Vitamin C reduces interleukin-6 plasma concentration: a systematic review and meta-analysis of randomized clinical trials

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Introduction
Inflammatory responses triggered by rapid viral replication and cellular destruction can employ macrophages and monocytes and provoke the release of cytokines and chemokines.Subsequently, cytokines and chemokines activate immune responses, leading to cytokine storms and other metabolic exacerbations [1].Among cytokines, Interleukin-6 (IL-6) is reported to be significantly associated with a high risk of the development of severe illness conditions [2e4].Furthermore, IL-6 is an important biomarker in Nutrition Risk in the Critically Ill (NUTRIC) Score [5,6] and stimulates the production of acute-phase proteins in different inflammatory conditions [7].Serum IL-6 levels in normal healthy people is 5e15 pg/ml [8], however, Yang and colleagues' study on rheumatoid arthritis patients showed that IL-6 levels can raise to 102 pg/ml in this inflammatory condition [9].In cytokine storm syndrome, the levels of IL-6 can raise to 1000 pg/ml [10], and clinical improvement has been observed following reducing the IL-6 levels with IL-6 receptor antagonist [11].After IL-6 is produced in the injury area in the initial phase of inflammation, it is conducted to the liver via the blood circulation, followed by the quick induction of a wide range of acute-phase proteins like C-reactive protein (CRP), serum amyloid A [12], fibrinogen, haptoglobin, and a1-antichymotrypsin [13].Also, this cytokine can elicit chronic inflammation by the employment of monocytes to the zone of inflammation [14].
However, to our knowledge, no meta-analysis has examined the impacts of the dose, duration, and administration route of vitamin C supplementation on IL-6 levels in patients with severe respiratory illness and other conditions.We conducted this systematic review and meta-analysis to evaluate this effect of vitamin C and determine the appropriate dose, duration, and administration route of vitamin C usage for this purpose.

Methods
We completed this study conforming to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines [27].This study sought to assess the probable mechanism of the advantageous effects of vitamin C through decreasing IL-6 concentrations in patients and to determine the appropriate dose, duration, and administration route of vitamin C usage for this purpose.PubMed, Google Scholar, Scopus, and Cochrane databases were searched from database inception to 3 rd July 2021.Using "AND" and "OR" Boolean operators, we searched the following search terms: ("Sodium Ascorbic Acid" OR "L-Ascorbic Acid" OR "Acid, L-Ascorbic" OR "L Ascorbic Acid" OR "Vitamin C" OR "Ascorbate" AND "Interleukin-6" OR "IL6" OR "B-Cell Stimulatory Factor" OR "B-Cell Stimulatory Factor-2" OR "IL-6").
Among the studies, all randomized clinical trials (RCTs) that were conducted in diverse population groups including children, schoolchildren, adults, males, and females; used vitamin C as intervention; had measured IL-6 levels (reported mean and standard deviation or standard error); and had placebo or control group were included in this study.No date, language, country, or route of administration restriction was applied.Animal studies, cell culture experiments, in vitro supplementation, secondary studies, studies that had used fruit juice instead of vitamin C, co-interventions of vitamin C with another nutrient or active substance, editorials, commentaries, case reports, and studies without the full-text accessibility were not reviewed.
Articles were reviewed based on the title, abstract, and full text, independently, by three authors (MG, SS, and FK), and any instances of disagreement were resolved by consensus with the senior author (KDj).We displayed the selection process in Figure 1.
Habbu's checklist [28] was applied for qualitative assessment of the studies.Nineteen items are included in this checklist.If all criteria were present, the maximum mark of nineteen was achieved.Studies with scores lower than twelve were excluded from the study.Therefore, the minimum and the maximum scores were twelve and nineteen, respectively.Statistical analyses were carried out using Stata version fourteen (Stata Corporation) software.The effect size was used for quantitative measurement of the magnitude of the mean difference between groups and 95% confidence intervals (CI) were calculated.Statistical heterogeneity was assessed with I 2 between 0 and 100%.We considered I 2 ¼ 0%, without heterogeneity; I 2 ≤ 25%, low heterogeneity; I 2 < 50%, moderate heterogeneity; and I 2 > 75%, high heterogeneity [29].To discern the source of heterogeneity, for groups, we performed subgroup analysis based on dose, duration, and conditions of vitamin C administration.To further assess causes of heterogeneity, a sensitivity analysis was done, in which the consequential deletion of individual studies was performed to examine the power of a single study on the overall effect of vitamin C usage on IL-6 levels.The risk of bias was evaluated using Stata software.Random effects meta-regression was used to explain the influence of age on the effectiveness of vitamin C for lowering IL-6 levels.

