Alpha-tocopheryl hydroquinone is an efficient multifunctional inhibitor of radical-initiated oxidation of low density lipoprotein lipids

Abstract

As the oxidation of low density lipoprotein (LDL) lipids may be a key event in atherogenesis, there is interest in antioxidants as potential anti-atherogenic compounds. Here we report that alpha-tocopheryl hydroquinone (alpha-TQH2) strongly inhibited or completely prevented the (per)oxidation of ubiquinol-10 (CoQ10H2), alpha-tocopherol (alpha-TOH), and both surface and core lipids in LDL exposed to either aqueous or lipophilic peroxyl radicals, Cu2+, soybean lipoxygenase, or the transition metal-containing Ham's F-10 medium in the absence or presence of human monocyte-derived macrophages. The antioxidant activity of alpha-TQH2 was superior to that of several other lipophilic hydroquinones, including endogenous CoQ10H2, which is regarded as LDL's first line of antioxidant defence. At least three independent activities contributed to the antioxidant action of alpha-TQH2. First, alpha-TQH2 readily associated with LDL and instantaneously reduced the lipoprotein's ubiquinone-10 to CoQ10H2, thereby maintaining this antioxidant in its active form. Second, alpha-TQH2 directly intercepted aqueous peroxyl radicals, as indicated by the increased rate of its consumption with increasing rates of radical production, independent of LDL's content of CoQ10H2 and alpha-TOH. Third, alpha-TQH2 rapidly quenched alpha-tocopheroxyl radical in oxidizing LDL, as demonstrated directly by electron paramagnetic resonance spectroscopy. Similar antioxidant activities were also seen when alpha-TQH2 was added to high-density lipoprotein or the protein-free Intralipid, indicating that the potent antioxidant activity of alpha-TQH2 was neither lipoprotein specific nor dependent on proteins. These results suggest that alpha-TQH2 is a candidate for a therapeutic lipid-soluble antioxidant. As alpha-tocopherylquinone is formed in vivo at sites of oxidative stress, including human atherosclerotic plaque, and biological systems exist that reduce the quinone to the hydroquinone, our results also suggest that alpha-TQH2 could be a previously unrecognized natural antioxidant.

Figure 1

Hydroquinones protect LDL’s cholesteryl esters from ROO^⋅-induced peroxidation. An LDL solution (1.2 μM in apoB) was incubated at 37°C with 2 mM of AAPH in the absence (A) or presence (B) of 10 μM of 2,5-DTBHQ or α-TQH₂ (C). At the time points indicated, aliquots were taken and analyzed for CoQ₁₀H₂ (▪), CoQ₁₀ (□), α-TOH (○), and CE-O(O)H (⊞) and, where applicable, for α-TQH₂ (▴) and α-TQ (▵). The data shown represent mean values of three separate experiments with a variation of <15%. The initial concentrations of CoQ₁₀H₂, CoQ₁₀, and α-TOH in the LDL solution were 0.73 ± 0.15, 0.43 ± 0.09, and 9.5 ± 0.9 μM, respectively. The initial levels of α-TQH₂ and α-TQ measured after addition of the freshly prepared hydroquinone to LDL varied between 8.9 to 9.1 and 1.1 to 1.6 μM, respectively. Note that the sum of CoQ₁₀H₂ plus CoQ₁₀ and that of α-TQH₂ plus α-TQ represent 100%.

Figure 2

The consumption of α-TQH₂ during LDL oxidation is dependent on the rate of ROO^⋅ production but independent of the α-TOH content of the lipoprotein. (A) LDL (1 μM in apoB), control or supplemented with 10 μM α-TQH₂, was oxidized with increasing concentrations of AAPH, and the rates of consumption of CoQ₁₀H₂ (▪) and α-TOH (○) in the control and α-TQH₂ (▴) in the supplemented LDL estimated from the linear portion of their respective depletion curves. R_g was calculated from R_g = 1.3 × 10⁻³ [AAPH] nM/s (34). The data shown are average values obtained from two or three (indicated by asterisk) independent experiments, with the range indicated by the vertical lines. (B) LDL (1.5 μM in apoB) from a FIVE patient depleted (circles), partially (squares), or fully re-supplemented with α-TOH (triangles) was incubated with 1 mM AAPH in the presence of 10 μM of α-TQH₂. At the time points indicated, an aliquot was taken and analyzed for α-TQH₂ (solid symbols) and α-TQ (open symbols). The data shown are mean values ± SD of three independent experiments performed with LDL prepared from plasma of one FIVE patient.

Figure 3

α-TQH₂ efficiently and stoichiometrically reduces CoQ₁₀ present in intact LDL but not LDL lipid extracts or organic solution. (A) In vivo CoQ₁₀H₂-enriched (30) LDL (1.1 μM in apoB) was allowed to autoxidize until all of the coenzyme Q was present as CoQ₁₀. The intact lipoprotein (○), its total lipid extract (▵), or an ethanolic solution of CoQ₁₀ (□) were then placed on ice before 10 μM of α-TQH₂ was added. The samples were placed at 37°C and aliquots were removed at the time points indicated and analyzed for CoQ₁₀H₂. The initial CoQ₁₀ concentration was 3.1 ± 0.25, 2.62 ± 0.20, and 10 ± 0 μM (mean ± SD; n = 3) for intact LDL, LDL lipid extract, and organic CoQ₁₀ solution, respectively. The data shown are average values derived from three independent experiments with <3% variation for all conditions. (B) In vivo CoQ₁₀H₂-enriched (29) LDL (0.93 μM in apoB) was preincubated at 37°C for ≈8 hr and then left at room temperature overnight for most of the CoQ₁₀H₂ to autoxidize. Such LDL preparation contained 0.3 and 3 μM of CoQ₁₀H₂ and CoQ₁₀, respectively, and was hydroperoxide-free as determined by HPLC (see text). Increasing amounts of α-TQH₂ were then added and the formation of CoQ₁₀H₂ monitored at 37°C. The data shown are mean values derived from a single experiment performed in triplicate, with variation <5%.

Figure 4

Direct interaction of α-TQH₂ with α-TO^⋅ in LDL. α-TO^⋅ was generated in LDL (7.2 μM in apoB) by incubation with SLO (1 mg/ml) at 37°C, and recorded by EPR spectroscopy before (A) and after (B) addition of 10 μM of α-TQH₂ at the same region of field as detailed in the text. (C) The changes in UV_{234 nm} absorbance associated with the conversion of linoleic acid (0.1 mM in PBS) to linoleic acid hydroperoxide by SLO (1 mg/ml) in the absence (dashed line) and presence (solid line) of 10 μM α-TQH₂. The data shown are from a single experiment, representative of three separate experiments.