Radicals In Membranes Research Articles

Vitamin and non-vitamin antioxidants and their interaction in food

Aerobic life has been challenged by the changing conditions appearing through evolution resulting in appearance of both hydrophilic and lipophilic antioxidants as an indispensable protection against the ubiquitous reactive oxygen- and nitrogen species. Interaction between hydrophilic and lipophilic antioxidants at interfaces often results in synergistic effects important for food stability. Such concerted actions by antioxidants also seem of relevance for human nutrition, but need a more mechanistic description. Non-vitamin antioxidants like the hydrophilic polyphenols including their glycosides thus show synergism through enhancing the lipophilic tocopherols as chainbreaking antioxidants. While this tocopherol/polyphenol synergism seems to be controlled kinetically, the synergism between the lipophilic tocopherols and the lipophilic carotenoids is rather thermodynamically controlled through a regeneration of the carotenoids by the tocopherols. Antioxidant interaction between carotenoids and polyphenols is less investigated and more complex, but may explain the special role of the non-vitamin carotenoids as antioxidants. Carotenoids act as electron donors reducing lipid radicals in membranes subsequently being regenerated at interfaces by hydrophilic polyphenols. Taking distribution phenomena into account, a kinetic description may be possible. Carotenoid radical cations of the less reducing carotenoids like astaxanthin is regenerated faster to form the parent carotenoids showing the importance of the keto group for electron transfer in favour of kinetic control as is further confirmed by linear-free-energy relationships.

Z-Isomers of (4α→6″, 2α→O→1″)-phenylflavan substituted with R′=R=OH. Conformational properties, electronic structure and aqueous solvent effects

Procyanidins are highly hydroxylated polymers known as antioxidant compounds, thereby exhibiting beneficial effects. These compounds are protective agents against oxidative stress and the damage induced by free radicals in membranes and nucleic acids. This paper describes a study of the conformational space of (4α→6″, 2α→O→1″)-phenylflavan substituted with R'=R=OH as part of a larger study of similar structures with different substitutions. The relationships between aqueous solution-vacuum variations of some properties were studied, as well as the stabilization and reactivity of (4α→6″, 2α→O→1″)-phenylflavan substituted with R'=R=H, R'=H, R=OH, R'=R=OH, and (+)-catechin. The variations in geometric parameters and electronic properties due to conformational changes, as well as the effects of substituents and polar solvents, were evaluated and analyzed. Bader's theory of atoms in molecules was applied to characterize intramolecular interactions, along with a natural bond orbital analysis for each conformer described. The molecular electrostatic potential was rationalized by charge delocalization mechanisms and interatomic intramolecular interactions, relating them to the structural changes and topological properties of the electron charge density. Molecular polarizability and permanent electric dipole moment values were estimated. The results show the importance of a knowledge of the conformational space, and values for each conformer. Based on our previous results, we showed the existence of electron charge delocalization mechanisms acting cooperatively as "delocalization routes", showing interactions between different rings not even sharing the same plane. These "delocalization routes" were more effective for (4α→6″, 2α→O→1″)-phenylflavan substituted with R'=R=OH than for (+)-catechin, and are proposed as adding insight into the structure-antioxidant activity relationship of flavans.

Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the reaction rates of vitamin E (α-, β-, γ-, δ-tocopherols, TocH) and tocol with aroxyl radical (ArO ) as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry. Egg yolk phosphatidylcholine (EYPC) vesicles were used as a membrane model. EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H 2O = 7:3, v/v) that gave the lowest turbidity in samples. The second-order rate constants ( k s) for α-TocH in MeOH/H 2O solution with EYPC vesicles were apparently 3.45 × 10 5 M −1 s −1, which was about 8 times higher than that (4.50 × 10 4 M −1 s −1) in MeOH/H 2O solution without EYPC vesicles. The corrected k s of α-TocH in vesicles, which was calculated assuming that the concentration of α-TocH was 133 times higher in membranes of 10 mM EYPC vesicles than in the bulk MeOH/H 2O solution, was 2.60 × 10 3 M −1 s −1, which was one-seventeenth that in MeOH/H 2O solution because of the lower mobility of α-TocH in membranes. Similar analyses were performed for other vitamin E analogues. The k s of vitamin E in membranes increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH. There was not much difference in the ratios of reaction rates in vesicles and MeOH/H 2O solution among vitamin E analogues [ k s(vesicle)/ k s (MeOH/H 2O) = 7.7, 10.0, 9.5, 7.4, and 5.1 for α-, β-, γ-, δ-TocH, and tocol, respectively], but their reported ratios in solutions of micelles and ethanol were quite different [ k s(micelle)/ k s(EtOH) = 100, 47, 41, 15, and 6.3 for α-, β-, γ-, δ-TocH, and tocol, respectively]. These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes, which increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH.

