**1. Introduction**

Vitamin C (ascorbate) is an essential water-soluble micronutrient in humans and is obtained through the diet primarily from fruits and vegetables [1]. *In vivo*, it acts as a cofactor for numerous biosynthetic enzymes required for the synthesis of amino acid-derived macromolecules, neurotransmitters and neuropeptide hormones [2], and for various hydroxylases involved in the regulation of gene transcription and epigenetics [3,4]. Vitamin C is concentrated from the plasma into the body's organs and is found in particularly high concentrations in the pituitary and adrenal glands and in the corpus luteum [5], although skeletal muscle, brain, and liver comprise the largest body pools [6]. Most animals can synthesize vitamin C from glucose in the liver [7]; however, humans and a small selection of animal species have lost the ability to synthesize vitamin C due to mutations in the gene encoding L-gulono-γ-lactone oxidase, the terminal enzyme in the vitamin C biosynthetic pathway [8]. Therefore, an adequate and regular dietary intake is essential to prevent hypovitaminosis C and the potentially fatal deficiency disease, scurvy [9].

In the mid 1700s the Royal Navy surgeon James Lind carried out controlled dietary trials and determined that citrus fruit could cure individuals with scurvy (reviewed in [10]). However, it wasn't until the early 1900s that experimental scurvy was first produced in guinea pigs through dietary restriction and shown to be prevented by feeding the animals fresh fruits and vegetables. In the early 1930s vitamin C was isolated from fruit and vegetables and adrenal cortex and was named "hexuronic acid", which was shown to cure scurvy in guinea pigs and was subsequently renamed ascorbic acid to reflect its anti-scorbutic properties. Vitamin C was first chemically synthesized in 1933 [10] and, since the mid 1930s, the question of the comparative bioavailability of synthetic *versus* natural, food-derived vitamin C in animal models and human subjects has been a point of consideration.

The bioavailability of dietary vitamin C represents the proportion of the micronutrient that is absorbed by the intestines and is available for metabolic processes within the body. *In vivo* vitamin C levels are a function of uptake, metabolism, and excretion (see [11] for an excellent review of these processes). Vitamin C is actively transported into the body via two sodium-dependent vitamin C transporters, SVCT1 and SVCT2 [12,13]. These transporters exhibit different tissue distributions and uptake kinetics. SVCT1 is expressed in epithelial tissues and is primarily responsible for intestinal uptake and renal reabsorption of vitamin C, the latter helping to maintain whole body homeostasis [13]. SVCT2 is expressed in specialized and metabolically active tissues and is required for delivery of vitamin C to tissues with a high demand for the vitamin either for enzymatic reactions [2] and/or to help protect these tissues from oxidative stress [13]. Both of these transporters show significantly more affinity for the L- *versus* D-isoform of vitamin C (Figure 1) [12,14], and this selectivity likely explains earlier observations of significantly lower tissue accumulation and anti-scorbutic activity of D-ascorbic acid in guinea pigs [15,16]. Although D-ascorbic acid is a commonly added food preservative [17], administration of D- and L-ascorbic acid together does not affect the bioavailability of the latter in humans [18].

Through its action as a reducing agent and antioxidant, ascorbate undergoes one and two electron oxidations to produce the ascorbyl radical and dehydroascorbic acid (DHA) (Figure 1). Recent research has shown that DHA can be taken up by the facilitative glucose transporters GLUT2 and GLUT8 in the small intestine [19]. Cells are also able to transport DHA via GLUT1 and GLUT3 [20,21], followed by intracellular reduction to ascorbate [22,23]. However, transport via the GLUTs is in competition with glucose which is at relatively high concentrations throughout the body and although different fruits and vegetables have been shown to contain relatively high amounts of DHA [24], the *in vivo* contribution of DHA is uncertain due to its minimal circulating and organ levels [25,26] (although white blood cells may be an exception to this) [27,28].

