**4. Conclusions**

Performing successful research on vitamin C, especially in the context of human health, requires specific focus and attention to detail. Although this review touched on many of the aspects of cell culture, animal, and human studies that are complicated by the use of ascorbic acid, it is not a complete guide to performing vitamin C research. Each study design is unique, so it must be critically analyzed with respect to ascorbate chemistry and biology to best understand whether and how it will make a contribution in the overall field. As described above, the effects of ascorbic acid within a biological system are potentially multifold, requiring extreme caution in implementation and interpretation.

As we change our view of vitamin C research, we must first address the most prevalent issues in frequently used model systems. The combination of high oxygen tension and the presence of reactive transition metals lead to a rapid oxidation of ascorbic acid in cell culture. The rapid loss of ascorbate in culture media promotes a pro-oxidant environment with the generation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals, as described above. Moreover, depriving cells of ascorbic acid for countless generations produces a cell culture environment that is no longer relevant to human health. Overall, steps must be taken to control ascorbate oxidation if vitamin C research is to continue in cell culture. The use of stable ascorbate derivatives, metal chelation, and low oxygen culture conditions show promise, but much additional work is needed. Understanding the effects of prolonged ascorbate deprivation of cells is also necessary to determine if certain cell lines must be eliminated from use with ascorbic acid.

The continued use of ascorbate-synthesizing animal models also complicates all studies on ascorbic acid *in vivo*. Currently, mouse and rat models that lack ascorbic acid synthesis are available, but show considerable differences from guinea pigs and humans in terms of ascorbic acid bioavailability and metabolism. For example, the apparent lack of intestinal absorption of ascorbic acid in rodent models does not appear related to the synthesis capacity, and must be considered in supplementation studies. Furthermore, the effects of vitamin C deprivation appear to have effects in one model system that are not recapitulated in others, although we do not know if strain differences or dietary factors may have influenced these observations. The use of animal models is especially troubling in light of genetic alterations in the regulation and expression of vitamin C transport proteins in animals without functional ascorbic acid synthesis. Further studies are required to determine if other genetic adaptations have occurred—specifically in primate evolution—that may determine the difference in response of animals and humans to vitamin C supplementation.

Currently, studies involving vitamin C consumption in human subjects are not held to a rigorous standard. Randomized controlled trials, normally considered the benchmark for determining the impact of a compound on human health, have been poorly designed in regard to the chemistry and biology of ascorbic acid. While remaining aware of the effects that different study populations may have on the outcome of vitamin C supplementation, there is a need to determine vitamin C status in subjects before, during, and after a supplementation trial. Evidence shows that methods to record vitamin C intake by using food frequency questionnaires or food diaries are insufficient means of achieving this goal. Therefore study design needs to include assessments of vitamin C status using measurements of plasma, and possibly tissue, ascorbic acid levels. Only with these more precise determinations can we begin to speculate on mechanisms of action and eliminate speculations surrounding the health effects of vitamin C supplementation. Furthermore, eliminating the technical issues surrounding the measurement of vitamin C in clinical settings are additional steps needed to preserve data integrity.

While all study designs deserve higher scrutiny, a note of caution must also be placed in the interpretation of vitamin C research. It is all too common for vitamin C to be assigned a role (as an antioxidant or a pro-oxidant, for instance) without any supporting evidence to validate those claims. It cannot be overemphasized that the primary role of ascorbic acid in biological systems is that of a reductant, and the most established health effects of this reductive power are related to ascorbate's role as an electron-donating enzyme cofactor used, e.g., for pro-collagen, carnitine, and catecholamine biosynthesis [3]. Ascorbic acid has many additional roles in the body beyond these enzyme functions, for example antioxidant protection, and likely more roles will be elucidated in the future. With a critical evaluation of all potential roles of ascorbic acid, the borders of vitamin C research can advance with balanced, evidence-based approaches. Already, emerging roles for vitamin C in various hydroxylase enzymes have recently placed a focus on HIF-1α-dependent gene expression [41,43] and changes in histone and DNA methylation [135,136], suggesting vitamin C may regulate global changes in gene expression. Further innovation has been demonstrated in studies on vitamin C bioavailability [69,108], metabolomics of vitamin C deficiency [137], genetic variation of the vitamin C transporters [103], and intravenous vitamin C infusions in cancer therapy [138,139].

As vitamin C research progresses into the 21st century, it has become clear that much more work still lies ahead of us. The experimental faults, artifacts, and myths currently afflicting vitamin C research limit the impact of many studies, making their contributions to general knowledge of the biological roles of ascorbic acid unremarkable at best, and confusing and detracting from the real issues at worst. This can also stretch beyond the realm of laboratory research, as the persistence of poorly controlled studies within this field often undermines efforts in the medical community to recommend vitamin C as a safe, effective means of promoting health. If nothing else, it weakens efforts to fund additional, well-designed RCTs necessary to establish definitive health claims that are desperately needed. In light of these issues, we must increase scrutiny of vitamin C studies in the past as well as the present, holding them to a higher standard based on the evidence discussed here if we are to make a lasting contribution to our understanding of vitamin C's impact on human health.
