Mechanisms of ascorbic acid antioxidant activity in vitro and in vivo

Reactive oxygen species can mediate the oxidation of low density lipoprotein (LDL): a critical step in the pathogenesis of atherosclerosis. Conversely, antioxidants can protect against such oxidative damage by directly scavenging radical species. Ascorbic acid is a water-soluble antioxidant and strong reducing agent that directly quenches reactive oxygen species and can inhibit oxidative modification of LDL.;To investigate the role of ascorbic acid in LDL oxidation and the pathogenesis of atherosclerosis, I isolated human LDL and showed that ascorbic acid prevented copper mediated oxidation of LDL. The incubation of ascorbic acid and copper with LDL resulted in 2-oxohistidine formation concomitant with the loss of copper binding to LDL. These results suggest a mechanism by which ascorbic acid covalently modifies LDL at transition metal binding sites on apo B, thereby inhibiting LDL oxidation.;To further investigate the role of histidine residues in copper induced LDL oxidation, I incubated LDL with diethylpyrocarbonate, a histidine specific modifying reagent. This modification of LDL inhibited subsequent oxidation by copper as reflected in a decreased rate of propagation and a lengthened lag phase. These results suggest that histidine residues in apo B play a critical role in propagating oxidative lipid damage in LDL. When LDL was incubated with diethylene-triamine-pentaacetic-acid (DTPA), a metal chelator, I found that DTPA binds copper in a redox inactive manner and prevents LDL from binding and reducing copper. In contrast, free histidine bound copper in a redox active manner but inhibited LDL oxidation by competing with LDL for copper binding.;In an extension of these studies, I also demonstrated antioxidant activity of ascorbic acid in vivo in the presence of transition metal ions. While ascorbic acid has been suggested to act as a pro-oxidant in the presence of iron by stimulation of the Fenton reaction, the results of this study, utilizing a guinea pig model system, show that ascorbic acid inhibits iron mediated damage in vivo, as measured by stable end products of lipid peroxidation, F2-isoprostane.;In total, these data support the hypothesis that ascorbic acid is an antioxidant both in vitro and in vivo, despite the presence of transition metal ions.