2.1.1. Ascorbate Stability in Cell Culture

The primary concern with the use of ascorbic acid in cell culture is the stability of the molecule under typical incubation conditions. Cell culture incubators use approximately 90%–95% air and 5%–10% CO2, resulting in oxygen levels approximately 10–100 times greater than those found in the circulation and in tissues. Increased oxygen tension can promote a pro-oxidant environment in cell culture media [6], possibly generating a wide variety of ROS that can react with, and hence deplete, ascorbate [7]. However, high oxygen levels alone are not sufficient to cause substantial ascorbic acid oxidation. Although the reduction potential of the reduced form of vitamin C, the ascorbate mono-anion (AscH<sup>−</sup> ), is sufficient to reduce molecular oxygen to superoxide radicals (Equation 1), the reaction kinetics make this process very slow (estimated second-order rate constant, k2 ≈ 10−4 M−1 s−1) [8].

$$\text{AscH}^- + \text{O}\_2 \xrightarrow{} \text{Asc}^- + \text{O}\_2^- \tag{1}$$

The stability of the ascorbate mono-anion is apparent in deionized water or simple salt solutions: ascorbate added to phosphate-buffered saline (PBS) shows minimal oxidation over a six-h time period in a cell culture incubator (Figure 1, PBS) or when exposed to ambient air at room temperature (data not shown). In contrast, ascorbate incubated in cell culture media under standard conditions is rapidly oxidized [9,10] (Figure 1, RPMI). The composition of the cell culture media plays an important role in the rate of ascorbate oxidation: in serum-free RPMI medium the half-life of ascorbate is about 1.5 h (Figure 1); however, a more rapid loss of ascorbate has been noted in other cell culture media formulations such as MEM or Williams E media (data not shown), including more complex solutions containing serum [6,11,12].

**Figure 1.** Ascorbate oxidation in buffer or cell culture medium. Ascorbate (100 μM) was added to RPMI 1640 or phosphate-buffered saline (PBS) and monitored over time. Chelated RPMI was used after overnight treatment with Chelex 100 resin and the addition of diethylenetriaminepentaacetic acid (DTPA, 1 mM) as described in Methods. RPMI RT represents media not incubated under 5% CO2 but under ambient air at room temperature.

Iron and copper are present in cell culture media, either added as part of the media formulation or appearing fortuitously, as they are required for normal cell growth and function [13,14]. However, the reduced forms of iron (ferrous iron, Fe2+) and copper (cuprous copper, Cu+ ) are able to reduce molecular oxygen to superoxide and, hence, participate in the production of ROS in cell culture systems. The Haber-Weiss reaction (also known as the superoxide-driven Fenton reaction) involves the reduction of the oxidized forms of iron (ferric iron, Fe3+) or copper (cupric copper, Cu2+) by superoxide (Equation 2) and the subsequent conversion of hydrogen peroxide (formed, e.g., by dismutation of superoxide radicals) to hydroxyl radicals and hydroxide by the reduced metal ions (Fenton reaction, Equation 3) (Scheme 1). Hence, the metal ions act as catalysts and are required only in trace amounts for the Haber-Weiss reaction (Equation 4).

**Scheme 1.** Metal-dependent and metal-independent production of reactive oxygen species by ascorbate in cell culture media.

One of the most important biological functions of ascorbate is the reduction of Fe3+ or Cu2+ in the active site of enzymes, providing electrons used either in the hydroxylation of the enzymes' substrates or the maintenance of the active-site metal ion in the reduced state [3]. In the case of enzyme-bound metals, the transfer of electrons occurs in a controlled manner, which minimizes deleterious side reactions. However, in a solution of non-protein bound iron or copper, added ascorbate will reduce these metal ions (Equation 5); replacing superoxide in Equation 2, leading to superoxide production and facilitating the flow of electrons into the Haber-Weiss reaction (Equation 4). Even if media formulations are carefully controlled, trace amounts of these transition metals may be found on cell culture glassware, plastic dishes, and reagents, fueling ascorbate oxidation and ROS production [8,15].

$$\text{Fe}^{3+}/\text{Cu}^{2+} + \text{O}\_2\text{"}-\text{Fe}^{2+}/\text{Cu}^{+} + \text{O}\_2 \tag{2}$$

$$\text{Fe}^{2+}/\text{Cu}^{+} + \text{H}\_{2}\text{O}\_{2} \rightarrow \text{Fe}^{3+}/\text{Cu}^{2+} + \text{OH}^{\cdot} + \text{OH}^{-} \tag{3}$$

$$\text{Sum:}\,\mathrm{O}\_{2}"\mathrm{"} + \mathrm{H}\_{2}\mathrm{O}\_{2}^{\mathrm{Fe}}\underline{\mathrm{O}}\_{2}^{\mathrm{H}} + \mathrm{OH}"+\mathrm{OH}"\tag{4}$$

$$\text{AscH}^- + \text{Fe}^{3+} / \text{Cu}^{2+} \rightarrow \text{Asc}^- + \text{Fe}^{2+} / \text{Cu}^+ + \text{H}^+ \tag{5}$$

