Vitamin C and Neutrophil Function: Findings from Randomized Controlled Trials

Vitamin C is known to support immune function and is accumulated by neutrophils to millimolar intracellular concentrations suggesting an important role for the vitamin in these cells. In this review, the effects of vitamin C, as a mono- or multi-supplement therapy, on neutrophil function were assessed by conducting a systematic review of randomized controlled trials (RCTs). Specifically, trials which assessed neutrophil migration (chemotaxis), phagocytosis, oxidative burst, enzyme activity, or cell death (apoptosis) as primary or secondary outcomes were assessed. A systematic literature search was conducted using the Cochrane Central Register of Controlled Trials, EMBASE, Embase Classic, Joanna Briggs Institute EBP, Ovid MEDLINE®, Ovid MEDLINE® In-Process & Other Non-Indexed Citations, Ovid Nursing Database, CINAHL and PubMed database, which identified 16 eligible RCTs. Quality appraisal of the included studies was carried out using the Cochrane Risk of Bias tool. Three of the studies assessed neutrophil chemotaxis in hospitalised patients or outpatients, two of which showed improved neutrophil function following intravenous vitamin C administration. Ten RCTs assessed neutrophil phagocytosis and/or oxidative burst activity; five were exercise studies, one in smokers, one in myocardial infarction patients and three in healthy volunteers. Two of the multi-supplement studies showed a difference between the intervention and control groups: increased oxidative burst activity in athletes post-exercise and decreased oxidant generation in myocardial infarction patients. Two studies assessed neutrophil enzyme activity; one showed deceased antioxidant enzyme activity in divers and the other showed increased antioxidant enzyme activity in athletes. One final study showed decreased neutrophil apoptosis in septic surgical patients following intravenous vitamin C administration. Overall, 44% of the RCTs assessed in this review showed effects of vitamin C supplementation on neutrophil functions. However, the studies were very heterogeneous, comprising different participant cohorts and different dosing regimens. There were also a number of limitations inherent in the design of many of these RCTs. Future RCTs should incorporate prescreening of potential participants for low vitamin C status or utilize cohorts known to have low vitamin status, such as hospitalized patients, and should also comprise appropriate vitamin C dosing for the cohort under investigation.


Introduction
Neutrophils are a vital component of the innate immune system, providing a first line of defense against invading pathogens [1]. Following microbial invasion, neutrophils migrate to the site of infection in response to pathogen-and host-derived pro-inflammatory mediators, known as chemotaxis [1]. The neutrophils then proceed to phagocytose, kill and digest the invading pathogens via both oxidative , CINAHL and PubMed databases using the following keywords: (1) vitamin C OR ascorbic acid OR ascorbate OR antiscorbutic factor OR l-ascorbate OR l-ascorbic acid; AND (2) neutrophils OR polymorphonuclear leukocytes OR immune system OR immunity OR immune function OR inflammation; AND (3) migration OR mobility OR chemotaxis OR apoptosis OR phagocytosis OR clearance OR necrosis OR necrotic cell death OR oxidative burst OR neutrophil enzyme activity. No restrictions were placed for the publication date, study location, age of participants, nature of participants, route of administration of vitamin C, and the source of vitamin C (supplemental or food-derived). However, only published RCTs and publications in English were included in this review.
The abstracts and titles of the papers obtained from the literature search were screened to ensure that all the PICO components were covered and also that the inclusion/exclusion criteria were met ( Table 1). Studies that did not meet the inclusion/exclusion criteria were rejected. Full-texts of the relevant papers were obtained using the University of Otago library and ResearchGate websites. Each of the full-text articles gathered for analysis were critically appraised for quality and risk of bias using the Cochrane Risk of Bias tool for RCTs [23]. For every criterion, each study was rated either low, unclear or high risk of bias and therefore appraised as good, fair or poor-quality research. The primary outcome measure for this review was the relationship between vitamin C and neutrophil function in humans.

Literature Search Outcome
The process of selection and screening of studies is illustrated in Figure 1 using the PRISMA flow chart [24]. Screening was based on the predetermined inclusion/exclusion criteria as well as the PICO components. After the screening process, sixteen papers were found to be eligible and were quality appraised for risks of bias using the Cochrane Risk of Bias tool ( Figure 2) [23]. Four publications were rated low-quality following the critical appraisal, of which two papers failed to meet allocation concealment and blinding requirements scoring as high-risk for selection bias and performance bias while the other two papers had flawed statistical analysis owing to missing data thus scoring as high-risk for attrition bias and other bias. Due to the limited number of studies available, these four publications were included in the analysis and results, however, the limitations of these papers were highlighted. Thus, sixteen papers were included in the synthesis.