**Figure 1.** Vitamin C in its reduced form (ascorbic acid), shown as both its L- and D-isomers, and its two electron oxidation form (dehydroascorbic acid, DHA). DHA can be readily reduced back to ascorbic acid *in vivo* via both chemical and enzymatic pathways [23].

Synthetic and food-derived vitamin C is chemically identical. However, fruit and vegetables are rich in numerous micronutrients (vitamins and minerals), dietary fiber, and phytochemicals (e.g., bioflavonoids), and the presence of some of these may affect the bioavailability of vitamin C. Vitamin C has long been known to interact with vitamin E by reducing the tocopheroxyl radical and regenerating native tocopherol [29]. Some fruit, such as kiwifruit, contain relatively high amounts of vitamin E and one animal study has indicated that vitamin E is able to preserve vitamin C *in vivo* [30]. Food-derived (and synthetic) vitamin C is well known to increase non-heme iron uptake and body status, likely via its ability to reduce iron from its ferric to ferrous state [31,32]. However, whether iron can affect vitamin C bioavailability is less clear [33–35]. Although iron has been shown to increase the uptake of vitamin C in cultured intestinal cells [33], human intervention studies have shown no effect of iron intake on vitamin C bioavailability [34,35]. One study has indicated that specific dietary fibers, such as hemicellulose and pectin, may affect the excretion of vitamin C [36], however, their influence on vitamin C uptake was not determined.

Plant-derived flavonoids have been of interest since the mid 1930s, when they were initially referred to as "vitamin P", primarily due to their effect on vascular permeability [37]. At the time, there was much debate in the literature regarding the role of "vitamin P" in experimental [38–42] and human scurvy [37,43–45]. Flavonoids can act as antioxidants via direct scavenging of free radicals [46,47] and/or chelation of redox-active metal ions [48,49]. As a result, it has been suggested that flavonoids may "spare" vitamin C and, thus, increase its bioavailability. Flavonoids have been shown to inhibit the *in vitro* oxidation of vitamin C [48–51], however, the *in vivo* relevance of metal-ion mediated oxidation of vitamin C is likely to be minimal as free metal ions are largely sequestered in the body [52]. Whether flavonoids can affect vitamin C uptake *in vivo* is uncertain due to the low plasma bioavailability of these compounds [53]. Thus, any interaction of flavonoids with vitamin C would be expected to occur primarily in the intestinal lumen prior to active uptake.

Of note, several *in vitro* studies have shown that various flavonoids can inhibit vitamin C and DHA uptake by their respective transporters. The flavonoid quercetin can reversibly inhibit SVCT1 expressed in *Xenopus* oocytes [54] and limited data from an animal model indicates that this may occur *in vivo* [54]. Quercetin and myricetin can inhibit the uptake of vitamin C and DHA into cultured monocytic (HL-60 and U937) and lymphocytic (Jurkat) cells via inhibition of GLUT1 and GLUT3 [55] and possibly also SVCT2, which is expressed in leukocytes [56]. Quercetin and phloretin can also inhibit the intestinal GLUT2 and GLUT8 transporters [19]. Thus, based on the above *in vitro* studies, it is unclear whether flavonoids will enhance *in vivo* vitamin C bioavailability through a sparing action, or decrease its bioavailability through inhibiting vitamin C transporters.

The effect of various purified flavonoids or flavonoid-rich fruits and vegetables on vitamin C bioavailability in different animal models and human subjects is discussed below. To test comparative vitamin C bioavailability, both steady-state and pharmacokinetic models have been used. The former monitors ascorbate levels in blood and/or urine following a number of weeks of supplementation, while the latter monitors transient changes in plasma levels and/or urinary excretion over the hours following ingestion of the vitamin C-containing test substance. The gold standard for analysis of vitamin C is HPLC with coulometric electrochemical detection due to its sensitivity and specificity [57]. Early studies, however, were limited primarily to colourimetric methods based on reduction of ferric iron compounds and are prone to interference by numerous other substances [57].
