Determination of plasma and leukocyte vitamin C concentrations in a randomized, double-blind, placebo-controlled trial with Ester-C ®
This crossover study examined vitamin C bioavailability and retention following a single oral administration of 1000 mg vitamin C provided by EC or AA as measured in plasma and leukocyte vitamin C concentrations. As expected, plasma vitamin C increased significantly from baseline at all time points when subjects were given EC and AA, but not when given PL. There was no significant difference in plasma vitamin C change between EC and AA, but significantly higher plasma Cmax was observed when subjects consumed EC compared to AA. EC lead to a sustained retention of leukocyte vitamin C during the investigational period, with percent changes from baseline that were significantly higher at 8 and 24 h post-dose compared to AA or PL. In contrast, AA resulted in poor leukocyte vitamin C retention, as indicated by a peak at 4 h postdose with a subsequent decline to baseline concentrations by 8 h. Additionally, the percent change in leukocyte vitamin C from baseline was significant at 4, 8, and 24 h with EC, while there were no significant increases with AA or PL. Area under the leukocyte concentration time curve was highest for EC and was 9 times higher than AA; however, values did not reach statistical significance due to subject variability.
Our study confirms that vitamin C from EC is more bioavailable in leukocytes than AA for male and female nonsmokers. These results are consistent with previous studies (unpublished observations) (Moyad et al. 2008; Wright and Kirk 1990). The most recent clinical trial found increased leukocyte vitamin C concentrations following a daily dose of 1000 mg vitamin C from EC compared to AA in healthy men (Moyad et al. 2008). Another double-blind, crossover trial with the same dose reported similar results (unpublished observations). A third trial found increased vitamin C leukocyte concentration at 8 and 24 h and also after 7–10 days of continuous ingestion of 3000 mg vitamin C as EC compared to AA (Wright and Kirk 1990). All three previous trials were done in men. To our knowledge, the present study is the first clinical trial comparing leukocyte vitamin C concentrations after EC and AA treatment in both male and female subjects. With the inclusion of female subjects, there is concern for a fluctuation in vitamin C concentration correlated with changing hormone levels throughout the menstrual cycle (Michos et al. 2006). To control for this potential variability, the trial was designed so that eumenorrheic women had 28 days between Test Periods, and vitamin C levels could be measured during the same phase of their menstrual cycles. Unexpectedly, only post-menopausal women were randomized, eliminating concerns about cyclic fluctuations.
Our study results also confirm that plasma vitamin C concentrations are tightly regulated; plasma vitamin C concentration with EC and AA had similar patterns, with an abrupt increase during the first 4 h and a peak at 4 h post-dose. Similarly, others have reported no difference in plasma vitamin C concentrations after a single dose of 1000 mg vitamin C given in the form of EC or AA in men (Moyad et al. 2008; Pancorbo et al. 2008). Interestingly, using a three-fold higher dose (3000 mg per day), Wright and Kirk (1990) found a higher serum vitamin C level when EC was consumed compared to AA, while Johnston and Luo (1994) reported a lower plasma vitamin C level when given a much lower EC dose (500 mg vitamin C per day). The latter study had a majority of female subjects under the age of 42 year with no evidence of controlling for vitamin C fluctuations during the menstrual cycle; in addition, subjects were pre-treated for two weeks with 1000 mg of vitamin C per day to saturate body stores before they entered the study, while no other studies used vitamin-C saturated subjects (Johnston and Luo 1994). From the evidence above, a dose–response curve may exist—a mega dose of EC, such as 3000 mg, may result in greater vitamin C concentration in plasma compared to AA, while 1000 mg of EC does not lead to a significant difference. Further studies are needed to clarify this dose–response relationship.
