Luciana Cabrini

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than α-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than α-tocopherol; (e) to be a weaker antiradical than α-tocopherol in the reduction of the stable radical DPPH·. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like α-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.

Solvent and pH effects on the antioxidant activity of caffeic and other phenolic acids.

The antioxidant activity of several phenolic acids and esters has been investigated both in organic solutions and in large unilamellar phosphatidylcholine vesicles. In solution these compounds behaved as good antioxidants, with the exception of protocatechuic acid, due to the presence of the catechol moiety. Because their antioxidant activity followed an inverse dependence on the magnitude of their O-H bond dissociation enthalpies (BDE), the key mechanism of the chain-breaking action was attributed to hydrogen atom transfer (HAT) from the phenolic OH to peroxyl radicals. In unilamellar vesicles the antioxidant activity was strongly dependent on the pH of the buffer solution. In acid media (pH 4) all of the examined phenolic acids or esters behaved as weak inhibitors of peroxidation, whereas, with increasing pH, their antioxidant activity increased substantially, becoming comparable to or even better than that of Trolox. At pH 8 they also gave rise to lag phases 2-3 times longer than that of Trolox. The increased activity being observed in proximity of the pK(a) value corresponding to the ionization of one of the catecholic hydroxyl groups, this effect has been attributed to the high antioxidant activity of the phenolate anion.

Polyamine binding to phospholipid vesicles and inhibition of lipid peroxidation

A study of the possible mechanism of inhibition by polyamines of lipid peroxidation was made utilizing vesicles prepared with mixed soy bean phospholipids. The results obtained can be summarized as follows: 1) Polyamines inhibit lipid peroxidation only when bound to the negative charges on vesicle surface. 2) Polyamines inhibit lipid peroxidation at concentrations lower than those required to cause precipitation of the vesicles and similar to those required for formation of the polyamine/phospholipid vesicle complex. 3) Spermine bound to vesicles, in contrast to free spermine, highly decreases the reactivity of both Fe2+ and Fe3+ versus superoxide.

Vitamin B6 deficiency affects antioxidant defences in rat liver and heart

We have evalued the effects of a diet containing normal amounts of lipids and a marginal content of vitamin B6 on lipid peroxidation. Pyridoxal phosphate concentrations of plasma and liver indicated that an initial deficiency state was reached. Vitamin B6 deficiency led to peroxidative stress: TBARS production was higher in the liver (+18·6%) and even more in the heart (+61%) of deficient rats as compared with controls. Furthermore, significant stimulation of glutathione‐dependent enzymes occurred in both heart and liver of deficient rats: glutathione peroxidase activity increased in heart (+144%) and liver (+505%); glutathione reductase increased in heart (+54·9%) and liver (+15·5%). No difference in the total glutathione content of the organs of the two groups was observed. The reduced glutathione/oxidized glutathione ratio was significantly lower in deficient rats. Although the activity of glutathione‐dependent enzymes was significantly greater in deficient rats than in controls, this stimulation was only partially able to counteract the peroxidative damage due to vitamin B6 deficiency.

Inhibition of phospholipase A2 and phospholipase C by polyamines.

Abstract The polyamines spermine, spermidine, and putrescine inhibit the activity of phospholipase A2 (Naja naja) and phospholipase C (Clostridium welchii) on phospholipid vesicles and mitochondrial membranes as sources of substrate phospholipids. The inhibitory effect is highest for spermine and lowest for putrescine. With both enzymes, inhibition is stronger when phospholipid vesicles rather than mitochondrial membranes are used as the substrate. No clear competition of polyamines with Ca2+, which is required for the activity of both enzymes, has been observed. The inhibition appears to be due to steric hindrance of enzyme-substrate interaction due to the binding of the organic polycations to the phospholipid bilayer.

Extraction of lipids and lipophilic antioxidants from fish tissues: a comparison among different methods.

1. Lipids, phospholipids and lipid soluble antioxidants were extracted from Sparus auratus liver and white muscle by three different methods and the yields obtained were compared. 2. None of the three procedures can recover the above components with the same efficiency. 3. For comparison the methods were also applied to rat liver homogenates. 4. The choice of the extraction procedures depends on the tissue investigated and on specific research requirements.

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe2+ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl2 and FeCl3 on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe2+ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl2/FeCl3 ratio when PC liposomes deprived of LOOH by triphenylphosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl2. The FeCl2 concentration at which Fe2+ oxidation was maximal, defined as critical Fe2+ concentration [Fe2+]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl3 addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe2+]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl3 addition. The experimental data obtained indicate the kinetics of LOOH-dependent lipid peroxidation depends on the Fe2+/Fe3+ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl3 does not affect the LOOH-independent but the LOOH-dependent lipid peroxidation; and suggest that the Fe3+ endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.

Antioxidant behaviour of Ubiquinone and β-Carotene Incorporated in model Membranes

Experiments with model membranes, in which ubiquinone was incorporated, were performed in order to clarify the mechanism by which ubiquinone can prevent or control chain lipid peroxidation in biomembranes. Comparing the behavior of ubiquinone-containing vesicles with beta-carotene containing vesicles we suggest that a possible explanation of the ubiquinone antioxidant effect could be to scavenge singlet oxygen and to affect structurally the lipid bilayer inhibiting hydroperoxide decomposition.

The antioxidant activity of ubiquinol-3 in homogeneous solution and in liposomes

With a view to determining the antioxidant effectiveness of ubiquinol, the autoxidation of egg phosphatidylcholine initiated by an azocompound was studied both in homogeneous solution and in liposomes, either in the presence or in the absence of ubiquinol-3. The results show that ubiquinol behaves as a chain-breaking antioxidant by trapping lipid peroxyl radicals, its inhibition rate constant being about one half of that of alpha-tocopherol in both systems under investigation. In organic solvents the stoichiometric factor was found approx. 2 and in liposomes approx. 0.5, i.e. one fourth of that of alpha-tocopherol. We suggest that the lower value found in model membranes is due to autoxidation of the quinol itself by a radical chain reaction taking place at the polar interface. Ubiquinol-3 exhibits a sparing effect toward alpha-tocopherol, both in liposomes and in tert-butanol. It is suggested, on a thermodynamic basis, that the regeneration of vitamin E from the corresponding radical is more likely to occur by reaction with the ubisemiquinone rather than with the ubiquinol. Although these results, obtained in in vitro systems, can not be directly extrapolated to an in vivo system, they may be useful to clarify the antioxidant role of ubiquinol in biomembranes.

Effect of endogenous ubiquinone on the interaction of exogenous Ubiquinone-1 with the respiratory chain of bovine heart mitochondria.

Abstract Pentane extraction of lyophilized mitochondria with depletion of up to 92% of endogenous ubiquinone (UQ) does not affect ubiquinol oxidase activity in terms of K m ; in certain preparations the V is decreased probably because the extraction is harmful to the membrane integrity. In such case dl -α-tocopherol is able to maintain enzymatic activity up to the normal values found in control mitochondria. On the other hand, NADH-UQ-1 reductase activity is greatly affected by pentane extraction with a large decrease in V but no change in K m , but this activity is protected by addition of dl -α-tocopherol to the extraction medium. The same conclusions can be drawn for succinate-UQ-1 reductase activity. In conclusion it appears that endogenous UQ does not mediate the interaction of exogenous UQ-1 with the redox sites for UQ in the respiratory chain.