Lipoxygenase, Seeding Peroxides and LDL Oxidation

Oxidation of the fatty acids in an LDL particle shares many of the characteristics associated with lipid peroxidation in other biological or chemical systems. Once initiated peroxyl radicals are formed and this results in the oxidation of a-tocopherol to give the a-tocopheroyl radical (Kalyanaraman etal., 1990). This can be demonstrated by e.s.r. techniques that allow the direct observation of stable radicals such as the a-tocopheroyl radical. After the a-tocopheryl radical is consumed, lipid-derived peroxyl radicals can be detected after reaction with spin traps (Kalyanaraman etal., 1990, 1991). 

The reactions described so far do not require the involvement of the apo-B protein, neither would they necessarily result in a significant amount of protein modification. However, the peroxyl radical can attack the fatty acid to which it is attached to cause scission of the chain with the concomitant formation of aldehydes such as malondialdehyde and 4-hydroxynonenal (Esterbauer et al., 1991). Indeed, complex mixtures of aldehydes have been detected during the oxidation of LDL and it is clear that they are capable of reacting with lysine residues on the surface of the apo-B molecule to convert the molecule to a ligand for the scavenger receptor (Haberland etal., 1984 Steinbrecher et al., 1989). In addition, the lipid-derived radical may react directly with the protein to cause fragmentation and modification of amino acids. 

The chemical adducts formed by reaction of aldehydes with lysine residues form highly immunogenic epitopes, and antibodies have been prepared specific for malondialdehyde- and 4-hydroxynonenal-conjugated LDL (Gonen et al., 1987 Yla-Herttuala et al., 1989 Jurgens et al., 1990). These antibodies cross-react with material in atherosclerotic lesions but not normal tissue, thus supporting the central role of lipid peroxidation in the patho nesis of atherosclerosis (Yla-Herttuala et al., 1989, 1991). 

An example of an experiment in which LDL has been treated with 15-lipoxygenase and the oxidation monitored by the formation of conjugated diene is shown in Fig. 2.2. In the absence of transition metal, a rapid increase in absorbance occurs, with no lag phase, which ceases after a period of about 90 min under these conditions. If copper is added to promote LDL oxidation at this point, LDL treated with lipoxygenase oxidizes at a faster rate with a short lag phase when compared to the control. During this procedure there is only a minimal loss of a-tocopherol and so we may ascribe the shortened lag phase to the increase in lipid peroxides brought about by lipoxygenase treatment. A similar result was found when LDL was supplemented with preformed fatty acid hydroperoxides (O Leary eta/., 1992). 

Rgure 2.3 The antioxidant activity of butyiated hydroxytoluene in the presence of exogenous iipid hydroperoxides. The oxidation of LDL was monitored by measuring the increase in absorbance at 234 nm as described in Fig. 2.2 and the lag phase (time before the phase of maximum rate of oxidation) estimated as described by Esterbauer et at. (1989). Samples of LDL were supplemented with the cortcentrations of 13-hydroperoxyoctadecanoic acid (13-HPODE) indicated and in the presence of 3 fM BHT. The lag phase in the absence of BHT for this preparation of LDL was 48 min. 

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