Antioxidants and total peroxyl radical-trapping ability of olive and seed oils.

The presence of the oxidized and reduced forms of ubiquinones Q(9) and Q(10) was determined in commercial extra virgin olive and seed oils, where the amounts of alpha- and gamma-tocopherols and beta-carotene were also quantitated. Very high concentrations of ubiquinones were found in soybean and corn oils. Furthermore, the total antioxidant capability of each oil was evaluated by measuring total radical-trapping antioxidant parameters (TRAP) in tert-butyl alcohol and using egg lecithin as the oxidizable substrate. These values decreased in the order sunflower > corn > peanut > olive; the highest TRAP, which was found in sunflower oil, was related to the very high amount of alpha-tocopherol. Olive oil, because of the low content of alpha-tocopherol, exhibited a TRAP value approximately one-third that of sunflower oil. TRAP values of corn and soybean oils, in which low amounts of alpha-tocopherol but very high contents of gamma-tocopherol and reduced ubiquinones were present, were intermediate. gamma-Tocopherol exhibited a poor ability of trapping peroxyl radicals in tert-butyl alcohol. This behavior was probably due to the effects of the solvent on the rate of hydrogen abstraction from this phenol.

Acute regulation of glucose transport in a human megakaryocytic cell line: difference between growth factors and H(2)O(2).

The present study was undertaken to: (i) compare the effect of some hematopoietic growth factors, like interleukine-3, thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, stem cell factor, and reactive oxygen species such as H(2)O(2) on glucose uptake in a human leukemic megakaryocytic cell line, M07; (ii) investigate the changes in kinetic parameters of the transport activity induced by these stimuli; and (iii) evaluate the effect of genistein, a tyrosine kinase inhibitor, on the glucose uptake activation by the cited agents. The results are as follows: (i) exposure of M07 cells to thrombopoietin, granulocyte-megakaryocyte colony-stimulating factor, and stem cell factor resulted in a rapid stimulation of glucose transport; interleukine-3-treated cells exhibited no increase in the rate of glucose uptake, although M07 proliferation is interleukine-3 dependent; a rapid glucose transport enhancement was also observed when M07 cells were exposed to low doses of H(2)O(2); (ii) the transport kinetic parameters point out that an important difference exists between the effect of cytokines and that of H(2)O(2): cytokines increased predominantly the affinity for glucose, while H(2)O(2) raised both the V(max) and K(m) values; (iii) the isoflavone genistein, at a very low concentration, inhibited the stem cell factor- or H(2)O(2)-induced stimulation of hexose transport, reversing the variations of K(m) and V(max), but it did not affect the transport activity of granulocyte-megakaryocyte colony-stimulating factor-treated cells; and (iv) catalase completely abolished the stimulatory action of H(2)O(2) on glucose transport and slightly prevented the effect of stem cell factor, while caffeic acid phenethyl ester was only able to affect the activation due to stem cell factor.

The effect of oxygen radicals on rat thymocyte glucose transport is independent of the site of their generation.

We studied the relationship between the site of production of oxygen radicals and their effect on a rat thymocyte functional activity, the glucose transport, measured using a radioactive analogue of glucose, 2-deoxy-glucose. We compared the effects of a hydrophilic thermolabile azo compound, mimicking a radical attack outside the cell, with the lipid-soluble cumene hydroperoxide, which initiates lipid peroxidation in cell membranes. Our results show that a low grade oxidative stress stimulated glucose uptake rapidly, independently of the site of radical generation. In the presence of the azocompound, glucose uptake increased smoothly, attaining its maximum extent within 1 h. In thymocytes treated with cumene hydroperoxide the rate of glucose transport increased suddenly and remained constant over 1 h. The effects of the radical donors on TBARS production and protein sulfhydryl groups content were also evaluated. In thymocytes treated with the azo derivative no lipid peroxidation was observed, but a slow decrease of protein thiol groups occurred; after the addition of cumene hydroperoxide sulfhydryl groups did not change and TBARS increased significantly. The water-soluble antioxidant Trolox was able to remove the glucose uptake increase induced by the hydrophilic initiator and to delay the loss of membrane integrity.

