Lipid hydroperoxide peroxidation

Scheme 2.1 The key reactions that occur during lipid peroxidation, in this scheme, X represents the initiating species, which must be a highiy reactive oxidant, in order to abstract a H atom from a poiyunsaturated fatty-acid chain LH, the iipid substrate LO2, the peroxyi radicai L, the alkyl radical LOOH, the lipid hydroperoxide.

In the previous section, we have described some of the mechanisms that may lead to the fijrmation of lipid hydroperoxides or peroxyl radicals in lipids. If the peroxyl radical is formed, then this will lead to propagation if no chain-breaking antioxidants are present (Scheme 2.1). However, in many biological situations chain-breaking antioxidants are present, for example, in LDL, and these will terminate the peroxyl radical and are consumed in the process. This will concomitandy increase the size of the peroxide pool in the membrane or lipoprotein. Such peroxides may be metabolized by the glutathione peroxidases in a cellular environment but are probably more stable in the plasma comjxutment. In the next section, the promotion of lipid peroxidation if the lipid peroxides encounter a transition metal will be considered. 

In this reaction scheme, the steady-state concentration of peroxyl radicals will be a direa function of the concentration of the transition metal and lipid peroxide content of the LDL particle, and will increase as the reaction proceeds. Scheme 2.2 is a diagrammatic representation of the redox interactions between copper, lipid hydroperoxides and lipid in the presence of a chain-breaking antioxidant. For the sake of clarity, the reaction involving the regeneration of the oxidized form of copper (Reaction 2.9) has been omitted. The first step is the independent decomposition of the Upid hydroperoxide to form the peroxyl radical. This may be terminated by reaction with an antioxidant, AH, but the lipid peroxide formed will contribute to the peroxide pool. It is evident from this scheme that the efficacy of a chain-breaking antioxidant in this scheme will be highly dependent on the initial size of the peroxide pool. In the section describing the copper-dependent oxidation of LDL (Section 2.6.1), the implications of this idea will be pursued further. 

Scheme 2.3 The inhibition of iipid peroxidation by antioxidants, in this scheme, AH represents an antioxidant A, the antioxidant-derived radicai LH, the lipid substrate LOj, the peroxyl radical L, the alkyl radical LOOM, the lipid hydroperoxide.

Extensive studies in vitro from many groups have confirmed that exposure of LDL to a variety of pro-oxidant systems, both cell-free and cell-mediated, results in the formation of lipid hydroperoxides and peroxidation products, fragmentation of apoprotein Bioo, hydrolysis of phospholipids, oxidation of cholesterol and cholesterylesters, formation of oxysterols, preceded by consumption of a-tocopherol and accompanied by consumption of 8-carotene, the minor carotenoids and 7-tocopherol. 

Esterbauer et cil. (1992) have studied the in vitro effects of copper on LDL oxidation and have shown that there are three distinct stages in this process. In the first part of the reaction, the rate of oxidation is low and this period is often referred to as the lag phase the lag phase is apparently dependent on the endogenous antioxidant content of the LDL, the lipid hydroperoxide content of the LDL particle and the fatty acid composition. In the second or propagation phase of the reaction, the rate of oxidation is much faster and independent of the initial antioxidant status of the LDL molecule. Ultimately, the termination reactions predominate and suppress the peroxidation process. The extensive studies of Esterbauer et al. have demonstrated the relative importance of the endogenous antioxidants within the LDL molecule in protecting it from oxidative modification. 

O Brien, P.J. (1969). Intracellular mechanisms for the decomposition of a lipid hydroperoxide I. Decomposition of a lipid peroxide by metals ions, haem compounds and nucleophils. Can. J. Biochem. 47, 485-492. 

Figure 17.2 Lipid peroxidation scheme. LH, a polyunsaturated fatty acid LOOM, lipid hydroperoxide LOH, lipid alcohol L, lipid radical LOO, lipid hydroperoxyl radical LO, lipid alkoxyl radical. Initiation the LH hydrogen is abstracted by reactive oxygen (e.g. lipid alkyl radical, lipid alkoxy radical, lipid hydroperoxyl radical, hydroxy radical, etc.) to produce a new lipid alkyl radical, L. Propagation the lipid alkyl, alkoxyl or hydroperoxyl radical abstracts hydrogen from the neighbouring LH to generate a new L radical.

Kaneko et al. (1993) have described a group of lipophilic ascorbic-acid analogues that have been studied in cultured human umbilical vein endothelial cells that were first incubated with test drug and then exposed to lipid hydroperoxides. Although ascorbate itself did not protect the endothelial cells, derivatives like CV3611 protected. Pretreatment was necessary. CV3611 was synergistic with vitamin E. The authors concluded that these lipophilic antioxidants incorporate into endothelial cell membranes where they are effective inhibitors of lipid peroxidation. In contrast, lipophobic antioxidants were not effective in their hands (Kaneko et al., 1993). 

Thiols are also important protection against lipid peroxidation. Glutathione (7-Glu-Cys-Gly) is used by several glutathione-dependent enzymes such as free-radical reductase (converts vitamin E radical to vitamin E), glutathione peroxidase (reduces hydrogen peroxide and lipid hydroperoxides to water and to the lipid alcohol, respectively), and others. In addition, the thiol group of many proteins is essential for function. Oxidation of the thiol of calcium ATPases impairs function and leads to increased intracellular calcium. Thiol derivatives such as the ovothiols (l-methyl-4-mercaptohistidines) (Shapiro, 1991) have been explored as therapeutics. 

As a reasonable biogenetie pathway for the enzymatic conversion of the polyunsaturated fatty acid 3 into the bicyclic peroxide 4, the free radical mechanism in Equation 3 was postulated 9). That such a free radical process is a viable mechanism has been indicated by model studies in which prostaglandin-like products were obtained from the autoxidation of methyl linolenate 10> and from the treatment of unsaturated lipid hydroperoxides with free radical initiators U). 

Moreover, redox cycling of free or weekly chelated iron may decompose a lipid hydroperoxide and thus initiate a chain of lipid peroxidation (Halliwell and Gutteridge, 2000). 

Luminol derivatives produce emission of light by oxidation with oxygen and hydrogen peroxide under alkaline conditions. By utilizing this reaction, peroxides such as hydrogen peroxide and lipid hydroperoxides can be determined after HPLC separation. Metal ions [e.g., iron(II), cobalt(II), etc.] catalyzing the luminol CL reaction can also be determined. 

Lipid peroxidation is probably the most studied oxidative process in biological systems. At present, Medline cites about 30,000 publications on lipid peroxidation, but the total number of studies must be much more because Medline does not include publications before 1970. Most of the earlier studies are in vitro studies, in which lipid peroxidation is carried out in lipid suspensions, cellular organelles (mitochondria and microsomes), or cells and initiated by simple chemical free radical-produced systems (the Fenton reaction, ferrous ions + ascorbate, carbon tetrachloride, etc). In these in vitro experiments reaction products (mainly, malon-dialdehyde (MDA), lipid hydroperoxides, and diene conjugates) were analyzed by physicochemical methods (optical spectroscopy and later on, HPLC and EPR spectroscopies). These studies gave the important information concerning the mechanism of lipid peroxidation, the structures of reaction products, etc. 

As mentioned earlier, MPO-hydrogen peroxide-chloride system of phagocytes induces the formation of lipid peroxidation products in LDL but their amount is small [167-169], It was proposed that HOCL can decompose the lipid hydroperoxides formed to yield alkoxyl radicals [170]. It was also suggested that chloramines formed in this process decompose to free radicals, which can initiate lipid peroxidation [171]. 

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