Under physiological conditions, vitamin C exists as both ascorbate (reduced form) and dehydroascorbate (DHA, oxidized form). It has been suggested that vitamin C is predominantly taken up by leukocytes in the form of DHA via a passive, energy-independent, gradient-driven process (Li and Schellhorn 2007). This gradient is influenced by the initial oxidation of vitamin C into DHA and the subsequent reduction into AA after entry into the cell. This allows leukocytes to store vitamin C at higher concentrations than those seen in plasma (Padayatty et al. 2007), which is important for proper cellular function (Bergsten et al. 1990). The vitamin C metabolites found in EC, generated after oxidation to DHA, are thought to further stabilize the extracellular oxidation and metabolism of DHA and/or enhance the passive, facilitated or active transport signaling mechanisms required for intracellular access (Moyad et al. 2008). Past research has indicated that the metabolites in EC help with the transport and utilization of vitamin C (Fay and Verlangieri 1991; Fay et al. 1994; Bush and Verlangieri 1987).
The increased retention of vitamin C in leukocytes is conducive to providing maximum cellular concentrations of vitamin C for optimum biochemical activity, including immune function. In a study with two groups of non-ascorbate synthesizing rats, EC fed animals showed higher vitamin C activity and body weight gain compared to the group fed AA (Verlangieri et al. 1991). In humans, the fundamental processes of leukocytes, especially neutrophil phagocytic capacity, can be depressed when leukocytes are low in vitamin C, however, repletion with vitamin C can help restore these important functions (Jayachandran et al. 2000). Diminished neutrophil function in subjects with furunculosis was improved following supplementation with 1000 mg vitamin C for 4–6 weeks, including increased neutrophil chemotaxis and phagocytosis (Levy et al. 1996). Enhancement of neutrophil motility, chemotaxis and phagocytosis has been observed after the ingestion of 250–3000 mg vitamin C in both healthy and diseased adults (Levy et al. 1996; Anderson et al. 1980). To our knowledge, only one published clinical trial has reported the disease treatment roles of EC—the study showed that compared to placebo, EC (1000 mg of vitamin C per day) can significantly reduce colds and shorten the duration of severe symptoms in winter over a 60-day period (Van Straten and Josling 2002). Further studies are needed to better understand how the level of vitamin C in leukocytes affects neutrophil function and is associated with disease prevention and treatment. The leukocyte vitamin C concentrations reported in our study can serve as future reference values.
While the current study did not measure urinary excretion of vitamin C, previous research is inconsistent. Wright et al. reported less urinary loss with 3000 mg of vitamin C from EC in the first 24 h and after 7–10 days of continuous ingestion when compared to AA (Wright and Kirk 1990). However, these results are not consistent with another study which found similar urinary excretion from EC and AA when only 500 mg of vitamin C was consumed daily (Johnston and Luo 1994). The latter study did not measure vitamin C levels in tissues, so it is possible that more vitamin C from EC moved into tissues (such as leukocytes) and thus yielded similar urinary loss and less plasma vitamin C when compared to AA (Johnston and Luo 1994).
There are a few limitations to our study. There was an observed variability in subjects’ vitamin C levels. Baseline plasma and leukocyte vitamin C concentrations showed a large standard deviation between subjects at study entry (Table 2); however, not significantly different between groups. This was anticipated as these values are dependent on many non-modifiable and modifiable factors (Li and Schellhorn 2007). To address this, measures were taken during this study to minimize the variability by using a crossover design, enrolling only non-smokers, providing meals during test periods and intensely counseling subjects to eliminate dietary vitamin C. The change from baseline and percent change from baseline in plasma and leukocyte vitamin C for each individual subject provided a more accurate assessment of the pharmacokinetics of the treatments. Further, we measured vitamin C levels after a single bolus dose. As shown in a previous trial, vitamin C concentrations in leukocytes were significantly higher at 8 and 24 h when treated with EC and the significant difference continued after 7–10 days of vitamin C consumption at 3000 mg (Wright and Kirk 1990). Future studies should investigate a continuous ingestion of various sources of vitamin C at a lower daily dose.