Study Characteristics
The 16 studies described were conducted in nine locations. These included Africa (n = 1), Austria (n = 1), New Zealand (n = 1), the Netherlands (n = 1), Poland (n = 1), Spain (n = 3), South Africa (n = 1), United States (n = 4), and the United Kingdom (n = 3). The 16 studies comprised nine that used vitamin C as monotherapy and seven that used combination supplementation. As illustrated in Tables 2a and b (vitamin C only and combination studies, respectively), participants were recruited from a number of different settings including schools, clinics, communities, and surgical departments of hospitals. However, some studies did not specify the trial settings. The time frames of the studies ranged from one day to six-month long trials. The sample size of the studies ranged from six to 131 participants garnering a total number of 497 participants. There was a wide variation in the methodologies used between studies, e.g., the neutrophil assays were either carried out with whole blood or the cells were isolated prior to analysis in buffer or culture media (Table 2a,b), the intervention was administered at different doses, via different routes of administration (four studies used intravenous vitamin C, with trauma, surgical, and septic cohorts), and the treatment periods also varied dramatically from one day to six months (Table 3a,b). The included studies also covered a wide range of age groups as illustrated in Table 4. Furthermore, the control measures instigated for potential confounding factors were also variable, e.g., not all studies measured the plasma vitamin C concentrations at baseline, and diet control as well as smoking status were often not taken into consideration or reported (Table 4). Overall, three studies assessed neutrophil chemotaxis (with one also assessing phagocytosis), ten studies assessed neutrophil phagocytosis and/or oxidative burst activity, two studies assessed neutrophil enzyme activity, and one study assessed neutrophil apoptosis.

Study Characteristics
The 16 studies described were conducted in nine locations. These included Africa (n = 1), Austria (n = 1), New Zealand (n = 1), the Netherlands (n = 1), Poland (n = 1), Spain (n = 3), South Africa (n = 1), United States (n = 4), and the United Kingdom (n = 3). The 16 studies comprised nine that used vitamin C as monotherapy and seven that used combination supplementation. As illustrated in Table 2a and Table 2b (vitamin C only and combination studies, respectively), participants were recruited from a number of different settings including schools, clinics, communities, and surgical departments of hospitals. However, some studies did not specify the trial settings. The time frames of the studies ranged from one day to six-month long trials. The sample size of the studies ranged from six to 131 participants garnering a total number of 497 participants. There was a wide variation in the methodologies used between studies, e.g., the neutrophil assays were either carried out with whole blood or the cells were isolated prior to analysis in buffer or culture media (Table 2a and Table 2b), the intervention was administered at different doses, via different routes of administration (four studies used intravenous vitamin C, with trauma, surgical, and septic cohorts), and the treatment periods also varied dramatically from one day to six months (Table 3a and Table 3b). The included studies also covered a wide range of age groups as illustrated in Table 4. Furthermore, the control measures instigated for potential confounding factors were also variable, e.g., not all studies measured the plasma vitamin C concentrations at baseline, and diet control as well as smoking status were often not taken into consideration or reported (Table 4). Overall, three studies assessed neutrophil chemotaxis (with one also assessing phagocytosis), ten studies assessed neutrophil phagocytosis and/or oxidative burst activity, two studies assessed neutrophil enzyme activity, and one study assessed neutrophil apoptosis.

Neutrophil Chemotaxis
Three of the included RCTs assessed neutrophil migration in response to chemoattractants; two were hospitalized cohorts (surgical premedication and trauma) and one from an outpatient clinic, and all three used intravenous vitamin C [25][26][27]. Anderson et al. [25] showed that administration of intravenous vitamin C (1000 mg once daily) to asthmatic children was associated with improved neutrophil chemotaxis compared with standard anti-asthma chemoprophylaxis (SAC) treatment alone. Charlton et al. [26] showed that premedication (papaveretum and hyoscine) used prior to surgery caused a decline in neutrophil chemotaxis in children undergoing elective surgical procedures. After the premedication was administered, neutrophil chemotaxis still fell significantly in the vitamin C group (single dose of 10 mg/kg) and was not significantly different to the placebo group. This suggests that vitamin C supplementation before premedication does not protect against depression of neutrophil chemotaxis in children as a result of the premedication given.
Neutrophil chemotaxis following serious blunt trauma was shown to be significantly decreased soon after injury, reaching maximum depression by day two of injury [27]. Plasma vitamin C concentrations also decreased to half initial levels. Neutrophil chemotaxis increased for those treated for a week with both vitamins C and E (200 and 50 mg/day, respectively). For the patients who received either vitamin C or E alone, there was a slightly better response than the placebo group, but this did not reach statistical significance, likely due to the low participant numbers (e.g., only three analysed in the vitamin C group). Thus, the combination of vitamins C and E together gave a better response than either vitamin C or E alone with regard to neutrophil chemotaxis.

