LDL is a spherical particle of 20 to 22 nm diameter consisting of a molecule of apoprotein B-100 (apo B) embedded in a monolayer "surface" of polar phospholipids and cholesterol that surrounds a "core" of neutral cholesteryl esters and triglycerides (Table 9.1). The oxidizability of LDL lipids is primarily dependent on their content of bisallylic hydrogen atoms. Cholesteryl esters contain ~ threefold more bisallylic hydrogens than phospholipids and as such are the major lipid substrates for peroxidation in LDL (Table 9.1). LDL lipid peroxidation is generally held to proceed, and to a certain extent cause, the oxidation of apo B33 and oxidized lipid possess various proatherogenic activities.8,34 Therefore, understanding the molecular events involved in LDL lipid peroxidation and how antioxidants prevent this process may provide important information in designing antioxidant strategies to attenuate oxidative modification of lipoproteins in vivo and perhaps atherosclerosis. Importantly, however, certain oxidants (e.g., HOCl35 and peroxynitrite31) directly oxidize apo B independent of lipid peroxidation. Therefore, different antioxidant strategies may be required to adequately protect both lipoprotein lipid and protein moieties from oxidative modification in vivo.
Before discussing the molecular mechanisms of lipoprotein lipid peroxidation (i.e., complex heterogeneous lipid emulsions) and the role of a-TOH and CoQ10H2 in this process, we will first briefly review the features of lipid oxidation and antioxidation occurring in homogeneous systems and liposomes.
9.2.1 Radical Scavenging Activity of a-TOH and CoQ10H2
Studies in homogeneous solutions and liposomes have established that a-TOH36 and CoQ10H237-39 are effective lipophilic chain-breaking antioxidants and as such effectively suppress lipid peroxidation. Thus, a-TOH and CoQ10H2 rapidly react with the chain-carrying lipid peroxyl radical (LOOO (Reactions 9.1 and 9.2). Alternatively, a-TOH and CoQ10H2 can react directly with the peroxidation initiating peroxyl radical (ROOO (Reactions 9.3 and 9.4). Radical scavenging by a-TOH and CoQ10H2 results in the formation of the relatively unreactive a-tocopheroxyl radical (a-TOO and the protonated ubisemiquinone radical (CoQ10H a para hydroxy substituted phenoxyl radical), respectively.
The chain-breaking action of a-TOH and CoQ10H2 results in a well-defined "lag period" during which less than one mole of lipid hydroperoxide (LOOH) is formed per mole of a-TOH or CoQ10H2 consumed (i.e., radical chain length v < 1.0). The length of the lag phase is increased when a-TOH and CoQ10H2 are added to liposomes in combination38,39 and CoQ10H2 is consumed before a-TOH in this system.38,39 As the rate constants for the reaction of peroxyl radicals and a-TOH or CoQ10H2 are comparable, a "sparing" effect of CoQ10H2 for a-TOH suggests a reduction of a-TO^ by CoQ10H2. Studies in organic solution,40 liposomes,41 or autoxidizing mitochondrial membranes42 have provided more direct support for the reduction of a-TO^ by CoQ10H2.
Various studies have reported that in liposomes, a-TOH prevents lipid peroxidation more efficiently than CoQ10H2.38,43,44 The lower antioxidant efficacy of CoQ10H2 is likely due to its greater propensity to autoxidize (via CoQ10H and the ubisemiquinone radical, CoQ^ ), a process that requires protons (H+ )45 (Reactions 9.5 and 9.6).
Such autoxidation likely explains why the stoichiometric number for CoQ10H2 in liposomes is ~1.38 The lower antioxidant efficiency of CoQ10H2 may also derive from a competing prooxidant activity of CoQ10H .4446 Thus, CoQ10H may autoxidize in an aprotic, lipophilic environment to give rise to the hydroperoxyl radical OOOH) (Reactions 9.6), which itself can oxidize lipids.47 Furthermore, a study in organic solvents has suggested that CoQ10H can promote homolysis of H2O2 or LOOH to form more highly reactive •OH and alkoxyl radicals, respectively.44
Although studies in homogeneous solution and liposomes can provide valuable information, caution is required in the extrapolation of such results to biological membranes or lipoproteins. First, the ratio of coenzyme Q to lipid employed in the liposomal systems is extremely high and nonphysiological. Second, the precise location and orientation of coenzyme Q in liposomes is unknown and may be different from that in biomembranes. In biomembranes, coenzyme Q can interact with proteins and there is evidence that at least in mitochondria, membrane proteins can bind and stabilize the ubisemiquinone radical.48
9.2.2 The Role of a-TOH and CoQ10H2 in LDL Lipid Peroxidation is Dependent on the In Vitro Oxidizing Conditions Employed
Similar to the situation in homogeneous solutions, a clearly defined initial period of low rates of lipid peroxidation is observed when isolated LDL is oxidized by exposure to high and nonphysi-ological concentrations of Cu2+.49 During this initial period CoQ10H2, a-TOH, and other compounds referred to as "antioxidants" (e.g., carotenoids) are consumed rapidly. Following complete consumption of these antioxidants, lipid peroxidation proceeds at high rates.49 These results suggested that a-TOH represents an effective chain-breaking antioxidant for LDL's lipids in vitro. Consistent with this, supplementation of the lipoprotein with a-TOH increases the length of the "lag phase," when LDL is exposed to these strongly oxidizing conditions.13 50 51
However, many studies have documented a lack of significant correlation between a-TOH content and duration of lag time when native LDL is exposed to high Cu2+ concentrations.19 20 52 53 Furthermore, when CoQ10H2-free lipoproteins and ascorbate- and CoQ10H2-free plasma are exposed to more mild oxidizing conditions, a-TOH promotes, and is even required for efficient initiation of lipid peroxidation.1754 Under such mild oxidizing conditions, lipoprotein lipid peroxidation in CoQ10H2-free lipoproteins (i) proceeds via a radical chain reaction of length > 1 in the presence of a-TOH; (ii) is accelerated by enriching the LDL with a-TOH, (iii) is markedly suppressed in LDL deficient in a-TOH; and (iv) is faster in the presence of a-TOH than immediately after its complete consumption.14,15 17,18,25,28,31 55 These findings are not consistent with the conventional view that vitamin E acts as a chain-breaking antioxidant for LDL lipids.
