Edit Schnitzer

Peroxidation of liposomal lipids

Free radicals, formed via different mechanisms, induce peroxidation of membrane lipids. This process is of great importance because it modifies the physical properties of the membranes, including its permeability to different solutes and the packing of lipids and proteins in the membranes, which in turn, influences the membranes’ function. Accordingly, much research effort has been devoted to the understanding of the factors that govern peroxidation, including the composition and properties of the membranes and the inducer of peroxidation. In view of the complexity of biological membranes, much work was devoted to the latter issues in simplified model systems, mostly lipid vesicles (liposomes). Although peroxidation in model membranes may be very different from peroxidation in biological membranes, the results obtained in model membranes may be used to advance our understanding of issues that cannot be studied in biological membranes. Nonetheless, in spite of the relative simplicity of peroxidation of liposomal lipids, these reactions are still quite complex because they depend in a complex fashion on both the inducer of peroxidation and the composition and physical properties of the liposomes. This complexity is the most likely cause of the apparent contradictions of literature results. The main conclusion of this review is that most, if not all, of the published results (sometimes apparently contradictory) on the peroxidation of liposomal lipids can be understood on the basis of the physico-chemical properties of the liposomes. Specifically: (1) The kinetics of peroxidation induced by an “external” generator of free radicals (e.g. AAPH) is governed by the balance between the effects of membrane properties on the rate constants of propagation (kp) and termination (kt) of the free radical peroxidation in the relevant membrane domains, i.e. in those domains in which the oxidizable lipids reside. Both these rate constants depend similarly on the packing of lipids in the bilayer, but influence the overall rate in opposite directions. (2) Peroxidation induced by transition metal ions depends on additional factors, including the binding of metal ions to the lipid–water interface and the formation of a metal ions-hydroperoxide complex at the surface. (3) Reducing agents, commonly regarded as “antioxidants”, may either promote or inhibit peroxidation, depending on the membrane composition, the inducer of oxidation and the membrane/water partitioning. All the published data can be explained in terms of these (quite complex) generalizations. More detailed analysis requires additional experimental investigations.

Kinetic analysis of copper-induced peroxidation of LDL

We have employed our recently developed spectroscopic method of continuous monitoring of lipid oxidation to study the formation and decomposition of hydroperoxides in the time course of LDL oxidation. The results show satisfactory agreement with simulated time courses based on the following assumptions: (a) Both the rates of formation and decomposition of hydroperoxides depend on the ratio of bound copper to LDL as computed under the assumption that each LDL particle has 17 equivalent copper binding sites characterized by a dissociation constant K = 1 microM. (b) Peroxidation is initiated by copper-catalyzed decomposition of hydroperoxides (LOOH) into peroxy radicals (LOO.) and other products, including dienals. Under these assumptions, the rate of accumulation of LOOH can be computed from the equation (equation in text). The agreement between the simulated and experimentally-observed kinetics supports the assumptions used for simulations. The close agreement between the values of lipid oxidizability (kp/square root 2kt) obtained for LDL (0.035 (Ms)[-1/2]) and previously published data on the oxidizability of linoleates (0.02-0.11 (Ms)[-1/2]) lends further support for these assumptions.