Phagocytosis and Oxidative Burst
Five studies investigated the effects of either vitamin C alone or antioxidant combinations on exercise-induced changes in neutrophil oxidative burst activity and/or phagocytosis [19,28,29,31,35]. Nieman et al. [19] showed that exercise caused a significant increase in granulocyte phagocytosis and a decrease in granulocyte oxidative burst post-exercise. The vitamin C group (1 g/day for eight days) did not differ significantly from the placebo group which suggests that vitamin C supplementation had no effect on the exercise-induced changes in granulocyte phagocytosis and oxidative burst activity. Krause et al. [31] reported decreased phagocytosis and bactericidal activity of neutrophils isolated after high-intensity exercise (biathlon), although no change in intracellular reactive oxygen species generation was observed. There were no differences between the vitamin C supplementation (2 g for one week) and the placebo groups in this study. Davison and Gleeson [28] investigated the influence of acute vitamin C supplementation (3.4 g single dose) and/or carbohydrate ingestion on neutrophil degranulation and oxidative burst activity after a prolonged exercise trial. They found that neutrophil degranulation was significantly decreased in the vitamin C and carbohydrate + vitamin C groups post-exercise but did not differ to the placebo group. In addition, neither vitamin C nor carbohydrate had an effect on the decrease in neutrophil oxidative burst activity post-exercise. Therefore, in this study, vitamin C supplementation had no effect on neutrophil degranulation or oxidative burst activity post-exercise. In a similar study conducted by the same investigators [29], it was reported that both oxidative burst activity and neutrophil degranulation was decreased post-exercise. Vitamin C supplementation (1 g/day for two weeks) was no different to the placebo group and thus did not influence neutrophil oxidative burst or degranulation.
In contrast to the vitamin C-alone supplementation studies, Robson et al. [35] showed that neutrophil oxidative burst was significantly higher in trained runners following prolonged exercise in the antioxidant group (with vitamin C 960 mg/day for one week) compared to the placebo group post-exercise. Thus, antioxidant combinations may provide improved neutrophil function in athletes compared to vitamin C monotherapy.
One study investigated the effects of vitamin C supplementation on neutrophil oxidative burst of smokers [30]. Smoking increases susceptibility to bacterial and viral infections and compromises the anti-microbial functions of neutrophils [40,41]. Furthermore, smoking is associated with lower plasma vitamin C concentrations [42][43][44][45]. Fuller et al. [30] supplemented healthy smokers with vitamin C (1 g/day for eight weeks) and/or vitamin E but found no difference in neutrophil superoxide production in any of the treatment groups compared with placebo, despite an increase in plasma vitamin C status (from 39 to 62 µmol/L).
Ischemia/reperfusion injury can also result in increased production of reactive oxygen species, inducing oxidative stress, and is common in disease states such as stroke and myocardial ischemia [46]. In a study of patients with acute myocardial infarction, Herbaczynska et al. [34] found that supplementation with vitamins C and E (600 mg/day) decreased oxygen free radical generation by neutrophils. Thus, the combination of vitamins C and E appeared to exhibit antioxidant functions in these patients.
Two studies investigated the effects of micronutrient supplementation on neutrophil phagocytosis and oxidative burst activities in healthy individuals [37,38]. Wolvers et al. [38] found that the consumption of micronutrient (with~375 mg/day of vitamin C for ten weeks) did not affect the phagocytosing or oxidative burst activities of neutrophils in any of the treatment groups of healthy volunteers. Nieman et al. [37] showed that breakfast cereal fortified with varying levels of micronutrients (low, medium and high groups containing 0.8, 20, and 100 mg/day of vitamin C respectively) given over two months to healthy children also did not affect the pattern of change in granulocyte phagocytosis or oxidative burst. Finally, Hunter et al. [36] supplemented healthy older adults with a vitamin C rich food (gold kiwifruit containing~360 mg vitamin C daily for four weeks), however, this had no effect on neutrophil phagocytosing activity. Thus, micronutrient supplementation or fortification, or vitamin C-rich foods, do not appear to influence neutrophil function in healthy individuals.