Results
According to the specified search criteria, 10504 articles were obtained, including 1208 articles from Scopus, 8296 articles from PubMed, 995 articles from Cochrane, and five articles from Google Scholar.After deleting duplicate articles, 10399 articles remained.Subsequently, screening was done on the remaining articles according to the form prepared, considering the Population, Intervention, Comparison, Outcome and Study design (PICOS) framework.Finally, twenty-four eligible articles were included in our systematic review and meta-analysis, eighteen articles for peroral and six articles for intravenous administration (Figure 1).

Characteristics of included studies
The studies included 801 participants, aged 20e68 years, from four continents; most studies were conducted in Europe and Asia.Baseline sample sizes ranged from 8 -68 participants.Specifications of included articles are described in Table 1.Six studies are included for IV and eighteen studies are included for PO, respectively.The places of studies were in Iran (n¼6), UK(n¼6), US (n¼3), Spain (n¼2), Africa (n¼2), Greece (n¼1), Mexico (n¼1), Palestine (n¼1), Egypt (n¼1), and Sweden (n¼1).The conditions of patients that studies were carried out were in athletes, diabetes mellitus (DM), DM with obesity, DM with cardiovascular disease (CVD), depression, atrial fibrillation (AF), coronary artery bypass surgery (CABG), and ischemic reperfusion injury.The duration of studies was from one day in athletes, before and after exercise, up to 60 days in depressed people.

Effect of vitamin C supplementation on plasma ascorbic acid levels
Seventeen out of twenty-four included studies had measured plasma ascorbic acid levels before and after vitamin C supplementation.Analysis showed a significant (P¼0.000)increase of plasma ascorbic acid levels after vitamin C supplementation (WMD ¼ 33.86 mm/L, 95% CI [33.80, 33.93], (Figure 2). in subgroup analysis by dosage, it showed that all supplemented doses of vitamin C (except 1500 mg/d) have increased significantly plasma ascorbic acid levels.

Effect of vitamin C supplementation on IL-6 levels in oral route
The primary analysis on peroral studies showed a significant effect (P<0.0001) of vitamin C on IL-6 levels (WMD ¼ -0.29 pg/l, 95% CI [-0.42, -0.16].Furthermore, we found low heterogeneity (I 2 ¼ 19.6%), (Figure 3).In the sensitivity analysis for finding the source of heterogeneity, there was no significant heterogeneity between studies (Figure 4).Therefore, subgroups analysis was done based on predefined criteria including the dose of vitamin C, duration of the treatment, and patients' conditions.
Moreover, the dose-response analysis showed that the lower doses of vitamin C have the greatest effect on IL-6.The trend of significance decreased by increasing the dosage of vitamin C. Furthermore, at doses higher than the highest dose of vitamin C (1500e2000 mg/d), the increase of vitamin C concentration did not change IL-6 plasma levels (Figure 5).Finally, the regression analysis by age did not illustrate a significant association between age variable and plasma IL-6 concentration (P¼ 0.23) (Appendix 4).
Egger and Begg's analysis did not demonstrate a significant bias to report (95%CI: -0.639, 0.417, P¼0.66).The funnel plot of studies did not show any significant publication bias (Figure 6).

