomes towards lipid peroxidation was further modified. An optimal further protection was observed with 0.02 nmol β-carotene/mg phospholipid or 0.06 nmol lycopene/mg phospholipid. At higher levels both these carotenoids exhibited prooxidant effects. Lutein inhibited lipid peroxidation in a dose-dependent manner between 0.02 and 2.6 nmol/mg phospholipid. With increasing levels of lycopene and lutein the consumption of encapsulated GSH decreased moderately, and high levels of β-carotene led to a more pronounced loss of GSH.

The data demonstrate that interactions between GSH and carotenoids may improve resistance of biological membranes towards lipid peroxidation. Different carotenoids exhibit specific properties, and the level for optimal protection varies between the carotenoids.