Vitamin C in Critically Ill Patients: An Updated Systematic Review and Meta-Analysis

Background: Vitamin C is a water-soluble antioxidant vitamin. Oxidative stress and its markers, along with inflammatory markers, are high during critical illness. Due to conflicting results of the published literature regarding the efficacy of vitamin C in critically ill patients, and especially the concerns for nephrotoxicity raised by some case reports, this meta-analysis was carried out to appraise the evidence and affirmation regarding the role of vitamin C in critically ill patients. Methods: We searched the database thoroughly to collect relevant studies that assessed intravenous vitamin C use in critically ill patients published until 25 February 2021. We included randomized controlled trials and observational studies with 20 or more critically ill patients who have received intravenous ascorbic acid (vitamin C). After screening 18,312 studies from different databases, 53 were included in our narrative synthesis, and 48 were included in the meta-analysis. We used the Covidence software for screening of the retrieved literature. Review Manager (RevMan) 5.4 was used for the pooling of data and Odds Ratios (OR) and Mean difference (MD) as measures of effects with a 95% confidence interval to assess for explanatory variables. Results: Pooling data from 33 studies for overall hospital mortality outcomes using a random-effect model showed a 19% reduction in odds of mortality among the vitamin C group (OR, 0.81; 95% CI, 0.66–0.98). Length of hospital stay (LOS), mortality at 28/30 days, ICU mortality, new-onset AKI and Renal Replacement Therapy (RRT) for AKI did not differ significantly across the two groups. Analysis of data from 30 studies reporting ICU stay disclosed 0.76 fewer ICU days in the vitamin C group than the placebo/standard of care (SOC) group (95% CI, −1.34 to −0.19). This significance for shortening ICU stay persisted even when considering RCTs only in the analysis (MD, −0.70; 95% CI, −1.39 to −0.02). Conclusion: Treatment of critically ill patients with intravenous vitamin C was relatively safe with no significant difference in adverse renal events and decreased in-hospital mortality. The use of vitamin C showed a significant reduction in the length of ICU stays in critically ill patients.


Introduction
Vitamin C (ascorbic acid) is a water-soluble essential vitamin synthesized by most plants and animals. Vitamin C and stress have long been associated, and previous research has shown that vitamin C reduces systemic inflammation, helps correct sepsis-induced coagulopathy, and prevents vascular injury [1][2][3]. Vitamin C has drawn substantial interest in critical care medicine, and several studies have been published, especially in the last five years, with intriguing but conflicting results. In a primary study by Fowler et al. [3] in 2014, vitamin C decreased the risk of organ failure in patients with a critical illness. However, the subsequent CITRIS-ALI trial failed to show benefit in organ dysfunction or suppress the inflammatory markers and vascular injury but reported a 28-day mortality benefit: 29.8% in the vitamin C group and 46.3% in the placebo group [4]. Marik et al. [5] showed a reduction in mortality and organ failure with vitamin C use among severe sepsis or septic shock patients. A study by Zabet et al. [6] in 2016 showed a reduction in the dose and duration of vasopressors medication use in surgical septic shock patients. A study by Nakajima et al. [7] in 2019 showed mortality benefit with high dose vitamin C use in patients with severe burn. Thus, vitamin C has been extensively studied in various critical care settings including medical, surgical, trauma and burn ICUs. Prior meta-analyses have shown a reduction in the duration of vasopressor and mechanical ventilation days [8,9], although a mortality benefit has not been seen [9][10][11][12]. Many studies have been published since the publication of the last meta-analyses by Wei et al. and Putzu et al. [10,11].
Thus, in order to fully appraise the available data, we sought to perform this systematic review and meta-analysis, including 53 studies with 352,395 critically ill patients, to evaluate the role of vitamin C, paying particular interest to the renal safety of vitamin C.

Methods
The protocol for this systematic review and meta-analysis was prepared and registered in PROSPERO, and the review was conducted according to the protocol's predefined criteria (CRD42020222906) [13]. We abide by our study as per the standard Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [14].

Search Strategy
We conducted a thorough literature search on PubMed, PubMed Central, Scopus and Embase to collect relevant studies published until 25 February 2021, reporting the use of intravenous vitamin C in critically ill patients. We used appropriate search words such as ascorbic acid, vitamin C, sepsis, septic shock, critical illness, intensive care unit, ICU, burn, ARDS and trauma using appropriate Boolean operators without any filters and language restrictions. Details of the electronic database search are available in Supplementary file S1.

