Table 2.

Summary of the most important design challenges in clinical studies of vitamin C in health and disease leading to potential misinterpretation of the results and erroneous conclusions.

Study type	Design challenges	Potential pitfall in interpretation
Observational studies	Confounding	Vitamin C deficiency is commonly accompanied by other micronutrient deficiencies, suboptimal lifestyle and other residual confounding that may potentially contribute to or even be responsible for the observed associations.
	Using vitamin C intake as surrogate marker for vitamin C status	Vitamin C intake is a poor surrogate for vitamin C status as the estimation of vitamin C intake is inherently inaccurate and the relationship between intake and status is highly complex.
Randomized controlled trials	Subjects already high in vitamin C at study start	Due to the saturation kinetics of vitamin C following oral administration, individual vitamin C status greatly affect the potential effect of supplementation. As vitamin C deficiency is most commonly limited to selected subpopulations, the potential efficacy will be effectively diluted if inclusion criteria are not taking this into account.
	Placebo group continues to take supplements	Allowing continued supplement intake in the placebo group will test two doses of vitamin C against each other rather than the effect of vitamin C supplementation per se. Because of saturation kinetics, this will further diminish the possibility of identifying effects of the vitamin C intervention.
	Both intervention and placebo groups have had a lifelong preload with vitamin C	The human diet typically contains from 0 to 250mg vitamin C per day not considering supplementation, i.e. a wide range and with the high end being within the range or even exceeding that of several of the large intervention studies. Thus, the potential for observing disease prevention with supplementation vs placebo during the study period should be compared to the lifelong vitamin C status of all study subjects.
	Vitamin C is not tested as a single supplement	Not testing vitamin C as a single supplement limits the possibility of extracting its effect per se.
All studies	Selection bias	Recruitment may favor health-conscious, self-motivated subjects eating a healthy diet already high in micronutrients and with a lower disease rate than background population. This will limit the possibility of identifying effects of supplementation.
	Using non-fasted blood samples	Oral vitamin C intake produces a transient albeit significant increase in plasma level depending on the vitamin C status of the individual (See Fig. 2D for example). This will lead to larger variation and may result in an artefactually high average vitamin C concentration.
	Inadequate sample handling	Inadequate sampling stabilization and handling leads to increased post sampling oxidation and artefactually low vitamin C concentrations regardless of methodology.