The Role of Copper Reduction by α-Tocopherol in Low-Density Lipoprotein Oxidation

Abstract

The oxidation of lipoproteins is thought to be an important early step in atherogenesis. The measurement of lipid peroxidation in low-density lipoprotein (LDL) challenged with Cu 2+ has become a widespread test to determine the “susceptibility” of LDL to oxidation. The determination of lag time to oxidation is thought to be a measure of the total antioxidant capacity of the LDL. However, we and others have failed to observe any correlation between lag time and the LDL content of its major lipid antioxidant, α-tocopherol. In fact, several studies now suggest a pro-oxidant role for tocopherol under some conditions of LDL oxidation. In the present study we sought to determine if there was a relationship between Cu 2+ reduction by LDL and kinetic parameters of LDL oxidation. LDL (0.3 mmol/l cholesterol, ∼0.1 mg protein/ml) was incubated at 30°C with 2 μM Cu 2+ and the formation of conjugated dienes measured over a 4-h period. Using neocuproine, an indicator molecule that specifically complexes Cu + but not Cu 2+, the reduction of Cu 2+ by LDL was monitored. The final Cu concentration in these assays was 100 μM and neocuproine 750 μM. Cu + formation was measured by absorbance at 454 nm. A strong negative correlation was observed between copper reduction by LDL and lag time to oxidation ( r = −0.66, p < .005, n = 16). Further experiments showed that (1) LDL was able to reduce Cu 2+ to Cu + in a time and concentration-dependent manner; (2) blocking of free —SH groups on LDL apoprotein B by preincubation with dithionitrobenzoic acid (DTNB) had no significant effect on the rate and extent of Cu 2+ reduction; (3) consumption of tocopherol in LDL undergoing oxidation with Cu was very rapid (rate = 6 × 10 −10 M s −1). When Cu + formed during incubation with LDL was complexed with neocuproine, there was significant inhibition of LDL oxidation, as indicated by lipid peroxide formation and mobility on agarose gel electrophoresis. Surprisingly, tocopherol consumption was even more rapid in the presence of neocuproine, consistent with a shift in Cu 2+/ Cu + equilibrium and faster reduction of Cu 2+ by α-tocopherol. These results indicate that under these conditions tocopherol is a major reducing agent in LDL, converting Cu 2+ to Cu +, and therefore, may play an important role in promoting LDL oxidation. However, there was no correlation between LDL tocopherol content and reduction of Cu 2+. Examination of the time course of Cu 2+ reduction in tocopherol enriched and depleted LDL indicates that tocopherol may determine Cu reduction at early time points but that the eventual capacity of LDL to reduce Cu may depend on more complex interactions between tocopherol and other LDL components.