Selection Criteria
Inclusion criteria: Type of studies: randomized controlled trials and other observational studies, namely, cohort studies, case-control studies, cross-sectional studies and case series with 20 or more critically ill patients.
Population: Adult patient with a critical illness. Intervention: Intravenous vitamin C alone or in combination with other antioxidants. The comparator includes standard of care or placebo.
Study outcome: Hospital mortality, 28/30-day mortality, ICU mortality, length of stay in ICU and hospital, the adverse renal outcome in the form of new-onset AKI and AKI requiring renal replacement therapy were our main outcomes of interest. We used Odds Ratios (OR) and mean differences (MD) as a measure of effects to assess the explanatory variables.
Exclusion criteria: Types of studies: Any form of review including meta-analysis, narrative reviews, case reports, opinions, editorials, letter to the editor with no primary data, study protocols, conference abstracts/presentations, thesis dissertation and animal studies.
Full-text articles that are not retrievable. Incomplete studies (still ongoing) or preliminary reports with no complete data available. COVID-19 related studies.

Data Extraction
We used Covidence software to screen the literature imported from databases for the eligibility assessment [15]. Every citation was reviewed independently by two reviewers (S.K. and S.S.) for eligibility, and conflicts were resolved by a third reviewer (S.M.). After two phases of screening (title and abstract and full-text review), the data from selected studies were extracted in a Microsoft Excel sheet. The number of manuscripts chosen was divided across the reviewers. Each reviewer curated the data from assigned articles, which was later verified for accuracy and any discrepancies by other reviewers. The internal review was carried out thoroughly to ensure the correctness of the data curation among the outcomes of interest with no subjective errors.

Risk of Bias
We used the Cochrane Risk of Bias (ROB) 2.0 tool for randomized control trials and Joanna Briggs Institute (JBI) critical appraisal checklist for observational studies for quality and risk of bias assessment ( Figure 1 and Table 1) [16,17]. We used Review Manager (RevMan) 5.4 for creating a summary of biases for RCTs [18].

Statistical Analysis
Revman 5.4 was used to pool the data using Odds Ratio (OR) to estimate dichotomous outcome with a 95% Confident Interval (CI). Based on the level of heterogeneities, we used the fixed/random-effects model to pool the result. We used the mean differences (MD) between vitamin C and control groups for continuous variables. Where mean was not available in the study, mean and standard deviation (SD) were estimated using the sample size, median value and inter-quartile ranges [40].

Assessment of Heterogeneity
Based on the established recommendation for the statistical heterogeneity, we stratified heterogeneity as low, moderate and high to I 2 values of 25, 50 and 75%, respectively [41]. Based on the calculated heterogeneity I 2 test value, we used the random/fixed effect model for the analysis. We conducted sensitivity analysis by re-running the analysis to assess the hidden differences where appropriate.

Results
After electronic database searches, we retrieved 18,312 studies imported in Covidence, and 1248 duplicates were removed. After the duplicate check, we screened the 17,064 studies in a title and abstracts review, and we excluded 16,720 studies for not meeting the inclusion criteria. A total of 344 studies were filtered for full-text review, of which 291 studies were excluded for definite reasons. After a full-text review, we included 53 studies in the narrative review and 48 studies (27 randomized controlled trials and 21 observational studies) in the meta-analysis ( Figure 2).

Narrative Summary
A total of 53 studies were included in the narrative review; of them, 23 were observational studies, while 30 were RCTs (Table 2). Basic study details are presented in Supplementary file S2.    IV hydrocortisone (50 mg every 6 h for 7 days or until ICU discharge, whichever occurred first), vitamin C (1.5 g every 6 h for 4 days or until ICU discharge, whichever occurred first), and IV thiamine (200 mg every 12 h for 4 days or until ICU discharge, whichever occurred first) Same frequency and volume of saline. In the treatment group, vitamin C (10 g/day) was given intravenously for 5 days, high dose.
In the control group (n = 44), vitamin C (1 g/day) was given intravenously for 5 days (Low dose).
Hospital LOS T = 9.34 ± 4.24 C = 13.45 ± 3.21 Given the same amount of placebo (normal saline).  Intravenous ascorbic acid 1000 mg intravenously in 100 mL of NS every 8 h, alpha-tocopherol 1000 IU (1 mL) via naso-or orogastric tube every 8 h, and selenium 200 mcg intravenously in 100 mL NS once daily. Ascorbic acid was administered as a bolus over 1 h and selenium as a bolus over 2 h, although both were permitted to be changed to an enteral dosage form once enteral access was established. The treatment course was 7 days or until discharge from hospital, whichever came first. Vitamin C (50 mg/kg, maximum single dose 3 g, daily dose 6 g) and thiamine (200 mg) were mixed in a 50-milliliter 0.9% saline bag, respectively, and intravenously administered to patients over 60 min every 12 h for a total of 48 h.
Identical volume of 0.9% saline from the placebo drug ampoule was administered to patients using the same protocol. Treatment group was treated with vitamin C protocol. The protocol consisted of intravenous vitamin C (1.5 g every 6 h for 4 days), hydrocortisone (50 mg every 6 h for 7 days followed by a taper over 3 days) and intravenous thiamine (200 mg every 12 h for 4 days). Treatment group was administered 6 g of vitamin C per day (divided into four equal doses).
The patients who were admitted to the same ICU between June 2016 and January 2017 were not treated with the vitamin C protocol and thus formed the control group.
Intravenously by 5% dextrose (100 mL/time, 2 times/day) as placebo.       Intravenous vitamin C (1.5 g q 6 hourly for 4 days or until discharge from the hospital), hydrocortisone (50 mg q 6 hourly for 7 days or until ICU discharge followed by a taper over 3 days) and intravenous thiamine (200 mg q 12 hourly for 4 days or until discharge from the hospital).  An injectable ascorbic acid solution was diluted 10-fold with distilled water. The ascorbic acid solution was administered as a continuous intravenous infusion.

