A Randomized clinical trial of antioxidant therapy in patients with septic shock. Reference study to propose adjuvant therapy in patients with critical organic damage by COVID-19

Background: Oxidative stress (OS) participates in the pathophysiology of patients with septic shock having multiple organ failure (MOF), ischemia-reperfusion injury and acute respiratory failure syndrome (ARDS). Antioxidants have been proposed in their therapy. Objective: To evaluate the effect of antioxidant treatment in patients with septic shock with MOF and levels OS after treatment. Research question: Will the administration of specic antioxidant therapy decrease deregulatory factors of oxidative stress and organ failure in patients with septic shock? Study design and Methods: Double-blind, placebo-controlled randomized clinical trial run in 2 ICU in Mexico City between May 2018 and January 2020. The random allocation sequence was generated using computer methods. Patients older than 18 years of either sex, with septic shock were included, were excluded when informed consent could not be obtained, they received chronic or recent use of steroids, statins, or antioxidants or if they had contraindications to the use of antioxidants. All antioxidants were administered by mouth or nasogastric tube during 5 days and were added to standard. Results: 97 patients were included with median age of 66 years. 20 were treated with MT and 18 with Vit C and they showed post-treatment decreased SOFA scores [p=0.007 and p<0.001 respectively]. Also, total antioxidant capacity (TAC) was improved by NAC. All patients had decreased basal levels of Vit C and patients that received Vit C had decreased levels of the NO 3− /NO 2− (p=0.02) and RCP levels [p=0.045]. Procalcitonin (PCT) levels were reduced by Vit E, [p=0.047], NAC; [p=0.001] and MT [p=0.045]. LPO was reduced in patients that received MT p=0.042 Conclusion: In septic shock, antioxidant therapy associated with standard intensive care unit therapy reduces MOF, the oxidative and inammatory state. These results could be a reference to use adjuvant antioxidant therapy in patients with septic shock in COVID19.


Introduction
Damage caused by OS participates in the pathophysiology of serious diseases like MOF due to sepsis.
Bacteria, fungi and viruses or a combination of them cause these diseases (1). Sepsis and septic shock are the largest cause of mortality worldwide in the Intensive Care Units (ICU), MOF (2) constituting a high cost to in health systems (3).
Studies in animal models and in patients with septic shock have shown an imbalance between the production of reactive oxygen species (ROS), reactive nitrogen species (RNS) and antioxidant defenses (4)(5)(6).
In sepsis, ROS are generated by phagocytic cells, by the increased activity of enzymes such as NAD(P)H oxidase, xanthine oxidase and inducible nitric oxide (iNOS) and by increased in ammatory mediators through the activation of nuclear factor κB (NFκB) (7). Mitochondrial damage caused by OS is a component of the pathophysiology of MOF secondary to sepsis (8).
Vit C prevents the excessive production of nitric oxide (NO), decreases vasoconstriction and loss of vascular permeability. (18). Decreased Vit C levels are related to severity of MOF and mortality (19). Vit C therapy decreases SOFA scores, PCT, C-reactive protein (CRP) and thrombomodulin leading to a lower mortality rate (20). E (Vit E) is an important lipophilic antioxidant in cell membranes. It protects them from lipid peroxidation (LPO), (21). In septic patients with decreased levels of Vit E and O 2 − overproduction, the administration of Vit E and simvastatin inactivates NAD(P)H oxidase (22).
MT lowers OS both at the plasma and intracellular membranes due to its hydrophilic and lipophilic properties. MT possesses ROS sequestration properties, thus protecting cell membrane lipids, cytosol proteins, and nuclear and mitochondrial DNA (23)(24)(25).
Recently, there has been an increase in the prevalence and incidence of sepsis and septic shock due to the current pandemic caused by SARS-CoV2 (26,27).
Although there is a marked increase in ROS and a decrease in endogenous antioxidant defenses in critically ill patients with sepsis (28), the usefulness of different antioxidants has not yet been evaluated through clinical-randomized trials. In this study we evaluated the antioxidant effect of Vit C, Vit E, NAC and MT in patients with septic shock through the Sequential Organ Failure Assessment (SOFA) score and the measurement of antioxidant markers and OS.

