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 H(2)O(2) 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 ( approximately 0.25-0.5 mg per gram of body weight). After i.v. injection, ascorbate baseline concentrations of 50-100 microM 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 microM 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(2)O(2) 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 H(2)O(2) 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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Proposed mechanism of preferential formation of Asc•− and H2O2 in extracellular fluid compared with blood. After oral and parenteral administration, ascorbic acid is proposed to achieve equivalent concentrations in blood (left side) and extracellular fluid (right side). In extracellular fluid, pharmacologic concentrations of ascorbic acid lose one electron and form Asc•−. The electron reduces a protein-centered metal: An example reaction is shown as reduction of Fe3+ to Fe2+. Fe2+ donates an electron to oxygen, forming active oxygen including superoxide (O2•−) with subsequent dismutation to H2O2 (17). In blood (left side), it is proposed that these reactions are damped or inhibited (dashed lines). Asc•− appearance will be inhibited by red blood cell membrane-reducing proteins (18) and/or by large plasma proteins that do not distribute to the extracellular space. Any formed H2O2 will be immediately destroyed by plasma catalase and red blood cell GSH peroxidase, so that no H2O2 will be detectable (–16). The identities of the metal-centered proteins are unknown.

Fig. 2.

Parenteral administration of ascorbic acid bypasses tight control of its intestinal absorption. A total dose of 0.5 mg of ascorbate per gram of body weight was given to rats by i.v. injection (circles) (two-thirds of the dose at 0 min and one-third at 30 min); by i.p. injection (stars) at 0 min; or by gavage (oral administration) (triangles) at 0 min. Blood was taken at each indicated time point. Extracellular fluid at the end of 30-min intervals was collected for ascorbic acid measurement (see Materials and Methods). Numbers of rats for each administration route are indicated. All data are displayed ± SD. (A and B) Ascorbic acid concentration in plasma (A) and extracellular fluid (B), measured in millimolar as a function of time in minutes. (A Inset and B Inset) Gavage administration of ascorbic acid, displayed as plasma concentration (micromolar) as a function of time (minutes). (C) Ascorbic acid concentration in extracellular fluid (millimolar) as a function of ascorbic acid concentration in plasma (millimolar) for all administration routes, all animals, and all time points (R2 = 0.93, P < 0.0001).

Fig. 3.

Asc•− concentrations in extracellular fluid and in blood measured by EPR, before and after i.v., i.p., and gavage (oral) ascorbic acid administration. Ascorbic acid dose was 0.5 mg per grams of body weight unless otherwise indicated and administered as described for Fig. 2. Numbers of animals for each condition are indicated. (A) EPR spectra of Asc•− in blood and in extracellular fluid before i.v. ascorbic acid injection at the time corresponding to peak plasma ascorbic acid (see Fig. 2) and 2 h after injection. Blood samples and normal saline samples with added ascorbic acid (2 mM) were measured without dilution. Extracellular fluid samples were diluted 4-fold due to microdialysis sample collection procedures and microdialysis membrane throughput. Normal saline with 2 mM ascorbic acid was a control for baseline Asc•− formation from possible ascorbate oxidation in the microdialysis perfusate buffer. Arrows indicate the expected positions of Asc•− doublet signal. (B) Asc•− concentrations (nanomolar) in extracellular fluid (blue) and blood (red) as a function of time (minutes) before and after ascorbate administration i.v., i.p., or by gavage. (C) Asc•− concentrations (nanomolar) in extracellular fluid (blue) or blood (red) and as function of ascorbic acid concentration (millimolar) in extracellular fluid (blue) or plasma (red) from all animals, all doses, and all time points. Ascorbate doses were 0.5 mg per gram of body weight for i.p. and gavage administration and 0.5 mg per gram of body weight and 0.25 mg per gram of body weight for i.v. administration. Two-thirds of the i.v. dose was given at time 0, and one-third was given at 30 min. For Asc•− in extracellular fluid, R2 = 0.96 and P < 0.0001.

Fig. 4.

H2O2 formation in extracellular fluid with parenteral administration of ascorbic acid. Administered doses were 0.5 mg per gram of body weight for all routes and were given as described for Fig. 2. Extracellular fluid at the end of 30-min intervals was collected for H2O2, ascorbic acid, and Asc•− measurement (see Fig. 2 and Materials and Methods). Numbers of rats for each administration route are indicated. All data are displayed ± SD. (A) Fluorescent spectra of PX1 in normal saline with added H2O2 at the indicated concentrations. Catalase (600 units/ml) was added to parallel samples to account for non-H2O2 fluorescence background. (Left Inset) Chemical structure of PX1 and its product after reaction with H2O2. (Right Inset) Typical H2O2 standard curve. (B) H2O2 formation in extracellular fluid as a function of time before and after i.v. (circles), i.p. (stars), and oral (triangles) ascorbic acid administration. (C) Correlation of H2O2 concentration with Asc•− concentration in extracellular fluid (R2 = 0.77, P < 0.0015). (D) Correlation of H2O2 concentration with ascorbic acid in extracellular fluid (R2 = 0.84, P < 0.0001) or in plasma (Inset; R2 = 0.87, P < 0.0001). Ascorbic acid concentrations in plasma were determined as in Fig. 2.

Fig. 5.

Pharmacologic ascorbic acid concentrations: mechanisms for selective cell death. Pharmacologic ascorbic acid concentrations produce extracellular H2O2, which can diffuse into cells, deplete ATP in sensitive cells, and thereby cause cell death. ATP may be depleted by three mechanisms. (i) DNA damage induced by H2O2 activates PARP. Activated PARP catabolizes NAD+, thereby depleting substrate for NADH formation and consequent ATP synthesis. (ii) H2O2 is catabolized by concurrent oxidation of GSH to GSSG. To reduce GSSG back to GSH, GSH reductase utilizes NADPH, which is provided by the pentose shunt from glucose. Glucose used to reduce NADP+ to NADPH cannot be used for glycolysis or NADH production so that ATP generation is decreased. (iii) H2O2 may directly damage mitochondria, especially ATP synthase, so that ATP production falls. Some cancer cells rely primarily on glycolysis rather than on oxidative phosphorylation respiration for ATP production (the Warburg effect). Compared with oxidative phosphorylation, ATP generation by glycolysis is inefficient. In glycolysis-dependent cancer cells, decreased glycolysis may lower intracellular ATP. Cancer cells that are glycolysis-dependent may be particularly sensitive to pharmacologic ascorbic acid concentrations, compared with cells that use oxidative phosphorylation. See text for additional details.