Neutrophil Enzyme Activities
The enzymes catalase, superoxide dismutase and glutathione peroxidase scavenge reactive oxygen species and are therefore markers of neutrophil antioxidant defence [47]. Two studies investigated the effect of vitamin C, either alone or in an antioxidant cocktail, on neutrophil enzyme activities [32,39]. A study of a group of divers showed that oxidative stress induced by hypoxia can affect neutrophil function, specifically neutrophil antioxidant defences [32]. They showed that a group of divers supplemented with vitamin C (1 g/day for seven days) had significantly lower catalase and glutathione peroxidase enzyme activities compared to the placebo group at post-dive as well as during recovery. One interpretation was that vitamin C supplementation may have resulted in a higher rate of non-enzymatic scavenging of reactive oxygen species and hence a decrease in the activity of the antioxidant enzymes. In contrast, in a group of athletes, Tauler et al. [39] showed that three months of antioxidant supplementation (which included 500 mg/day vitamin C for the last fifteen days) significantly increased neutrophil enzyme activities, particularly of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase, relative to the placebo group. Once again, an antioxidant cocktail appeared to be more effective than monotherapy in athletes.

Neutrophil Apoptosis
One study investigated the effects of vitamin C supplementation on apoptosis in neutrophils isolated from the peripheral blood of septic abdominal surgery patients [33]. Although these investigators did not assess the vitamin C status of their patients, it is well known that low vitamin C status is a common feature of patients with sepsis [9]. Ferron-Celma et al. [33] found that administration of intravenous vitamin C (450 mg/day for six days) resulted in significantly lower levels of the pro-apoptotic factors caspase-3 and poly-ADP-ribose polymerase (PARP) in the vitamin C group compared to the placebo group. In addition, significantly higher levels of the anti-apoptotic factor (Bcl-2) were observed in the vitamin C group compared to the placebo group. Therefore, this study indicated that vitamin C supplementation may exert anti-apoptotic effects on the peripheral blood neutrophils of septic patients.

