2.1.2. "Cellular Scurvy"

Even under conditions of severe vitamin C deficiency, *i.e.*, scurvy, some ascorbate is still present in cells and tissues of humans *in vivo*. As discussed above, cell culture media are not usually supplemented with ascorbic acid, due to its inherent instability in these media. As a consequence, many researchers have reported that cells in culture are devoid of any detectible amounts of ascorbate, even with the use of extremely sensitive HPLC techniques [24,33–38]. Similarly, many complete cell culture media containing fetal bovine serum (FBS) have no detectable amounts of ascorbate [33,34,36,38], and our own analysis of various commercial sources of media and FBS has shown identical results (unpublished observations). The effects of these ascorbate-free conditions are not well defined or understood. However, it should be recognized that many immortalized cell lines likely have been maintained under scorbutic conditions for generations. In this manner, a whole host of cell culture artifacts may be expected when ascorbate is reintroduced into the system.

Cells in culture can be maintained without ascorbic acid because it is not essential to cell growth and division. The biological functions of ascorbate as an electron donor in enzymatic synthesis pathways do not have an absolute requirement for ascorbate [3,39,40]. These enzymes can use other reducing substrates as sources of electrons [39,41], and enzyme activity can still occur in the absence of ascorbate, albeit at a far decreased rate [42]. In particular, the α-ketoglutarate-dependent dioxygenases, such as those involved in collagen synthesis and regulation of hypoxia-inducible factor 1α (HIF-1α), do not require ascorbate as part of the normal catalytic cycle; ascorbate is only needed to rescue the enzyme should an uncoupled enzymatic reaction occur [3]. It has also been suggested that ascorbate may function to maintain intracellular iron in the ferrous state, making it available to replenish or replace ferric iron in the active site of these enzymes [43]. It is possible that cells in culture adapt by increasing ferrous iron uptake and turn-over of iron-containing proteins, partially circumventing the need for ascorbic acid. Regardless of the mechanism, it is evident from cell culture studies that "ascorbate-requiring" enzymes, such as those involved in collagen synthesis [44], degradation of HIF-1α [43], norepinephrine and α-amidated peptide synthesis [45], and histone and DNA demethylase activity [46], still exhibit some residual activity in the absence of ascorbate. However, these enzymes have diverse effects in different tissues, and their activity in an ascorbate-free environment may not be reflective of their roles *in vivo*.

On the other hand, normal physiological functioning of cells can be recapitulated when ascorbate is provided. Ascorbate appears to play an important role in the normal function of cultured endothelial cells, raising antioxidant protection, reducing oxidative stress and damage, and increasing eNOS activity when compared to cells devoid of vitamin C [33,38]. These effects on eNOS, at least, appear dependent on the ability of ascorbate to enhance the stability of tetrahydrobiopterin [34] and influence AMP-activated kinase (AMPK) activity [47]. In addition, ascorbate supplementation of cultured endothelial cells tightens cell-to-cell junctions that are critical for maintaining an endothelial barrier *in vivo* [48] and regulates NADPH oxidase activity [49], a critical component of the inflammatory response.

It is important to note that the effect of ascorbate supplementation may also greatly vary by cell type. Many of the aforementioned effects of ascorbate are observed in primary cell lines. Although propagated in the absence of ascorbate, the response to ascorbate supplementation in these cells reflects responses seen *in vivo*. Cancer cell lines and other immortalized cells, however, often show cytotoxic effects in response to ascorbate addition that are not observed in primary cell lines [24]. This may be the result of adaptations that have accumulated in these cells due to the "culture shock" that alters the normal physiological responses to stimuli [6], possibly involving iron dysregulation or aberrant cell signaling responses.