Methods
This was a double-blind study in 2 ICU in Mexico City with a parallel randomized group.

Study Population
Patients were admitted to the ICU with a primary diagnosis of septic shock. All diagnostic criteria for septic shock were based on the Sepsis-3 consensus (29) which had to be ful lled within a maximum of 24 hours prior to enrollment, with an acute increase of at least 2 points in the SOFA score (30), lactate level greater than 2 mmol/L and patients were dependent on a vasopressor for at least 2 hours at the time of enrollment. Exclusion occurred when they were younger than 18 years, not able to grant an informed consent, refused to be included, if they were under chronic use (last 6th months) or recent use of steroids, statins or antioxidants, any contraindication for the use of Vit C, Vit E, NAC o MT of if pregnant or breastfeeding.
Ethical approval was obtained from the local ethics committee (PT 10-0-76; . Written informed consent for enrollment or consent to continue and use patient data was obtained from each patient or their legal surrogate.

Randomization, Masking, and Drug Administration
The random allocation sequence was generated at the coordinating center using computer-generated random program. (Fig. 1). Blinding was maintained by the investigational pharmacy at each institution. Investigators were blinded from the onset until the analysis outcomes were completed.
Administration of all antioxidants was by mouth or nasogastric tube for 5 days. Tablets of 600 mg every 12 hours of NAC were used. MT capsules of 5 mg were given to patients 50 mg once a day. Vit C 1 gm tablets every 6 hours were given. We used Vit E capsules of 400 UI every 8 hours. Patients of the control group did not receive any type of therapy. All data entry was monitored at the coordinating center, with site visits for source data veri cation. ventilator-free days, ICU-free days, and hospital-free days. Ventilator-free days were de ned as the number of days a patient was extubated from mechanical ventilation, after ICU admission and requiring reintubation were subtracted from the total days. If the patient died in the hospital, a value of zero was assigned to postextubation. ICU-free days began the moment the patient was transferred out of the ICU to day 28. Hospital-and ICU-free days were calculated similarly.

Study Measurements and Procedures
To evaluate the organ dysfunction, SOFA score (neurologic, respiratory, hemodynamic, hepatic, and hematologic) was calculated at admission and during all days of treatment. The CRP and the PCT determinations were performed on admission before the beginning of the antioxidant therapy and during the next 7 days.
Sampling for the determination of oxidative stress and antioxidant state The measurement of OS markers was done before the beginning of the antioxidant therapy and 48 hours after its initiation.

Sample obtention and storage
Blood samples were obtained from each patient entered to the draw, before initiation of the treatment and 48 hours after it began. The plasma of the samples was placed in 3 or 4 aliquots and was stored at -70 °C.

