Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo

Abstract

Ascorbate (ascorbic acid, vitamin C), in pharmacologic concentrations easily achieved in humans by i.v. administration, selectively kills some cancer cells but not normal cells. We proposed that pharmacologic ascorbate is a prodrug for preferential steady-state formation of ascorbate radical (Asc •؊ ) and H2O2 in the extracellular space compared with blood. Here we test this hypothesis in vivo. Rats were administered parenteral (i.v. or i.p.) or oral ascorbate in typical human pharmacologic doses (Ϸ0.25-0.5 mg per gram of body weight). After i.v. injection, ascorbate baseline concentrations of 50 -100 M in blood and extracellular fluid increased to peaks of >8 mM. After i.p. injection, peaks approached 3 mM in both fluids. By gavage, the same doses produced ascorbate concentrations of <150 M in both fluids. In blood, Asc •؊ concentrations measured by EPR were undetectable with oral administration and always <50 nM with parenteral administration, even when corresponding ascorbate concentrations were >8 mM. After parenteral dosing, Asc •؊ concentrations in extracellular fluid were 4-to 12-fold higher than those in blood, were as high as 250 nM, and were a function of ascorbate concentrations. By using the synthesized probe peroxyxanthone, H 2O2 in extracellular fluid was detected only after parenteral administration of ascorbate and when Asc •؊ concentrations in extracellular fluid exceeded 100 nM. The data show that pharmacologic ascorbate is a prodrug for preferential steady-state formation of Asc •؊ and H2O2 in the extracellular space but not blood. These data provide a foundation for pursuing pharmacologic ascorbate as a prooxidant therapeutic agent in cancer and infections. ascorbic acid ͉ cancer ͉ vitamin C ͉ pharmacokinetics

Proposed Insights in the Pharmaco-Dynamic Theory of Ascorbate Pro-Oxidant Activity

“…Besides, another application field is the in vitro modeling of OS damage. In contrary to Chen et al (2007) who argued that the immunodeficient mice with a xenograft is the most characterized method of predicting potential activity of a given molecule in humans, we propose a simple cellbased in vitro model (Chen et al, 2005). In fact, Chen's in vivo model induces a high level of ROS after hypoxic ischemia and cold ischemia of the xenograft and stimulates endothelial cell which might up-regulate AA transporters.…”

“…Based on these data, the most cogent explanation of ascorbate action in inducing lipid peroxydation is by its intracellular mechanisms. Several data highlight that the most common theory of cytotoxic effect of high dose of AA is the intracellular and extracellular induction of H 2 O 2 without demonstrating whether this will translate to a clinical benefit (Chen et al, 2007;2005). Morever, despite the catalyst could not be identified; Deubzer et al (2010) suggested that it could be a metallo-protein secreted by some tumor cells (Deubzer et al, 2010).…”

“…6). Chen et al (2008;2007;2005) suggested that ascorbate, at pharmacological concentrations, is a pro-drug for H 2 O 2 delivery to tissues with major therapeutic implications. However, such conclusions may be criticized viewing the limits of the experimental model and don't concord with our data that showed that AA alone (model 0) has a low impact on the first oxidative pathway compared to PFEM+Cu ++ and PFEM+H 2 O 2 +Cu ++ .…”

“…They showed that even in the presence of redox-active iron or copper and hydrogen peroxide, ascorbate acts as an antioxidant to prevent lipid peroxidation and protein oxidation (Suh et al, 2003). However, Chen et al (2007) has demonstrated in an animal model that H 2 O 2 induced by Ascorbic Acid (AA) in presence of a divalent cation are strongly destroyed by the plasmatic defense although it still highly reactive in tissues (Chen et al, 2007). These two pilot studies have evoked more questions than answers and suggested that high doses of AA are prooxidant but without any damage effects on human plasma because of plasmatic H 2 O 2 scavengers.…”

α-Tocopheryl succinate promotes selective cell death induced by vitamin K3 in combination with ascorbate

“…Pharmacological doses of AA (40.2 mM) are required to induce oxidation-dependent cytotoxicity both in vitro (Beck et al, 2009) and in vivo (Tareen et al, 2008). Cells convert ascorbate to the ascorbyl radical (A À ) with concomitant formation of H 2 O 2 in extracellular fluids (Chen et al, 2007), resulting in toxicity towards tumour cells (Rhee, 2006). However, this approach faces a possible complications in that the therapeutic synergy of the drug combination could be accompanied by deleterious side-effects, and by the possibility that the extracellular levels of H 2 O 2 main drop below 1 mM, at which concentration proliferation rather than cell death may be promoted (Rhee, 2006).…”