Sensitivity Analysis among RCTs
To further analyze the hospital mortality outcome among RCTs, we re-run the analysis based on the subgroups of critically ill patients as surgical and sepsis/septic shock. Neither among patients with sepsis/septic shock (OR, 0.98; 95% CI, 0.76-1.26; n = 1297; I 2 = 0%; p = 0.86) nor among surgical patients (OR, 1.08; 95% CI, 0.63-1.86; n = 860; I 2 = 4%) was a significant difference for hospital mortality observed while comparing vitamin C and placebo or SOC groups (Supplementary file S3, Figure S1).
Based on the blinding of RCTs, the odds for mortality was shown to be slightly higher in double blinded trials among the vitamin C group (OR, 1.04; 95% CI, 0.76-1.43; n = 938; I 2 = 0%; p = 0.80), while for single blinded/open, odds were slightly reduced (OR 0.92, 95% CI 0.65 to 1.29; n = 1199; I 2 = 0%; p = 0.61) compared with the placebo or SOC group, but it could not reach statistical significance (Supplementary file S3, Figure S2).
Considering mild-moderate heterogeneity and pooling the results using randomeffect model also showed no significant differences (Supplementary file S3, Figure S6).  Figure S7).
Running analysis for LoHS taking RCTs based on the blinding of trials showed a significant reduction in the length of hospital stay of the vitamin C group among both double blinded trials (MD, −0.54; 95% CI, −1.43 to 0.36; n = 1247; I 2 = 34%; p = 0.24), and the single blinded/open RCTs' reduction in LoHS among the vitamin C group could not reach statistical significance (MD, −0.78; 95% CI, −3.77 to 2.20; n = 557; I 2 = 73%; p = 0.61) compared with the placebo or SOC group (Supplementary file S3, Figure S9).

Length of ICU Stay
Polling data from 30 studies reporting the length of ICU stay outcome using a random effect model showed an overall mean difference of −0.76 days, indicating a 0.76-day shorter ICU stay among the vitamin C group compared with placebo/SOC (95% CI, 95% CI, −1.34 to −0.19; n = 252,897; I 2 = 96%; p = 0.009). Similarly, further subgrouping of the length of ICU stay based on the type of study also showed a small but significant reduction in the length of ICU stay among the vitamin C group comparing with placebo/SOC among RCTs (MD, −0.70; 95% CI, −1.39 to −0.02; n = 1712; I 2 = 86%; p = 0.04), while the observational studies could not reach the level of significance (MD, −0.82; 95% CI, −1.75 to 0.10; n = 251,185; I 2 = 98%; p = 0.08) ( Figure 6).  Pooling the new onset AKI outcomes using a random-effect model also showed no significant difference in new AKI outcomes (OR, 1.22; 95% CI, 0.94 to 1.58; n = 5330; I 2 = 0%; p = 0.14). Similarly, the type of study could not show significant changes among observational studies and RCTs (Supplementary file S3, Figure S13).