Oxidative stress markers
Nitrates and nitrites The NO 3 − was reduced to NO 2 − by the nitrate reductase enzyme reaction. At the end of the incubation period 200 µl of sulfanilamide1% and 200 µl of N-naphthyl-ethyldiamine 0.1% were added and the total volume was adjusted to 1 ml. The absorbance was measured at 540 nm.
Lipid Peroxidation 50 µl CH3-OH with 4% BHT plus phosphate buffer pH 7.4 was added to 100 µl of plasma. It was incubated, centrifuged at 4000 rpm at room temperature for 2 min. Then the n-butanol phase was extracted, the absorbance was measured at 532 nm.
Evaluation of total antioxidant capacity 100 l of plasma were suspended in 1.5 mL of a reaction mixture prepared as follows: 300 mM acetate buffer pH 3.6, 20 mM hexahydrate of ferric chloride, and 10 mM of 2,4,6-Tris-2-pyridil-s-triazine dissolved in 40 mM chlorhydric acid. These reactives were added in a relation of 10:1:1 v/v, respectively. After mixing samples were incubated at 37 ∘ C for 15 min in the dark. The absorbance was measured at 593 nm.
Carbonylation 100 µl of plasma were added to 500 µl of HCl 2.5 N in parallel with another sample with 500 µl of 2, 4dinitrophenylhydrazine (DNPH), and incubated. At the end of the incubation period, they were centrifuged at 15,000 g for 5 min. The supernatant was discarded. Two washings were performed. The mixture was incubated again at 37 °C for 30 min. Absorbance was read in a spectrophotometer at 370 nm, using water bi-distilled as blank and a molar absorption coe cient of 22000 M − 1 cm − 1 .
Vitamin C 100 l of 20% trichloroacetic acid was added to 100 L of plasma., centrifuged at 5000 rpm for 5 min. 200 l of Folin-Ciocalteu reagent 0.20 mM was added to the supernatant. The mixture was incubated for 10 min. The absorbance was measured at 760 nm.

Statistical Analysis
Based on a SD of 2.9 of the SOFA score, the study estimated to require 55 (11 per group) patients to have 84% power (2-sided with an α = 0.05) and 160 (32 per group). In accordance with these calculations, our study enrolled 97 patients to allow for 10% dropouts, providing a statistical power of 99%, with an α < .05. Testing was 2 sided. Effects are reported with a point estimate and 95% CIs in addition to P values.
Group comparisons were made using χ2 tests for equal proportions, t tests for normally distributed data, Kruskal Wallis and Wilcoxon rank sum tests otherwise, with results presented as frequencies with percentages, means with SDs, and medians with minimum and maximum, respectively.
The primary end point SOFA score and secondary end points (PCR and PCT) were analyzed with a mixed linear model and t to repeated-measures analysis of variance. days]), and the interaction between group and time, testing the hypothesis that differences between treatment groups are the same over time. Because of a potential for type I error caused by multiple comparisons, ndings for analyses of secondary end points should be interpreted as exploratory.
Statistical analysis was performed with Stata version 15.1.

Characteristics of the Patients
From July 2018 to November 2019 a total of 1695 eligible patients were identi ed, of whom 1598 were excluded (reasons listed in Fig. 1). Ninety-seven patients were randomized, with 18 assigned to each antioxidant and 21 to the control group. Of all patients included none was lost in the follow up. Baseline demographic data (eg, age, sex) were similar between the groups (Table 1).   Patients receiving Vit C had a signi cant decrease in CRP levels per day of treatment (Fig. 3). PCT levels were signi cantly decreased in patients receiving Vit E, NAC, and MT (Fig. 4). Vit E showed a tendency to reduce levels before and after treatment of LPO and of carbonylation.
Regarding the secondary outcomes, 13 patients (13.68%) required renal replacement therapy, 63 (65.63%) mechanical ventilation and 17 (17.89%) died. There was no statistically signi cant difference in days free of renal replacement therapy, mechanical ventilation, ICU stay length or hospitalization at 28 days. There was also no statistically signi cant difference in intrahospital mortality.

Undesired side effects
A patient receiving Vit C presented abdominal pain and another patient underwent a skin rash. Only one patient who received MT reported drowsiness. No adverse events were reported in patients with NAC or Vit E.