Heat inactivation of fetal calf serum is not required for in vitro measurement of lymphocyte functions.

This study was undertaken to test whether fetal calf serum (FCS) must be heat inactivated before use in tissue culture. We tested various immune functions of lymphocytes growing in medium containing non-treated and heat-inactivated FCS. The data clearly show that heat inactivation of the serum is not mandatory. In some cases, the addition of untreated FCS resulted in elevated response levels, while maintaining immune function specificity.

Medium effects on the antioxidant activity of dipyridamole.

Very strong medium effects have been observed when testing the antioxidant activity of dipyridamole (DP) in different media such as benzene, tert-butanol, methanol solutions and egg yolk lecithin unilamellar and multilamellar vesicles. Actually, dipyridamole behaves as a very poor antioxidant in benzene while its ability to inhibit the lipid peroxidation reaction increases with increasing solvent polarity, being the highest in lipid vesicles. This behavior can not be rationalized on the basis of the classical chain breaking mechanism which operates in the case of phenolic and amine antioxidants and involving the transfer of a hydrogen atom to peroxyl radicals. An explanation is instead given in terms of an electron transfer reaction which leads to the oxidation of DP by the chain carrying peroxyl radical to give the dipyridamole cation radical, DP+*, and the peroxyl anion LOO-, and whose rate constant is expected to increase in strongly polar media. EPR and electrochemical data supporting this interpretation have been collected.

Vitamin B6 deficiency affects antioxidant defences in rat liver and heart.

We have evaluated 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.

HPLC and light scattering detection allow the determination of phospholipids in biological samples and the assay of phospholipase A2.

Some applications to biological samples of a method for the separation and the quantitative analysis of phospholipids by high performance liquid chromatography (HPLC) and light scattering mass detection are described. Results obtained in the determination of phospholipid classes from rat tissues such as liver, heart and kidney have been compared with data from the literature. The method has been applied to the evaluation of phospholipids in human low-density lipoproteins (LDL), about which little is known. The procedure is also suitable for a rapid and reliable assay of the water-soluble phospholipase A2 activity; the relationship between the aggregation state of substrate phospholipids (mixed micelles, multilamellar and unilamellar vesicles) and the enzyme activity has been studied.

Antioxidant activity of reduced menadione in solvent solution and in model membranes.

The antioxidant activity of reduced menadione was investigated and compared with that of alpha-tocopherol both in solvent solution and in large unilamellar vesicles by using azocompounds as free radical generators. The results show that: i) reduced menadione behaves as a chain-breaking antioxidant; ii) its inhibition rate constant is similar to that of alpha-tocopherol in homogeneous solution, whereas it is 4 times larger in egg yolk lecithin vesicles; iii) the stoichiometric factor is found lower than 1 in both systems, since a substantial portion of menadiol is consumed by autoxidation and does not contribute to radical trapping; iv) when both alpha-tocopherol and menadiol are present in vesicles, reduced menadione can spare alpha-tocopherol. Data presented here suggest that the reduced form of vitamin K may protect, when present, cellular membranes from free radical damage.

DT-Diaphorase maintains the reduced state of ubiquinones in lipid vesicles thereby promoting their antioxidant function.

The activity of purified DT-diaphorase in the reduction of ubiquinone homologues of different side-chain length incorporated in uni- and multilamellar vesicles was determined. The direct relationship between the reduced state of ubiquinones and the inhibition of lipid autoxidation induced by thermolabile azocompounds was also demonstrated. Results demonstrate that DT-diaphorase is able to generate and to maintain the reduced, antioxidant form of ubiquinones in both types of vesicles. Furthermore, the results reported herein show that, in the presence of nicotinamide adenine dinucleotide (NADH) and DT-diaphorase, ubiquinol-containing multilamellar vesicles exposed to a lipophilic azocompound did not undergo lipid peroxidation, whereas in vesicles lacking either NADH or DT-diaphorase, thiobarbituric acid reactive substances (TBARS) formation occurred. It is suggested that DT-diaphorase may be responsible for maintaining the reduced state of ubiquinones in various nonmitochondrial cellular membranes.