A kinetic analysis of LDL lipid peroxidation initiated by ROO^ resulted in the formulation of TMP15 as a general model to explain the molecular events involved in lipid peroxidation and antioxidation in isolated, a-TOH-containing LDL exposed to radical oxidants (Figure 9.1). The TMP model of lipid peroxidation encompasses the physical constraints and consequences of the radical reactions taking place in emulsions of peroxidizing lipoproteins. The model predicts that a-TOH, in the absence of CoQ10H2 and other low-molecular weight antioxidants (see below) can promote LDL lipid peroxidation. Principally, this is due to both the phase-transfer activity of a-TOH (Reaction 9.1, Figure 9.1) and the chain-transfer activity of a-TO^ (Reaction 9.2, Figure 9.1).'15,25,56,57 TMP and the molecular action of vitamin E in oxidizing lipoproteins have been reviewed recently.23,24,58-61,62
The in vitro oxidizing conditions employed determine whether a-TOH acts as an antioxidant or a prooxidant for lipids in CoQ10H2-free, isolated LDL. Under conditions of high radical flux, radical-radical termination reactions between a-TO^ and the oxidation-initiating radical predominate (Reaction 9.5, Figure 9.1), such that a-TOH exhibits an overall antioxidant activity. This readily
FIGURE 9.1 Model of TMP for LDL lipid oxidation and antioxidation by CoQ10H2. A solution of radical oxidizing lipoprotein is an aqueous emulsion of lipid particles where the radical in one oxidizing particle, present predominantly as a-TO', is segregated from a-TO' in other oxidizing particles, and oxidation of the lipids proceeds via TMP.15 TMP (solid lines) is initiated by Reaction 9.1, reflecting the phase-transfer activity of a-TOH. Lipid peroxidation initiation (Reaction 9.2), followed by the propagation Reactions 9.3 and 9.4, reflect the chain-transfer activity of a-TO'. This is a feature relevant for LDL exposed to mild radical fluxes. Inhibition of TMP (anti-TMP, broken lines) can be achieved by reaction of a second aqueous radical oxidant with a-TO' (Reaction 9.5), resulting in both formation of nonradical product(s) (NRP) and consumption of a-TOH. This is a feature particularly relevant to high radical flux conditions, where a-TOH appears to act as a conventional antioxidant. Alternatively, anti-TMP is achieved by LDL-associated CoQ10H2, (or other coan-tioxidants), which reduces a-TO' (Reaction 9.6) resulting in the formation of CoQ10H', which may undergo one of two reactions. First, CoQ10 /CoQ10H may scavenge a-TO' resulting in the formation of CoQ10 and a-TOH (not shown). Second, CoQ10H' at the lipophilic/aqueous interface may deprotonate and the resulting CoQ10 autoxidize to form the chargedO2 that escapes to the aqueous environment (Reaction 9.7). It is assumed that lipid peroxyl radicals (LOO') and a-TO' move freely within though do not readily escape from oxidizing lipoprotein particles.57 L', carbon-centered lipid radical; LOOH, lipid hydroperoxide.
explains the "lag-phase" observed during the commonly employed Cu2+/LDL oxidation test system.49 However, under low radical flux conditions a-TO' is not "eliminated" so that chain-transfer (Reaction 9.2, Figure 9.1) predominates and hence a-TOH exhibits prooxidant activity. For Cu2+ as the oxidant, a-TOH switches from a prooxidant to an antioxidant at a Cu2+ to LDL ratio of ~3.28 With peroxynitrite added as a bolus, the switching point occurs at oxidant to LDL ratios of 100:1 to 200:1.31 Whether vitamin E exhibits pro- or antioxidant activity is also determined by the reactivity of the oxidant.17 For highly reactive oxidants (e.g., 'OH), a lower radical flux is required to achieve a prooxidant activity when compared to less reactive oxidants (e.g., ROO').17 Thus, the point at which a-TOH switches from a pro- to an antioxidant is reached at a radical flux of ~130 and 250 nM/min for 'OH and ROO', respectively.17 Extensive studies have confirmed that TMP is relevant for oxidizing conditions that promote formation of free radicals. These include Cu2 +, human monocytes or macrophages cultured in the transition metal containing Ham's F-10 medium, 15-lipoxygenase, hydroxyl radical ('OH), peroxynitrite (either added as a bolus or delivered in a time-dependent manner by the simultaneous generation of O2' and 'NO), MPO-derived tyrosyl radicals, myoglobin and horseradish peroxidase/H2O2.15,17,18,25,28-31,61 In contrast, two-electron oxidants such as HOCl do not induce substantial lipid peroxidation.63 However, among the oxidized amino acid adducts formed by treatment of LDL with HOCl are chloramines that break down to yield "secondary" radicals64 that induce LDL lipid peroxidation via TMP.65 Similar to HOCl, apo B (rather than lipids) is the major target for peroxynitrite-induced oxidation reactions.31 However, peroxynitrite also induces one-electron reactions66 and hence LDL lipid peroxidation via TMP.31
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