Determination of Intra- and Extra-cellular Vitamin C Dynamics
A Simple, Label-free Chromatographic Technique for Studying Vitamin C
- Cellular uptake of dehydroascorbic acid (DHA, purple color) via glucose transporter 1 (GLUT1, green color) and accumulation of its reduced form, ascorbic acid (ASC, blue color) in the cell. Here, a chromatographic protocol to instantly determine these vitamins in the cell is reported.
- Fig. 2: Flow chart of (A) extraction from the cells and (B) analytical conditions for determination of cellular ASC and DHA by HPLC-DAD. 
The redox-sensitive inter-conversion of ascorbic acid (ASC) and its oxidized form dehydroascorbic acid (DHA) in intracellular and extracellular environments is of exceptional interest at the forefront of metabolomics and pharmaceutical research, including high-dose vitamin C cancer therapy. A chromatographic protocol to instantly determine these vitamers in both forms from cellular extracts, without any labeling or pretreatment has been reported earlier. 
Vitamin C is one of the most fundamental nutrients to sustain life. Distributed ubiquitously and the most abundant of all vitamins (vitamin A, B, C, D, E and K), it plays a vital role in regulating redox balance in the body.  Vitamin C involves several vitamers that are inter-convertible depending on the redox state, among which ascorbic acid (ASC) and its oxidized form dehydroascorbic acid (DHA) represent two dominant species.  ASC is oxidized in the extracellular space by reactive oxygen species (ROS), producing ascorbate radical, which can be oxidized to DHA. Unlike ASC, DHA is transported via glucose transporters (GLUTs). Among 12 different GLUTs, GLUT1 and GLUT3 have higher affinity for DHA than for glucose.  DHA is taken up by cells via GLUTs or degraded to 2,3-l-diketoglutonate (2,3-DKG), which is further degraded into oxalic acid and threonic acid.
The spatiotemporal pattern of this vitamer pair underlies both specificity and kinetic aspects for several important cellular events and, therefore, quantitative analysis of their inter-conversion is of continuous research interest. A debate on high-dose (at the millimolar scale) vitamin C cancer therapy may best illustrate the importance of accurate determination of the dynamics of the intracellular DHA–ASC pair. That is to say, the contradictory clinical data (some studies have indicated anticancer activity of vitamin C while others have shown little effect ) have, at least in part, been attributed to differences in dose, implying the existence of a threshold vitamin C value at the millimolar concentration range exerting effective cytotoxicity, which is only achievable via intravenous administration, not via oral administration.
High-dose vitamin C kills cancer cells by increasing oxidative stress via two controversial possible mechanisms (Fig. 1). In the first proposed mechanism, extracellular H2O2 directly kills cancer cells by generating •OH via the Fenton reaction (Fig. 1, Hypothesis (1)).  Alternatively, in the second possible mechanism, DHA was recently reported to efficiently enter cells via GLUT1 and consume the intracellular reducing potential of reduced glutathione (GSH) and NADPH, resulting in increased levels of intracellular ROS (Fig. 1, Hypothesis (2)).  If hypothesis (2) is feasible, high-dose vitamin C therapy could be extended to a variety of cancers presenting high GLUT1 expression and high glycolytic activity. A recent study showed that DHA treatment gave stronger anticancer effects in gastric cancer cells with high GLUT1 expression than in cells with low GLUT1 expression.  However, owing to the unstable nature of DHA and the chemical and biological equilibrium between ASC and DHA, it is difficult to quantify the exact amount of DHA generated from ASC. Thus, exploring intracellular and extracellular DHA–ASC dynamics is of exceptional interest at the forefront of metabolomics, pharmaceutical, and clinical research, including high-dose vitamin C therapy.
Isotope labeling and mass-spectroscopic techniques are the gold-standard methods for monitoring the dynamics of ASC and DHA in biological samples. [7, 8, 9] However, these methods need costly instrumentation and complicated parameter optimization. A more cost-effective and widely accessible alternative is based on high-performance liquid chromatography coupled with diode array UV detection (HPLC-DAD). However, the striking similarity in the size and polarity of DHA and ASC along with the fact that UV absorption of DHA is much weaker compared to ASC makes this technique poorly reliable, especially for discrimination purposes. Alternatively, taking advantage of the poor UV sensitivity of DHA, reductant pretreatment using glutathione or dithiothreitol to convert DHA into ASC has been widely applied; the initial amount of DHA in the sample extract can then be estimated from the increase in the total vitamin C amount (ASC + DHA). However, this indirect assay (“reduction method”) lacks true specificity and is prone to interference by other factors, such as biological compounds and the conditions of the reduction reaction.  It is only feasible under the assumption of quantitative DHA–ASC conversion, which is not always the case; in fact, it has been found that the presence of a widely used vitamin C stabilizer (metaphosphoric acid: MPA) and variation of the pH can severely interfere with the reductant’s efficiency. 
A modified and remarkably simple HPLC-DAD protocol has been that enables, to our knowledge for the first time, simultaneous absolute quantitative measurement of the DHA–ASC pair dynamics in the intracellular and extracellular environment, thus eliminating the abovementioned reductant pretreatment (“reduction method”).  Our protocol (hereinafter referred to as the “direct method”) commences with a cellular vitamin C extraction process using MPA, a commonly used stabilizer during vitamin C extraction from food samples (Figure 2). [1, 10, 11] This treatment not only ensures the stability of vitamin C in the sample but also facilitates the simple removal of proteins from samples by centrifugation. Of note, there are no other reported protocols harnessing MPA as the cellular vitamin C extraction reagent because of its severe interference with the “reduction method”; instead, existing protocols usually involve organic solvents as the extraction media. The MPA-compatible and organic solvent-free protocol, hence, provides striking advantages over the “reduction method” in terms of the robustness of measurement, sample storage, and ease of purification. It has been demonstrated that this “direct method” can be readily applied to in vitro assay both on erythrocytes, the most extensively studied target, as well as pancreatic cancer cell line (MIA PaCa-2), to the authors’ knowledge the first-ever study on nucleated cell types, to trace in detail their GLUT1 (glucose transporter)-dependent or DHA-specific cellular uptake and, concomitantly, time- and dose-dependent intracellular conversion into ASC.  Yun et al. recently reported oxidative stress synchronized with intracellular DHA–ASC conversion, which eventually causes glutathione depletion, as a proposed modality for cell death, supporting Hypothesis (2). [7, 8] Indeed, also such intracellular ASC-induced cytotoxicity was observed. Of note, any preceding reports only provided up to 30 minutes of kinetic information. In contrast, owing to the simplicity of our “direct method”, the researchers were able to readily continue the monitoring for hours, the timescale over which the intracellular ASC conversion takes place in connection with the resultant cytotoxicity. Therefore, the presented technique should aid in providing quantitative bases for those therapeutic approaches.
Taiki Miyazawa1, Akira Matsumoto 1,2 and Yuji Miyahara 1
1 Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan
2 Kanagawa Institute of Industrial Science and Technology (KISTEC-KAST), Kawasaki, Japan
Tokyo Medical and Dental University
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