The two-electron quinone reductase DT-diaphorase generates and maintains the antioxidant (reduced) form of coenzyme Q in membranes.

The experiments reported here were undertaken to test the hypothesis that the antioxidative, reduced form of hydrophobic phase coenzyme Q (CoQ) may be generated and maintained by the two-electron quinone reductase, DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] by catalyzing formation of the hydroquinone form of CoQ. This enzyme was isolated and purified from rat liver cytosol and its reduction of several CoQ homologs incorporated into large unilamellar vesicles (LUVETs) was demonstrated. The addition of NADH and DT-diaphorase to LUVETs and to multilamellar vesicles (MLVs) containing CoQ homologs, including CoQ9 and CoQ10, resulted in essentially complete reduction of the CoQ. Incorporation of either CoQ9H2 or CoQ10H2 and the lipophylic radical generator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) into MLVs in the presence of DT-diaphorase and NADH maintained the reduced state of CoQ and inhibited lipid peroxidation. The reaction between DT-diaphorase and CoQ was also demonstrated in isolated rat liver hepatocytes in which incorporation of CoQ10 provided protection from adriamycin (adr)-induced mitochondrial membrane damage. The role of DT-diaphorase in the antioxidant activity of CoQ was demonstrated by the co-incorporation of dicoumarol (dic), a potent inhibitor of DT-diaphorase, resulting in a loss of protection by incorporated CoQ10. These results support the antioxidant function of DT-diaphorase in both artificial and natural membrane systems by acting as a two-electron CoQ reductase which forms and maintains CoQ in the reduced state.

The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.

Injury of rat thymocytes caused by exogenous peroxyl radicals in vitro.

The aim of this study was to investigate oxidative cell injury in rat thymocytes under conditions of radical generation exterior to the cell utilizing the thermolabile azocompound 2,2'-azobis(2-amidinopropane) dihydrochloride to generate peroxyl radicals at a constant and reproducible rate. This initiator, being water-soluble and endowed with a positive charge, is suitable for studies on oxidative damage of biomembranes induced in the external water environment. The relationship between cell viability, lipid and thiol oxidation and chain-breaking antioxidant depletion was studied. During the first hour of treatment cell viability decreased slightly, protein sulfhydryl groups were consumed slowly and no significant production of conjugated dienes occurred. After 90 min of incubation, when thymocyte permeability started to increase, the concentration of alpha-tocopherol decreased gradually, significant changes of polyunsaturated fatty acids occurred and a rapid phase of thio oxidation commenced. It can be concluded that, under conditions of an exogenous oxidant challenge, initially the cell membrane provides a physical barrier to the entrance of radicals to the thymocyte. When peroxyl radicals gain access to the membrane and the molecular barrier begins to disorganize, the oxidizable cellular components become susceptible to massive attack.

Characterization of large unilamellar vesicles as models for studies of lipid peroxidation initiated by azocompounds.

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.

Autoxidation and antioxidant activity of ubiquinol homologues in large unilamellar vesicles.

The antioxidant activity of ubiquinol homologues with different side-chain length such as ubiquinol-3 and ubiquinol-7 was compared with that of alpha-tocopherol when peroxidation was induced by the water-soluble initiator 2,2'-azobis-(2-amidinopropane hydrochloride). In large unilamellar vesicles containing equal amounts of alpha-tocopherol, ubiquinol-3 and ubiquinol-7 the rates of inhibition were very similar but the stoichiometric factor of quinols was approximately 1. To explain this low value, which is one-half of that found when the autoxidation was performed in apolar solvents (Chem. Phys. Lipids (1992) 61, 121-130), the oxidation of alpha-tocopherol and ubiquinol-3 initiated by the azocompound was studied both in methanol and in dimiristoyl-lecithin vesicles. The results obtained show that the ubiquinol homologues undergo a radical chain reaction taking place at the polar interface and suggest that the average preferred location of both quinol headgroups is near to the outer surface of the bilayer.