Impact of oxidized lipids and antioxidants, such as vitamin E and lazaroids, on the structure and dynamics of unsaturated membranes

The structural and dynamic effects of well defined, synthesized lipid peroxidation products (PLPC-OH and PLPC-OOH), the natural membrane-bound antioxidant vitamin E (α-tocopherol and β-tocopherol) and three synthetic membrane-bound antioxidants (the lazaroids U-74006F, U-75412 and U78518E) on bilayer lipid membranes have been investigated by time-resolved fluorescence depolarization and angle-resolved fluorescence depolarization (AFD) techniques and by electron paramagnetic resonance utilizing probe molecules. The lipid peroxidation products strongly disorder the investigated unsaturated lipid membranes, but they do not affect the lipid dynamics. The disordering effect is compatible with a model where the lipid hydroperoxy of hydroxy moieties reside closer to the polar head-group region of the membrane lipids. Fluorescence depolarization data obtained from the intrinsic fluorescence of α-tocopherol indicate that the trolox moiety of α-tocopherol (the fluorophore and antioxidant part of the molecule) can have different orientations in DOPC membranes depending on the α-tocopherol concentration. These orientational possibilities and the location of the lipid hydroperoxy moieties in lipid bilayers may account for the high reaction efficiency of α-tocopherol with lipid hydroperoxy radicals in membranes. Vitamin E in low and in high concentrations does not affect the molecular orientational order in membranes, but it does affect the dynamics. Because of the lack of structural effects of vitamin E on unsaturated lipid membranes without or with lipid peroxidation products, the hypothesis of vitamin E functioning as a membrane-stabilizing agent is questionable.In contrast with vitamin E, the three lazaroids as synthetic antioxidants have different effects on membrane structure and dynamics. The steroid-based lazaroids U-74006F and U-75412 have a disordering effect in the majority of the lipid systems investigated whereas the effects on dynamics vary depending on the surrounding lipids. The trolox derivative U-78518E, structurally closely related to α-tocopherol, has no or small ordering effects while the effects on dynamics also vary depending on the surrounding lipids. The effects of the different compounds on the molecular orientational order of membranes may be understood by their overall molecular shape. The main question that as yet remains unanswered is: which factors determine the dynamic effects of molecules in membranes?

Peroxyl radical trapping and autoxidation reactions of .alpha.-tocopherol in lipid bilayers

A phospholipid liposome system was employed to model peroxyl radical trapping reactions of alpha-tocopherol (1) in biological membranes. Peroxyl radicals generated by thermolysis of 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) at 37 degrees C oxidized 1 to 8a-[(2,4-dimethyl-1-nitrilopent-2-yl)dioxy]tocopherone (3a), 8a-(hydroperoxy)tocopherone (3b), alpha-tocopherol quinone (4), 4a,5-epoxy-8a-hydroperoxytocopherone (6), 2,3-epoxy-alpha-tocopherol quinone (7), and 5,6-epoxy-alpha-tocopherol quinone (8). The products were purified by high-performance liquid chromatography and characterized by UV-vis spectroscopy, mass spectrometry, and cochromatography with authentic standards. Products accumulated in approximately constant proportion as 1 was consumed. Tocopherones 3a/3b decomposed in the bilayer primarily by hydrolyzing to produce 4. Tocopherone decomposition also produced small amounts of epoxides 6-8, apparently by unimolecular tocopherone decomposition rather than by peroxyl radical dependent oxidation, since neither AMVN nor 1 affected the rate of 3a loss or the distribution of products. Epoxides 6-8 appear to be formed primarily by autoxidation reactions that compete with the peroxyl radical trapping reactions that form tocopherone 3a. Epoxide products may thus serve as biochemical markers for irreversible oxidation of 1 by peroxyl radicals in membranes.

A method to evaluate the antioxidant system for radicals in erythrocyte membranes

The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When a oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in eryhrocyte membranes, using fatty acid spin labels with cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37°C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3. ± 1.8 × 10 −3/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occuring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.

Protection against carbon tetrachloride hepatotoxicity by 5,10-dihydroindeno[1,2-b]indole, a potent inhibitor of lipid peroxidation

The influence of 5,10-dihydroindeno[1,2-b]indole (indenoindole) on carbon tetrachloride (CCl 4)-mediated hepatotoxicity and lipid peroxidation were examined. Indenoindole (25 mg/kg body weight) ameliorated the increase in liver enzymes appearing in the plasma 24 hr after CCl 4 administration, with about a 63% reduction for alanine transaminase, 56% for ornithine transcarbamylase and 84% for alkaline phosphatase. Indenoindole also partially prevented, in a dose-dependent fashion, the decrease in hepatic cytochromes P-450, total tissue reducing equivalents and hepatic ascorbate levels resulting 4 hr after CCl 4 administration. In a homogeneous chemical system consisting of purified soybean phospholipid substrate in chlorobenzene, azobisisobutyronitrile-initiated lipid peroxidation was inhibited by indenoindole, with 50% inhibition occurring at about 17 μ m. Inhibition by indenoindole of iron-ascorbate-initiated lipid peroxidation in aqueous buffer containing phospholipid vesicles was about tenfold more efficient, with 50% inhibition occurring at about 1.5 μ m. Presumably, this was due to the increased concentration of indenoindole in the membrane of the phospholipid vesicle. The efficiency of inhibition of lipid peroxidation was in the order of indenoindole = butylated hydroxytoluene (BHT) > α-tocopherol ⪢ indole > indene. These 50% inhibition values of lipid peroxidation for these compounds were similar in an assay system composed of NADPH-fortified mouse-liver musomes initiated with CCl 4. For indenoindole, the 50% inhibition value (1.3 μ m) was more than two orders of magnitude less than the spectral binding constant for indenoindole to mouse-liver cytochrome P-450 ( K s = 236 μ m ), implying that the partial inhibition of metabolic activation of CCl 4 was not responsible for the inhibition of lipid peroxidation observed with indenoindole in this system. It appears that indenoindole may trap reactive radicals and inhibit lipid peroxidation in vitro. Regardless of whether inhibition is at the level of scavenging CCl 4 metabolite radicals, or lipid radicals in membranes, radical trapping provides a plausible mechanism by which this compound inhibited CCl 4 hepatotoxicity.