2.3.2. Technical Issues of Human Studies

Vitamin C research is heavily dependent on accurate assessment of ascorbate in biological samples. Unfortunately, there is no standard method for measuring vitamin C that has been applied across the field. It is generally acceptable practice to preserve samples in acid or methanol after sample extraction. Furthermore, it is preferred that a direct measure of ascorbic acid be made, such as HPLC separation followed by electrochemical detection (ECD). Other chromatographic methods are generally avoided, especially those that require oxidation of the sample followed by derivatization as these procedures can generate erroneous results. The detection of dehydroascorbic acid cannot be achieved with ECD unless the sample is first treated with a reductant, obtaining total ascorbate levels from which dehydroascorbic acid levels can be inferred [112,113]. However, the value and interpretation of dehydroascorbic acid measurements in biological samples is questionable (see below).

The labile nature of ascorbic acid outside the body underscores the need for controlled conditions during collection, processing, and storage of biological samples. In many human studies, poor standards in obtaining blood or tissues specifically for vitamin C analysis are complicated by a lack of controlled sample handling in many clinical settings. Phlebotomy is common practice, but many factors can contribute to the instability of ascorbic acid in biological samples. Although the use of vacutainers for plasma samples is generally acceptable for vitamin C analysis, the choice of vacutainer type and anticoagulants can influence the results obtained [114]. As an example, plasma ascorbate levels were determined from five individuals using various anticoagulants or no anticoagulant (serum) as a control. Despite individual variability in plasma ascorbate levels reflected in the standard error, the data (Figure 2) suggest that the use of K2 EDTA vacutainers results in a significant loss of ascorbate compared to both sodium and lithium heparin containers. Consistent with these data, EDTA has been shown to accelerate the oxidation of ascorbate in whole blood and plasma [115,116] and be unable to prevent the loss of ascorbate in the presence of iron or copper [8]. In addition, it has been suggested that fluoride and serum vacutainers should be avoided for vitamin C analysis [114].

**Figure 2.** Vacutainer effects on plasma ascorbate levels. Plasma ascorbate concentrations were determined from different anticoagulant-containing or untreated vacutainers as described in Materials and Methods. Plasma ascorbate means are the average and standard error from five different subjects. ANOVA analysis shows a significant (*p* < 0.05) decline in plasma ascorbate levels in K2 EDTA vacutainers when compared to lithium or sodium heparin vacutainers as denoted by asterisk (\*).

The oxidation of vitamin C in plasma is accelerated by heat, light, and elevated pH, similar to cell culture media as described above. Sample mishandling can cause the aberrant generation of dehydroascorbic acid in the sample and, over time, will cause a loss of total ascorbate. For instance, by careful preparation of the sample under nitrogen and limited exposure to heat and light, dehydroascorbic acid levels can be minimized (Figure 3). More reasonable, standard preparation methods with brief exposures to air, light, and heat result in little change in plasma ascorbate levels (Figure 3b). On the other hand, exposing samples to room temperature for hours not only can result in a significant decline in (reduced) ascorbic acid, but a loss of total ascorbic acid as well (Figure 3a). Not only does this result in an inaccurate estimate of dehydroascorbic acid levels in the exposed sample (Figure 3c) but the loss of total ascorbate suggests degradation of dehydroascorbic acid has also occurred.

The implication of these data (Figure 3) and others [114–116] is that care in sample handling with concern for ascorbate oxidation is crucial for accurate ascorbate analysis. Study designs must incorporate specific handling conditions of samples intended for ascorbate analysis (ideally by immediate plasma isolation, rapid acidification, and freezing below −20 °C) to avoid misinterpretations compounded by the use of poorly preserved samples. Furthermore, it also suggests that the presence of dehydroascorbic acid in clinical samples is more a measure of sample handling than a biologically relevant marker of *in vivo* oxidative stress.

**Figure 3.** Effects of sample handling on plasma ascorbate and dehydroascorbic acid concentrations. Vacutainers with blood samples were prepared under oxidation controlled, standard and exposed conditions as described in Materials and Methods. Plasma total ascorbate (**a**) and reduced ascorbate (**b**) levels are highest in controlled samples, showing declines under standard and exposed preparations that are reflected in calculated dehydroascorbic acid levels (**c**). Significant changes were observed in the exposed group when compared to controlled or standard samples as determined by ANOVA, and denoted with an asterisk (\*).

2.3.3. Health Effects of Vitamin C: Reality *versus* Mythology

Many excellent evidence-based reviews have summarized the health effects of vitamin C, focusing on vitamin C's possible role in the prevention or treatment of cardiovascular disease, cancer, diabetes, and other diseases [1,2,4,117]. However, there are a number of health effects attributed to vitamin C supplementation that are not supported by controlled trials with fundamental, mechanism-based endpoints. Opponents of vitamin C supplementation have sensationalized studies that have suggested deleterious health claims, most of which are of little consequence to the general population. For example, since vitamin C promotes iron absorption [118], this led to claims that vitamin C supplements were detrimental in individuals suffering from iron overload and hemochromatosis. On the contrary, in animals and humans with iron overload, high plasma ascorbate levels are protective against oxidative damage induced by excess iron [18,19]. Indeed, the recommendation for these individuals is to not avoid ascorbate, but instead limit dietary iron intake.

Another commonly cited risk of ascorbate supplementation is the formation of kidney stones, as oxalate is a product of dehydroascorbic acid breakdown. The evidence linking ascorbate supplement use and incidence of kidney stones in otherwise healthy individuals is mixed [119–121]. Large amounts of oxalate derived from ascorbate would require excessive vitamin C oxidation, but the mechanism underlying this oxidation has not been explored. Studies that suggest an increased risk observed it in individuals taking more than 1000 mg of vitamin C per day, in far excess of the amount that can be obtained from food sources. Without further study, it is prudent to caution individuals with kidney disease or a history of kidney stones against taking large amounts of vitamin C supplements. Unfortunately, this has also resulted in practitioners to advise individuals undergoing dialysis to severely restrict vitamin C consumption, leading to deficiencies [122].

The consumption of large doses of vitamin C supplements has also been occasionally associated with skin rash, heartburn, nausea, and diarrhea. These are usually the result of the formulation of the vitamin C tablets but may also be caused by excessive consumption of vitamin C in a short period of time. Large doses of vitamin C have been anecdotally associated with vitamin B12 deficiency and systemic conditioning (also known as "rebound scurvy"), conditions that have never been documented clinically [123]. Patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency have also been cautioned against taking vitamin C supplements, due to reports of hemolytic anemia that have also not been substantiated [124].

One of the most persistent health claims for vitamin C supplementation is the prevention and treatment of the common cold. Recent meta-analyses of the data from 70 years of placebo-controlled trials demonstrated an overabundance of poorly controlled trials with no apparent focus on the mechanism and biological relevance of vitamin C supplementation [125,126]. Despite the large number of research studies, the evidence supporting the effects of vitamin C supplements on cold incidence and duration has been relatively weak, with the exception of marathon runners, skiers, soldiers in subarctic conditions [125], or individuals with chronic gastritis [127]. A tendency to include older data is a common pitfall for the analysis of vitamin C research, performed when our understanding of vitamin C in biology was limited, and thus allowing for a bias toward poorer designed studies. Our understanding of vitamin C's role in biology has improved over time, leaving a re-analysis of these studies unable to provide any substantial conclusions with respect to the common cold and other proposed health effects of vitamin C supplementation. To resolve the controversies, a modern approach of evaluating rigorously designed, mechanism-based studies is necessary.