In biological fluids and inside cells *in vivo*, a combination of low oxygen levels and protein-bound metal ions greatly reduce ascorbate-mediated pro-oxidant effects [16–18]. Consequently, ascorbate in human plasma does not get readily oxidized [19]. By contrast, ascorbate addition to cell culture media results in the production of ROS, including superoxide radicals, hydrogen peroxide (from the dismutation of superoxide radicals), and hydroxyl radicals (Scheme 1) [6,15]. For this reason, ascorbic acid is often mistaken for inducing a pro-oxidant environment in cell culture systems [17,20], although the effects of hydrogen peroxide production specifically may be masked by other media components, such as serum proteins, pyruvate, or α-ketoglutarate [6,11,21,22]. A more accurate description may be that ascorbate unmasks the presence of catalytic transition metals in the cell culture environment [15]. Treating cell culture media with metal chelating agents slows the rate of ascorbate oxidation (Figure 1, Chelated RPMI), increasing the half-life from approximately 1.5 h in standard media (RPMI) to about 2.7 h. Interestingly, metal chelation does not completely prevent ascorbate oxidation (Figure 1), suggesting that other media components also contribute to ascorbate loss [6].

One such contributing factor may be ascorbate auto-oxidation: the direct reaction of ascorbate with molecular oxygen [8]. As stated above, the direct interaction between the ascorbate mono-anion and oxygen is highly unfavorable; however, the ascorbate di-anion (Asc2−) rapidly reacts with oxygen (Equation 6). At pH 7.0, the concentration of Asc2− is low, but its contribution to the rate of ascorbate oxidation can become considerable under conditions when pH rises or with increasing concentrations of added ascorbate (Scheme 1) [23]. Cell culture media pH is often stabilized by the addition of sodium bicarbonate to offset the acidic effects of the high CO2 environment. However, when this media is exposed to air outside the incubator, the pH value can rise rapidly to 8.0 or higher [11]. RPMI media equilibrated to ambient air accelerates ascorbate oxidation when compared to media in a cell culture incubator (Figure 1, RPMI RT *versus* RPMI), decreasing the half-life from about 1.5 h to less than one hour. As the Asc2− present in solution reacts with oxygen to form dehydroascorbic acid, additional Asc2− ions are generated to re-establish the equilibrium driven

by the pH (Scheme 1 and Equation 7). This continuous production of Asc2− would fuel auto-oxidation, which is prevalent when supraphysiological levels of ascorbate are added to cell culture media, since the Asc2− concentration would increase proportionally and greatly enhance oxidation effects. Although concentrated ascorbic acid will lower the pH of cell culture media, investigators usually offset this by the addition of sodium hydroxide to limit the impact of the acidic pH on cells. However, any shift toward a neutral or slightly alkaline environment will increase the metal-independent, pH-driven pro-oxidant effects of ascorbate, which may explain the limited effect of metal chelators to reduce the cytotoxicity of millimolar concentrations of ascorbate towards cancer cells [24].

$$\text{Asc}^{2-} + \text{O}\_2 \rightarrow \text{Asc}^{\bullet-} + \text{O}\_2^{\bullet-} \tag{6}$$

$$\text{AscH}\_2 \leftrightarrow \text{AscH}^- + \text{H}^+ \leftrightarrow \text{Asc}^{2-} + 2\text{H}^+ \tag{7}$$

Ascorbate levels can be maintained in cell culture media by frequent addition of vitamin C [9], but the persistent oxidation will continuously generate dehydroascorbic acid [25] and breakdown products, such as oxalate and threonate [26]. Extracellular concentrations of dehydroascorbic acid in excess of 1–2 μM are considered non-physiological, given its short half-life [25] and rapid uptake and by cells [27–29]. Although cells can reduce the dehydroascorbic acid to ascorbic acid, it is currently unclear what effects constant exposure of cells to high levels of dehydroascorbic acid or its breakdown products may have. For example, dehydroascorbic acid exposure has induced stress signaling and cytotoxicity in some cell types, probably due to the loss of NADPH or glutathione needed for dehydroascorbic acid reduction [30]. Oxalate has been shown to exert cytotoxic effects [31], and threonate can impact cell signaling pathways [32]. If anything, these exposures are more likely to generate artifacts as a consequence of the cell culture environment.
