beta-carotene and 2-2--azobis(2-4-dimethylvaleronitrile)

There is growing evidence that high levels of the macular xanthophyll carotenoids lutein and zeaxanthin may be protective against visual loss due to age-related macular degeneration, but the actual mechanisms of their protective effects are still poorly understood. We have recently purified, identified and characterized a pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye which specifically and saturably binds to the two forms of zeaxanthin endogenously found in the foveal region. In this report, we studied the synergistic antioxidant role of zeaxanthin and GSTP1 in egg yolk phosphatidylcholine (EYPC) liposomes using hydrophilic 2,2'-azobis(2-methyl-propionamidine) dihydrochloride (AAPH) and lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) as lipid peroxyl radical generators. The two zeaxanthin diastereomers displayed synergistic antioxidant effects against both azo lipid peroxyl radical generators when bound to GSTP1. In the presence of GSTP1, nondietary (3R,3'S-meso)-zeaxanthin was observed to be a better antioxidant than dietary (3R,3'R)-zeaxanthin. This effect was found to be independent of the presence of glutathione. Carotenoid degradation profiles indicated that the zeaxanthin diastereomers in association with GSTP1 were more resistant to degradation which may account for the synergistic antioxidant effects.

Topics: Amidines; Antioxidants; Azo Compounds; beta Carotene; Drug Stability; Glutathione; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Isoenzymes; Lipid Peroxidation; Liposomes; Nitriles; Oxidants; Phosphatidylcholines; Retina; Stereoisomerism; Xanthophylls; Zeaxanthins

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) 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, beta-carotene and lycopene in preventing lipid peroxidation. Lipid peroxidation was initiated with 2,2'-azobis-[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 degrees 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 beta-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 beta-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 beta-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.

Topics: Antioxidants; Azo Compounds; beta Carotene; Carotenoids; Chemical Phenomena; Chemistry, Physical; Glutathione; Lipid Peroxidation; Liposomes; Lutein; Lycopene; Malondialdehyde; Microscopy, Electron; Nitriles; Oxidants

Based on the recognized capacity of (+)-catechin (CTCH) to prevent free radical-mediated damage in different biological systems, its role in the protection of human plasma from oxidation was investigated. Samples of human blood plasma were incubated with 50 mM AAPH [2,2'-azobis-(2-amidinopropane) clorhidrate] or AMVN [2,2'-azobis(2,4-valeronitrile)], in the absence or the presence of CTCH (0.01 to 1 mM). Lipid oxidation was evaluated measuring the formation of 2-thiobarbituric acid reactive substances (TBARS). Alpha-tocopherol (AT), beta-carotene (BC), and CTCH were measured by reverse phase HPLC with electrochemical detection. TBARS formation was dependent on incubation time and on the nature of the azocompound, yielding 4.8 +/- 0.9, and 14.9 +/- 3.4 microM MDA, after 4 h, in AAPH and AMVN-exposed plasma, respectively. Plasma AT and BC were extensively depleted under these oxidant conditions. The addition of CTCH prevented or delayed the formation of TBARS, and the depletion of AT and BC in a dose dependent manner. This antioxidant effect was dependent on the concentration of CTCH and on the physical characteristics of the radical initiator. CTCH supplementation modified not only the lag time for the antioxidants depletion, but also the consumption rate. These results indicate that CTCH was an effective antioxidant in human blood plasma, delaying the consumption of endogenous lipid soluble antioxidants (AT and BC) and inhibiting lipid oxidation.

Topics: Amidines; Antioxidants; Azo Compounds; beta Carotene; Blood; Catechin; Chromatography, High Pressure Liquid; Humans; Kinetics; Lipid Peroxidation; Nitriles; Oxidants; Thiobarbituric Acid Reactive Substances; Vitamin E

Topics: Antioxidants; Azo Compounds; beta Carotene; Chromans; Fatty Acids, Unsaturated; Free Radicals; HL-60 Cells; Humans; Nitriles; Oxidative Stress; Peroxides; Phenol; Vitamin E

