Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide

The combination of ascorbate, transition metal ions, and hydrogen peroxide (H2O2) is an efficient hydroxyl radical generating system called “the Udenfriend system.” Although the pro-oxidant role of ascorbate in this system has been well characterized in vitro, it is uncertain whether ascorbate also acts as a pro-oxidant under physiological conditions. To address this question, human plasma, used as a representative biological fluid, was either depleted of endogenous ascorbate with ascorbate oxidase, left untreated, or supplemented with 25 μM–1 mM ascorbate. Subsequently, the plasma samples were incubated at 37°C with 50 μM–1 mM iron (from ferrous ammonium sulfate), 60 or 100 μM copper (from cupric sulfate), and/or 200 μM or 1 mM H2O2. Although endogenous and added ascorbate was depleted rapidly in the presence of transition metal ions and H2O2, no cholesterol ester hydroperoxides or malondialdehyde were formed, i.e., ascorbate protected against, rather than promoted, lipid peroxidation. Conversely, depletion of endogenous ascorbate was sufficient to cause lipid peroxidation, the rate and extent of which were enhanced by the addition of metal ions but not H2O2. Ascorbate also did not enhance protein oxidation in plasma exposed to metal ions and H2O2, as assessed by protein carbonyl formation and depletion of reduced thiols. Interestingly, neither the rate nor the extent of endogenous α-tocopherol oxidation in plasma was affected by any of the treatments. Our data show that even in the presence of redox-active iron or copper and H2O2, ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in human plasma in vitro.