Discussion
Herein, we report a comprehensive systematic review and meta-analysis including 48 previously published studies. We have included 27 randomized controlled trials and 21 observational studies that have studied the role of intravenous vitamin C in critically ill patients. We found a reduction in overall hospital mortality among critically ill patients who received vitamin C, in contrast to the findings of an earlier meta-analyses by Langlois et al. and Wei et al. [11,12]. However, this finding could have been impacted by the inclusion of observational studies in the cumulative analysis. These findings could not be replicated when a subgroup analysis was performed for the randomized controlled trials alone. Mortality benefit was observed when a cumulative analysis was performed for the observational studies alone.
Oxidative stress is thought to play a central role in the pathophysiology of the inflammatory syndrome post-operative state after surgery and sepsis. Vitamin C deficiency is commonly seen in surgical patients and patients in intensive care units [43,71]. The intense inflammatory process leads to an increased turnover of vitamin C, further resulting in severe deficiency [43,71]. Parenteral vitamin C has been investigated for its potential anti-inflammatory, antioxidant effects [3]. Vitamin C has also been studied for its properties to improve immune function, regulate microcirculation and prevent thrombosis [3]. Although the therapeutic efficacy of vitamin C is seen in individual studies, we found no difference in mortality among patients receiving intravenous vitamin C while performing a subgroup analysis of RCTs including surgical patients or patients with sepsis/septic shock. A reduction in mortality was observed among surgical patients in observational studies. We found no difference in the length of hospital stay in the analysis of the cohort and subgroup analyses. A reduction in the length of ICU stay was observed while analyzing studies reporting ICU stay and also among RCTs only separately, although a level of statistical significance could not be achieved while analyzing observational studies separately. Prior meta-analyses conducted by Langlois et al., Putzu et al. and Wei et al. found no difference in the duration of hospital stay and the length of ICU stay [10][11][12]. However, in a subgroup analysis, Putzu et al. found decreased lengths of hospital and ICU stays among patients undergoing cardiac surgery [10]. Vitamin C has shown to decrease the risk of postoperative atrial fibrillation in patients undergoing cardiac surgery, a further benefit observed in terms of the deceased length of ICU stay suggests that vitamin C may have beneficial effects from mechanisms yet to be recognized [72,73].
Vitamin C is metabolized into oxalate compounds that can accumulate in the kidneys and trigger acute kidney injury [44]. In our meta-analysis, we found no increase in the new-onset acute kidney injury or requirement for renal replacement therapy among the patients receiving vitamin C. This finding is consistent with the results of a prior metaanalyses conducted by Putzu et al. and Wei et al. [10,11]. Collectively, this suggests that vitamin C is potentially more renally safe than perceived.
We have sought to perform the most comprehensive meta-analysis, including 48 studies in quantitative analysis evaluating the vitamin C use in critically ill patients and their clinical outcomes. We have only included studies in which vitamin C was administered parenterally. High dose oral vitamin C saturates the intestinal absorption mechanism; thus, a higher plasma concentration cannot be achieved by a higher oral dose administration. We assumed that the intravenous administration makes it possible to achieve the higher plasma concentration required to scavenge oxidizing radicals and protect from their deleterious effects. Prior meta-analyses conducted in the role of vitamin C among critically ill patients have included fewer studies than our analysis [10][11][12]. They have included studies using both oral and parenteral formulations of vitamin C, which adds to the strength of our study [10][11][12]. Our analysis is further strengthened by the subgroup analyses conducted based on the clinical settings, medical and surgical etiology of hospitalization, sepsis and septic shock, and the type of studies.
Our study has several limitations. The included RCTs and observational studies have their own inherent limitations. The baseline characteristics of the population studied and the assessment of organ dysfunction were not individually reported across the studies. The formulations, dosing and duration of vitamin C use are comparable and offer granularity of data in assessing individual influence. Further, the patients could have been enrolled in different stages, and the severity of sepsis and ARDS, and clinical outcomes could have been influenced by the stage of the pathology.
ICU pathophysiology is complex and clinical outcomes are determined several factors relating to patients' general health and acute physiological state. This includes factors that cannot be reversed by vitamin C therapy alone and results can be confounded. Various studies in our analysis included treatment with other antioxidants such as thiamine and steroids, which can act as potential confounders. Nevertheless, our observation of no significant difference across the two groups in acute kidney injury and need for renal replacement therapy is reassuring in terms of the renal safety of intravenous vitamin C therapy and can guide future investigators.

Conclusions
The treatment of critically ill patients with vitamin C did not offer mortality benefits in terms of a reduction in in-hospital mortality, 28/30-day mortality and ICU mortality based on the cumulative analysis of the randomized controlled trials. However, vitamin C did reduce the length of hospitalization, although it did not reduce the number of ICU days. In addition, vitamin C therapy was relatively safe, especially from a renal standpoint, given the no significant group difference in the incidence of acute kidney injury and the need for renal replacement therapy. Funding: This article did not receive any specific grant from funding agencies in the public, commercial, or other sectors.