The ability of carotenoids to protect egg-yolk phosphatidylcholine (EYPC) lipids against oxidation by peroxyl radicals generated from azo-initiators was studied. In homogeneous organic solution, all the carotenoids tested ameliorated lipid peroxidation by AMVN, but none was as effective as alpha-tocopherol. Beta-ring carotenoids showed a correlation between protective effect and rate of carotenoid destruction. Beta,beta-Carotene and zeaxanthin, which react with peroxyl radicals at similar rates, gave a similar degree of protection in organic solution. The reactivity and protective ability of the 4,4'-diketocarotenoids, astaxanthin and canthaxanthin was less. Carotenoids incorporated into ordered membrane systems (EYPC liposomes) displayed different protective efficacies. Zeaxanthin and beta-cryptoxanthin were more effective than beta,beta-carotene against oxidation initiated in the aqueous and lipid phases. Astaxanthin and canthaxanthin afforded less protection to the liposomal lipids. Lycopene was destroyed most rapidly but was least effective as an antioxidant. Located in the hydrophobic inner core of the bilayer, the hydrocarbons lycopene and beta,beta-carotene would not be in a position to readily intercept free-radicals entering the membrane from the aqueous phase. Carotenoids with polar end groups span the bilayer with their end groups located near the hydrophobic-hydrophillic interface where free-radical attack from AAPH first occurs. Hydrogen abstraction from C-4 may be one of the mechanisms of carotenoid antioxidant activity in this system. The chemical reactivity of a carotenoid is not the only factor that determines its ability to protect membranes against oxidation. The position and orientation of the carotenoid in the bilayer is also of importance.

Topics: Azo Compounds; beta Carotene; Carotenoids; Cryptoxanthins; Drug Stability; Free Radicals; Kinetics; Lipid Peroxidation; Liposomes; Nitriles; Oxidation-Reduction; Peroxides; Phosphatidylcholines; Solutions; Structure-Activity Relationship; Vitamin E; Xanthophylls; Zeaxanthins

Antioxidant mechanisms have been proposed to underlie the beneficial pharmacological effects of EGb 761, an extract from Ginkgo biloba leaves used for treating peripheral vascular diseases and cerebrovascular insufficiency in the elderly. In vitro evidence has been reported that EGb 761 scavenges various reactive oxygen species, i.e. nitric oxide, and the superoxide, hydroxyl, and oxoferryl radicals. However, the ability of EGb 761 to scavenge peroxyl radicals (reactive species mainly involved in the propagation step of lipid peroxidation) has not been investigated. To characterize further the antioxidant action of EGb 761, we measured the protective effects of EGb 761 during: (1) the oxidation of B-phycoerythrin by peroxyl radicals generated in aqueous solution by 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH); and (2) the reaction of luminol or cis-parinaric acid with peroxyl radicals generated from 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) in liposomes or in human low density lipoprotein (LDL), respectively. To evaluate the peroxyl radical scavenging activity of EGb 761 in a more physiologically relevant model of damage to lipid-containing systems, we also analyzed the effect of the extract on the oxidation of human LDL exposed to the azo-initiators in terms of: (1) accumulation of cholesterol linoleate ester hydroperoxides, (2) depletion of alpha-tocopherol and beta-carotene, and (3) changes in intrinsic tryptophan fluorescence. EGb 761 afforded protection against oxidative damage in all the systems we analyzed; thus, it is an efficient scavenger of peroxyl radicals. This result extends the oxygen radical scavenging properties of the extract and supports the hypothesis of an antioxidant therapeutic action of EGb 761.

Topics: Amidines; Antioxidants; Azo Compounds; beta Carotene; Carotenoids; Free Radical Scavengers; Ginkgo biloba; Humans; Lipid Peroxidation; Lipoproteins, LDL; Nitriles; Peroxides; Phycoerythrin; Plant Extracts; Tryptophan; Vitamin E

In the present in vitro study we compared the antioxidative efficiency of the 9-cis to that of the all-trans beta-carotene. The 9-cis isomer was isolated from the alga Dunaliella bardawil. The experimental system consisted of 80 mM methyl linoleate, 4 mM azo-bis-2,2'-dimethylvaleronitrile (AMVN) as a free radical generating agent, and 200 microM beta-carotene (synthetic all-trans, 9-cis or a mixture of the 9-cis and all-trans isomers, having a ratio of 2.3). During the incubation at 37 degrees C the mixtures were analyzed for methyl linoleate hydroperoxides, total beta-carotene concentration, and its isomeric composition. The content of 9-cis beta-carotene in the various systems was negatively correlated to the level of the hydroperoxides accumulated, and positively related to the residual beta-carotene amount. The HPLC analysis of the system containing both isomers revealed a continuous decrease in the 9-cis to all-trans isomer ratio. The results suggest that the 9-cis beta-carotene has a higher antioxidant potency than that of the all-trans isomer and, therefore, it protects the methyl linoleate, as well as the all-trans isomer, from oxidation. This isomeric difference might be explained by the higher reactivity of cis, compared to trans, bonds.

Topics: Antioxidants; Azo Compounds; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Eukaryota; Free Radicals; Linoleic Acids; Lipid Peroxidation; Nitriles; Stereoisomerism