Discussion
Overall, nine of the 16 RCTs included in this review reported no effect of supplementation with vitamin C alone, or in combination with other micronutrients or antioxidants, on various neutrophil functions [19,26,[28][29][30][31][36][37][38]. The seven studies which did show effects of supplementation on the neutrophil functions assessed (i.e., chemotaxis, oxidative burst activity, antioxidant enzyme activity and apoptosis) were in hospitalized patients or outpatients [25,27,33,34] or athletes [32,35,39]. None of the other studies carried out with healthy volunteers showed any effects of additional supplementation.
Eight of 10 RCTs showed no effect of supplementation on neutrophil phagocytosis and/or oxidative burst activity [19,[28][29][30][31][36][37][38]. The two studies that showed an effect on oxidative burst activity used combination supplements and they showed opposite effects [34,35]. Herbaczynska-Cedro et al. [34] showed decreased oxidant burst activity in acute myocardial infarction patients treated with a combination of vitamins C and E. It is well known that vitamin C can recycle vitamin E and vitamin C has been proposed to interact with vitamin E in vivo, thus they appear to work synergistically [48]. Although another study also tested a combination of vitamins C and E, they saw no effect on oxidative burst activity [30]. However, this study was carried out in smokers and it is known that smoking causes enhanced oxidative stress and smokers require significantly higher intakes of vitamin C to reach the same circulating concentrations as non-smokers [49]. In contrast, Robson et al. [35] showed an increase in oxidative burst activity in trained runners supplemented with an antioxidant combination following prolonged exercise. Since the baseline vitamin C status of the participants was already at saturating levels (i.e., 70 µmol/L), this suggests that the observed effects may have been due to other components of the antioxidant mixture, although it does not rule out vitamin C acting synergistically with these components.
Eight of the 10 RCTs investigating oxidative burst activity and/or phagocytosis were carried out in healthy participants or athletes [19,28,29,31,[35][36][37][38]. However, only one of these studies assessed baseline vitamin C status, which was already saturating [35]. It should also be noted that neutrophils saturate at lower vitamin C levels than plasma [50]. Therefore, it is unlikely that supplementing healthy volunteers or athletes with additional vitamin C over and above their normal baseline levels would have an effect on neutrophil function [51]. Exercise trials can also be complicated in that, depending on the exercise intensity, the effects on the immune system can vary such that moderate exercises may have immunopotentiating effects while intense exercise is potentially immunosuppressive [52]. Although two combination trials showed positive effects of supplementation on neutrophil function in athletes [35,39], with combination studies, it is not always possible to determine which of the component(s) is having the effects, or if there are synergistic interactions occurring between the components.
Four of the RCTs administered intravenous vitamin C in hospital or outpatient settings, three of which saw an effect of the intervention on neutrophil chemotaxis and apoptosis [25][26][27]33]. Intravenous vitamin C is known to provide significantly higher plasma concentrations of vitamin C than oral administration [53]. Anderson et al. showed that administration of intravenous vitamin C (1000 mg once daily) was associated with improved neutrophil chemotaxis in asthmatic patients. Maderazo et al. [27] reported an increase in plasma vitamin C status and enhanced neutrophil chemotaxis in trauma patients administrated the combination of vitamins C and E. Ferron-Celma et al. [33] observed a decrease in pro-apoptotic enzymes and an increase in anti-apoptotic proteins in septic patients administered vitamin C alone. The forth study was carried out in healthy children undergoing elective surgery and although their baseline vitamin C levels were not assessed, these may have already been adequate [26]. Furthermore, the vitamin C was administered as a single dose only (10 mg/kg bodyweight), which is less likely to have an effect than repeated dosing [54].
There are a number of limitations of using the RCT paradigm to assess the effectiveness of nutrients such as vitamin C [55]. RCTs were specifically developed and designed to test the safety and efficacy of pharmaceutical drugs, not nutrients. For example, it is not possible to have a true placebo group in RCTs of nutrients as all of the participants will be consuming variable amounts of the nutrient of interest. Of note, a number of the assessed studies did not control for dietary intake or the participants were asked to maintain their normal diet. More than half of the RCTs included in this review did not assess baseline plasma vitamin C status, and a majority of the studies were carried out in healthy individuals, who likely already had adequate plasma vitamin C status prior to beginning the supplementation, thus negating any effect of supplementation. Only one study measured the vitamin C content of the neutrophils pre-and post-supplementation [32]. There was also variability in the analysis of the neutrophil functions, with the chemotaxis, enzyme activity, and apoptosis assays being carried out with isolated cells in buffer or culture media, and the phagocytosis and/or oxidative burst assays being carried out with either whole blood or isolated cells. Furthermore, two of the studies used only a single dose of the vitamin [26,28]. Because vitamin C is water soluble, it is cleared rapidly from circulation by the kidneys, with a half-life of approximately two hours, therefore a regular intake is required to maintain adequate levels [53]. Finally, smoking status is well known to impact on vitamin C status and requirements, due to enhanced oxidative stress [43,49], however, this was not taken into account in a majority of the studies.

Conclusions
Overall, 44% of the RCTs assessed in this review showed effects of vitamin C supplementation on various neutrophil functions. The studies were very heterogeneous, comprising different participant cohorts (athletes, hospitalized patents or healthy volunteers) and different dosing regimens (oral or intravenous, monotherapy or multi-supplements, synthetic or food-derived, and from one-off to many months in duration). There were also a number of limitations inherent in the design of many of these RCTs. Unlike drug trials, evidence indicates that RCTs of vitamin C supplementation will be more likely to have a positive effect in participants who are suboptimal or deficient in the vitamin at baseline [55]. Therefore, future RCTs should incorporate prescreening of potential participants for low vitamin C status or utilize cohorts known to have low vitamin status, such as hospitalized patients. The effects of vitamin C administration on more recently discovered functions of neutrophils, such as the formation of neutrophil extracellular traps, should also be explored in future studies based on promising in vitro and preclinical data [16]. Meta-analyses have indicated that vitamin C intakes of at least 200 mg/day can decrease the risk of acquiring respiratory infections [56,57], however, gram doses of vitamin C are required once an infection has taken hold, due to increased requirements for the vitamin [10,58]. Therefore, future RCTs should also comprise appropriate vitamin C dosing for the specific cohort under investigation.