Fe(2+)-induced lipid peroxidation kinetics in liposomes: the role of surface Fe2+ concentration in switching the reaction from acceleration to decay.

Kinetics of malonyldialdehyde (MDA) accumulation, Fe2+ oxidation, and chemiluminescence (CL) at different initial iron ([Fe2+]) and liposome ([L]) concentrations were measured in liposome suspension. Above certain critical Fe2+ concentrations ([Fe2+]*) the latent period (LP) of LPO development was observed. The method of [Fe2+]* estimation by the dependence of LP value (tau) on [Fe2+] was elaborated. The increase of [L] resulted in decrease of tau and increase of delta MDA as well as SF CL amplitude. [Fe2+]* value changed from 10 to 50 microM with change of [L] from 1 to 4 mg/ml, so that the ratio [Fe2+]*/[L] was kept constant. This may be explained under the assumption that the major part of Fe2+ is bound by the membranes. At concentrations of Fe2+ higher than the critical one, iron chelators (desferrioxamine, o-phenanthroline, and EDTA) and cations (Eu3+, Ca2+, and Fe3+) decreased tau without any essential influence on the CL "slow flash" amplitude (h). Apparently, the only result of iron complexones and cations on LPO is the decrease of Fe2+ ion concentration on the membrane surface. Thus, [Fe2+]* value and surface concentration of Fe2+ are the main parameters determining both kinetics and efficiency of Fe(2+)-induced LPO in membrane systems.

Coumarin derivatives enhance the chemiluminescence accompanying lipid peroxidation.

The effect of laser dyes, derivatives of 1,2-benzopyrone (coumarin), on the chemiluminescence (CL) accompanying Fe(2+)-induced lipid peroxidation (LPO) in liposomes prepared from egg yolk phospholipids has been investigated. It was found that quinolizin (9a,9,1-gh)-substituted coumarins enhanced CL at the stages of "fast" and "slow" flashes (abbreviated as FF and SF, respectively), which are known to accompany lipid hydroperoxide decomposition (FF) and chain LPO reaction development (SF). On the other hand, these compounds did not virtually change the shape of CL curve (in particular, lag phase duration) and accumulation of the LPO products reacting with 2-thiobarbituric acid (TBARS). The dependences of FF and SF amplitudes on the concentration of coumarins exhibited for some compounds an effect of saturation with subsequent decrease of CL at high concentrations of the dyes. The highest degree of CL amplification was reached with the compound 2,3,5,6-1H,4H-tetrahydro-9-(2'-benzoimidazolyl)-quinolizin- (9,9a,1-gh)coumarin (C-525), which enhanced CL at the stages of FF and SF by a factor 1600 at a dye concentration of 8 nmoles/mg of phospholipid. On the other hand, C-525 did not increase the intensity of CL associated with the decomposition of H2O2 by Fe2+ ions (Fenton's reaction). Apparently, these coumarin sensitizers may be used for selective enhancement of CL associated with LPO both in experimental and clinical investigations.

Carotenoid mixtures protect multilamellar liposomes against oxidative damage: synergistic effects of lycopene and lutein.

Antioxidant activity of carotenoids in multilamellar liposomes assayed by inhibition of formation of thiobarbituric acid-reactive substances was in the ranking: lycopene> alpha-tocopherol > alpha-carotene > beta-cryptoxanthin > zeaxanthin = beta-carotene > lutein. Mixtures of carotenoids were more effective than the single compounds. This synergistic effect was most pronounced when lycopene or lutein was present. The superior protection of mixtures may be related to specific positioning of different carotenoids in membranes.

Hypochlorite induces lipid peroxidation in blood lipoproteins and phospholipid liposomes.

The accumulation of lipid peroxidation products reacting with 2-thiobarbituric acid (TBARS) has been observed both in very low density blood lipoprotein (VLDL) and suspensions of liposomes prepared from VLDL phospholipids incubated with hypochlorite. Butylated hydroxytoluene (BHT) completely inhibited TBARS formation at a concentration of 100 microM, at which it decreased the concentration of hypochlorite in the absence of liposomes only by 7%. The formation of lipid peroxidation products in course of the incubation of egg yolk phospholipid liposomes with hypochlorite has been revealed using three methods: (1) measurement of TBARS, (2) measurement of additional amounts of TBARS resulting from the introduction of excess Fe2+ to peroxidized liposomes (delta TBARS), and (3) measurement of the chemiluminescence flash amplitude appeared upon the addition of Fe2+ to the suspension. The results obtained by all these methods were similar: Lipid peroxidation products were accumulated during the first 2 to 3 h of liposome incubation with 100 microM hypochlorite, and the amount of lipid peroxidation products accumulated after incubation was directly proportional to the initial hypochlorite concentration. These data suggest that hypochlorite can initiate lipid peroxidation both in lipoproteins and phospholipid liposomes.

Two processes responsible for chemiluminescence development in the course of iron-mediated lipid peroxidation.

The kinetics of chemiluminescence (CL) accompanying Fe(2+)-induced lipid peroxidation (LPO) in liposome suspension has been investigated. A sequence of stages was observed, namely: (1) fast CL flash (FF); (2) latent period (LP); (3) slow CL flash (SF) and (4) stationary chemiluminescence (SL). The first three stages are known to reflect the Fe(2+)-mediated LPO process. In spite of the fact that at the stage of SL Fe2+ has completely oxidized and MDA has not accumulated, CL intensity was found to increase and after 0.5-1 h reached a value that was several times higher than SF amplitude. The maximal SL level was linearly dependent on the initial Fe2+ concentration and was not dependent on liposome concentration in the suspension. The nature of the processes responsible for CL emission at the stage of SL has been investigated using free radical reaction inhibitors and measurement of CL spectra. The SL spectrum was observed in the red region (lamda > 590 nm) in contrast to the SF CL spectrum (maximum at 540nm). SL amplitude was strongly inhibited by sodium azide (40%), superoxide dismutase (SOD) (30%), desferrioxamine and EDTA (30%), whereas mannitol, ethanol, alpha-tocopherol and butylated hydroxytoluene were ineffective. The data obtained indicate that CL at the stage of SL is not directly related to LPO process, i.e. lipid free radical recombination. The mechanism of stationary CL generation is discussed.