Discussion
This systematic review and meta-analysis evaluated the current evidence base relating to vitamin C intervention on decreasing IL-6 levels in inflammatory diseases, and determined the appropriate dose, duration, and administration route of vitamin C usage for this purpose.We found that vitamin C has a prominent and statistically significant lowering effect on IL-6 levels via the oral route in Diabetic Mellitus patients, athletes, and inflammatory conditions.The most effective dosage was 200e500 and 1000 mg/d via the oral route.Also, in the intravenous route, we found a significant effect in dosages lower than 1500 mg/d in sepsis, ischemic reperfusion surgery, and BDD conditions.
In dosage subgroup analysis, a significant inverse association was observed at low doses.A D. Gutierrez et al. performed a study in DM patients, and found that 500 up to 1000 mg/d vitamin C supplementation in short durations has an anti-atherosclerotic effect in diabetic patients [31].Moreover, I Ferron-Elma et al. found that 450 mg/d vitamin C supplementation significantly can decrease plasma IL-6 concentration in patients with abdominal surgery and sepsis conditions [32].Furthermore, Hiedra R et al. administered vitamin C intravenously at a dose of 1 g every 8 h for 3 days and observed a significant decrease in inflammatory markers in COVID-19 patients [33].Jouybar et al. performed a study on Coronary Artery Bypass Graft Surgery patients with 3-gram vitamin C continuously over 12e18 hours, however they did not see any association between vitamin C and IL-6 levels in higher dosages [37].Nevertheless, our finding did not confirm the finding of Cheng et al. [34] who administered high doses of intravenous vitamin C (10e20 g/day and 1500 mg/kg/day) [34].Notably, overdoses of vitamin C can have adverse effects, such as kidney stones and diarrhea [35].Moreover, oxalate nephropathy due to administration of high doses of vitamin C has been reported in 2 patients with COVID-19 [36].The average daily requirement for vitamin C is 75 mg for females and 90 mg for males, whilst the tolerable upper level of vitamin C is 2 gr orally per day for adults [37].Due to poor absorption of vitamin C orally, it is assumed that higher doses must be administered intravenously.Intravenous administration is suitable for intensive care patients because near all of them have pre-existing intravenous lines.On the other hand, gastrointestinal problems and difficulty in swallowing are common among ICU patients, which can interfere with drug absorption [38].
Subgroup analysis based on duration demonstrated the highest effect in durations <1 week and 8 weeks' duration, respectively.Contrary to our findings, short durations (less than one week) of vitamin C treatment were carried out by Nakhostin et al. and were not effective on lowering IL-6 levels in inflammatory states [39].Our results showed that, in athletes, DM, and patients that were undergoing surgical procedures, vitamin C had a significant reducing effect on IL-6 levels.This finding shows that the indication of vitamin C for lowering IL-6 levels may be more effective in some medical conditions than others.
The mechanism of decrease of IL-6 levels by vitamin C is attributed to its antioxidant properties and inhibitory function on IL-6 producing monocytes.During lipopolysaccharide (LPS)-induced sepsis, which is brought about by oxidative damage, production of NF-kB is increased, and this transcription factor plays an important role in the overexpression of pro-inflammatory cytokines during sepsis.Indeed, vitamin C can lead to a decreased production of pro-inflammatory cytokines [23].
Accessible data shows that increased levels of IL-6 are notably coupled with adverse clinical consequences, admission in ICU, ARDS, and death in COVID-19 patients [40].Regarding the possibility of the development of cytokine storm syndrome in the course of the COVID-19 [41,42], and the beneficial effects of vitamin C on the alleviation of inflammation in cytokine storm syndrome [43e45], the results of this study could be extrapolated to COVID-19 patients.Undoubtedly, among the currently available drugs, vitamin C is a suitable and logical option for use to alleviate ARDS [34,46].Moreover, IL-6 is a biomarker that reduces appetite [47] and increases cachexia in sepsis conditions [48].
To our knowledge, this is the first meta-analysis on the effects of vitamin C administration on IL-6 levels.Additionally, included studies in our meta-analysis were from nearly all continents of the world and performed predefined subgroup analysis.However, this meta-analysis possesses some limitations.While most of the studies have measured plasma levels of IL-6 by ELISA method, only eight studies have reported the type of anticoagulant (EDTA) used in the test tubes for collecting blood samples.A study by Biancotto et al. showed a variation in cytokines levels between serum and plasma samples, and that measurement of some cytokines has been affected by diverse anticoagulants used in preparing plasma samples [49].Not measuring vitamin C plasma concentration before and after the intervention and not defining the time interval between the last dose of vitamin C and measuring plasma IL-6 levels were limitations that were defined in some included studies.However, this is beyond the control of the present study, but does, nevertheless, highlight the need for adequate time interval definitions in subsequent studies.

Figure 1 .
Figure 1.Literature search and filtering of studies according to the PRISMA flow diagrams.

Figure 2 .
Figure 2. Effect of vitamin C supplementation on plasma ascorbic acid levels.

Figure 3 .
Figure 3. Effi cacy of vitamin C supplementation on reducing IL-6 levels in studies that supplemented by peroral.

Figure 4 .
Figure 4. Sensitivity analysis for finding the weight of studies.In subgroup by duration, 7 up to 10 days supplementation pooled together.

Figure 5 .
Figure 5. Efficacy of dose-response of vitamin C supplementation on reducing IL-6 levels.

Figure 6 .
Figure 6.Funnel plot analysis for finding possible bias in the report.

Figure 7 .
Figure 7. Efficacy of vitamin C on reducing IL-6 levels in studies that supplemented Vitamin C intravenously.

Table 1
Characteristic of enrolled studies in the meta-analysis.