The effect of albumin on copper-induced LDL oxidation

In an attempt to gain deeper understanding of the mechanism or mechanisms responsible for the protective effect of serum albumin against Cu(2+)-induced peroxidation of low density lipoprotein (LDL), we have examined the influence of the concentrations of bovine serum albumin (BSA), Cu2+ and LDL on the kinetics of peroxidation. Since the common method of monitoring the oxidation by continuous recording of the absorbance of conjugated dienes at 234 nm cannot be used at high BSA-concentrations because of the intensive absorption of BSA, we have monitored the time-dependent increase of absorbance at 245 nm. At this wavelength, conjugated dienes absorb intensely, whereas the background absorbance of BSA is low. Using this method, as well as the TBARS assay for determination of malondialdehyde, over a large range of BSA concentrations, we show that in many cases the influence of BSA on the kinetics of oxidation can be compensated for by increasing the concentration of copper. This reconciles the apparent contradiction between previously published data. Detailed studies of the kinetic profiles obtained under different conditions indicate that binding of Cu2+ to albumin plays the major role in its protective effect while other mechanisms contribute much less than copper binding. This conclusion is consistent with the less pronounced effect of BSA on the oxidation induced by the free radical generator AAPH. It is also shown that the copper-albumin complex is capable of inducing LDL oxidation, although the kinetics of the latter process is very different from that of copper-induced oxidation. Nevertheless, when compared to copper induced oxidation at similar concentration of the oxidation-promotor, the kinetics of oxidation induced by copper-albumin complex is very different and is consistent with a tocopherol mediated peroxidation, characteristic under low radical flux. Similar kinetics was observed for copper-induced oxidation only at much lower copper concentrations.

LDL-Associated Phospholipase a Does Not Protect LDL Against Lipid Peroxidation In Vitro

The irreversible proteinase inhibitor Pefabloc (4-[2-aminoethyl] benzenesulfonyl fluoride) inactivates LDL-catalyzed hydrolysis of the short-chain fluorescent phospholipid C6-NBD-PC (1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine). The dose-dependence of this inactivation is similar to that obtained previously for the inhibitory effect of Pefabloc on the hydrolysis of platelet activating factor (PAF) by the LDL-associated PAF acetylhydrolase (PAF-AH), in agreement with the notion that the hydrolysis of C6-NBD-PC and PAF is catalyzed by the same enzyme (LDL-associated phospholipase A; LDL-PLA). This conclusion is also supported by the finding that hydrolysis of C6-NBD-PC by LDL becomes inactivated by LDL oxidation only at late stages of the oxidation, similar to the effect of oxidation on the hydrolysis of PAF by the LDL-associated PAF-AH. Under conditions of complete inactivation of this enzyme towards C6-NBD-PC, the kinetics of lipid peroxidation, induced either by copper ions or by the free radical generator AAPH at varying doses of the prooxidant, was similar to that observed when the PLA was active (i.e., in the absence of Pefabloc). Hence, LDL-associated PLA (PAF-AH) does not protect LDL lipids from peroxidation. Similar results were obtained with fractionated LDL in albumin-containing buffer and for non-fractionated serum, in which copper-induced peroxidation was also not influenced by inactivation of the enzyme responsible for hydrolysis of C6-NBD-PC. Phospholipolysis of short chain phospholipids by LDL-PLA may still play a protective role against the toxic effects of oxidized phospholipids by reducing their internalization into cells (Schmitt et al. 1995).

Ranking antioxidants based on their effect on human serum lipids peroxidation

Evaluation of the activity of antioxidants is commonly based on measurements of the effect of a specific antioxidant on redox reactions conducted in a solution. Given the difference between reactions that occur in homogeneous solutions and those that occur at lipid-water interfaces, as in biological membranes and lipoproteins, the relevance of the commonly-used assays (such as TEAC and ORAC) to the antioxidative activity in biological systems is questionable. The aim of the present investigation is to develop a more relevant assay. Based on our results, we propose an assay based on prolongation of the lag preceding fast peroxidation of serum lipids. The assay employs our previously developed procedure for determination of susceptibility of serum lipids to peroxidation. The effect of antioxidants is expressed in terms of the relative prolongation of the lag preceding peroxidation. It can be considered reliable because it is only marginally dependent on the specific sera used for the assay. The resultant ranking of antioxidants may be expressed either as the relative prolongation of the lag per 1μM of antioxidant or as the concentration of antioxidant required to double the lag. As expected, the observed ranking order is very different from that reported for TEAC or ORAC assays, undermining the relevance of these assays for oxidation that occurs at interfaces.