Ubiquinol-3 and ubiquinol-7 exhibit similar antioxidant activity in model membranes.

This study was undertaken to compare, on a kinetic basis, the antioxidant efficiency of an ubiquinol homologue having a short isoprenoid side-chain length, such as ubiquinol-3, with that of the long chain ubiquinol-7, by determining their rate constants of inhibition with respect to alpha-tocopherol. To this purpose we incorporated ubiquinol-3, or ubiquinol-7, or alpha-tocopherol into liposomes of egg yolk lecithin, and triggered lipid peroxidation with the thermal decomposition of a lipophilic azocompound. The results show that: i) the rate constants of inhibition for the two quinols are similar and slightly lower than that of alpha-tocopherol; ii) the length of the radical chain obtained in the presence of the two quinols is almost the same. From these data we concluded that the two homologues tested behave as chain-breaking antioxidants with quite similar effectiveness.

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.

The influence of phospholipid polar head on the lipid hydroperoxide dependent initiation of lipid peroxidation.

In a buffer (Mes) and at a pH (6.5) where Fe2+ is very stable, we have studied the peroxidation of liposomes catalyzed by FeCl2. The liposomes studied, prepared by sonolysis, contained either phosphatidylcholine or 1:1 molar ratio of phosphatidylcholine and phosphatidic acid. The presence of the negatively charged phospholipid causes: 1) rapid Fe2+ oxidation and oxygen consumption; 2) increased generation of lipid hydroperoxides; 3) decreased generation of thiobarbituric acid-reactive materials; 4) very low inhibition of Fe2+ oxidation and lipid hydroperoxide generation by BHT; 5) inhibition of the termination phase of lipid peroxidation at high FeCl2 concentrations. A hypothesis is proposed to explain the results obtained.

Reactions of oxygen radicals with the quinone ring of coenzyme Q.

Coenzyme Q, besides its role in electron transfer reactions, may act as a radical scavenger. The effect of oxygen radicals produced by ultrasonic irradiation on the quinone ring was investigated. Aqueous solutions of a Q homologue, completely lacking the side chain, were irradiated and the modifications were spectrophotometrically followed. The experimental results show that both degradation and reduction of the benzoquinone ring took place when the irradiation was performed in water. Data obtained when ultrasonic irradiation was carried out in the presence of OH. scavengers, as formate, organic and inorganic buffers, suggest: a) the responsible species for most the ubiquinol generated by sonication appeared to be the superoxide radical b) addition reactions of OH. radicals with the aromatic ring led probably to the degradation of Coenzyme Q molecules.

Ubiquinol prevents alpha-tocopherol consumption during liposome peroxidation.

In this study we investigated whether alpha-tocopherol can be spared by ubiquinol-3 during autoxidation of multilamellar liposome. A lipophilic azocompound, 2,2'-azobis-(2,4-dimethyl-valeronitrile), was chosen to initiate liposome autoxidation. The effect of either alpha-tocopherol, ubiquinol-3, or a mixture of them was compared. Rates of conjugated diene formation and concomitant disappearance of the two antioxidants was measured. Since the inhibition rate constant for the scavenging of peroxyl radical for alpha-tocopherol was higher than that for quinol-3, it was concluded that alpha-tocopherol is regenerated by ubiquinol-3.

The role of the side chain in the antioxidant activity of ubiquinones.

The question has been addressed whether the side chain contributes to the antioxidant activity of Ubiquinones. The length, the chemical composition and structure of the chain have been considered. The effect of the actual concentration of the quinone in egg lecithin vesicles has been investigated by means of both UV spectroscopy and time resolved fluorescence quenching experiments of 12-AS. The results indicate that the antioxidant properties of the quinone do not seem to depend on the side chain.