Discussion
Treatment with antioxidants as an adjuvant in the standard management of patients with sepsis, septic shock and infection with COVID-19 has been suggested (31)(32)(33)(34). We studied critically ill patients with septic shock regardless of the etiology and site of infection. All patients had initial low levels of Vit C, which was related with the severity of organ failure and mortality (18). The decrease in Vit C levels con rms the reported hypovitaminosis (< 0.23 µM ascorbic ac/mL) in septic shock (34)(35)(36)(37). It may be due to augmented metabolic demand, since intestinal absorption is not compromised in the patients in our study. (36). Vit C restored the normal values of this vitamin and organ function was improved. The best result was found in subjects with pneumonia with signi cant difference. This nding is in agreement with previous results (38)(39)(40). The combined use of Vit C, thiamine and steroids has recently been suggested. It is still necessary to compare if the use of Vit C alone has worse effects than the combinations (41).
In patients with septic shock, the administration of Vit C and MT improved the organ dysfunction assessed by the SOFA score. This nding could be associated to a decrease in the NO 3 − /NO 2 − and LPO levels.
The CITRIS-ALI study in patients with acute respiratory failure syndrome, ARFS, and organ failure showed no improvement with Vit C (42). The median time before starting treatment with Vit C was of 5 hours in this study, and markers such as CPR were signi cantly decreased, which was similar to another study (43). The possible difference with our results could be related to the fact that in the CITRIS-ALI study they started the therapy with Vit C later.
The VITAMINS trial showed no signi cant difference in the SOFA score, or in days without ventilation at 28 day; however, the use of Vit C lowered mortality (44). In that same study, CRP levels were not decreased, which was probably due to the late administration of Vit C in advanced stages of sepsis before developing ARDS (42). In contrast, we found a decrease in the levels of NO 3 − /NO 2 − which is relevant since Vit C inhibits the production of superoxide (O 2 − ) and peroxynitrite, thus preventing abundant NO synthesis, inhibiting mRNA expression and decreasing pathological vasoconstriction (17). These effects might be underlying the clinical bene t. A shorter time of use of vasopressors and a decreased intrahospital mortality was found in patient receiving Vit C (45).
This is the rst study of the use of MT in humans with septic shock. Recently MT has been applied in subjects with COVID 19 and it has a high safety pro le limiting this virus-related disease. Experimental and clinical studies are required to con rm this hypothesis (31). MT possesses free radical scavenging properties thus protecting cell membrane lipids, cytosol proteins, and nuclear and mitocondrial DNA (23,24). In our ndings, LPO was signi cantly decreased in the group of patients who received MT which was similar to results in the Galley's study (25). MT has bene cial effects in experimental cells, plants, and animals; however, its mechanisms of action remain unknown. The functions of the MT receptor relate to its ability as a detoxi cation agent, thus protecting molecules from the destructive effects of OS in ischemia/reperfusion (stroke, heart attack), ionizing radiation and drug toxicity. In sepsis, the protective effects of MT are associated with the inhibition of the apoptotic processes and reduction of OS.
Production of ROS is increased in an animal model of septic shock (46,47). This coincides with a lowering of the TAC and a reduction of the activity of superoxide dismutase and GSH peroxidase (48)(49)(50)(51)(52)(53)(54)(55). MT reversed morphological damage and increased the activities of antioxidant enzymes (46,48,61,49,53,(55)(56)(57)(58)(59)(60). Therefore, research through blinded clinical trials (62,63) and multicenter studies with adequate amounts of MT are needed to determine the potential of MT. In this clinical trial, we found a reduction of LPO and its potentially bene cial effect in organ dysfunction. Its use as an adjuvant in septic shock reduces in ammation and oxidation in animal models with respiratory damage induced by infection. MT has positive physiological actions, it is effective and safe for patients with septic shock of any etiology including those infected with SARS-CoV2 (31).
The use of NAC improved the antioxidant capacity and tended to increase GSH although the difference was not statistically signi cant. This con rms its antioxidant effect through the replacement of GSH deposits (12). NAC was related to decreases in organ failure, con rming previous ndings (14).
Other antioxidants such as polyphenols, MT, β-glucan, antioxidants targeting mitochondria, selenium salts, and selenium organ compounds are effective for improving OS in sepsis. The study of their pathophysiological implications justi es the combined therapy with antioxidants and standard treatments.
Vit E tended to decrease LPO and carbonylation. This vitamin protects cell membranes from LPO, ending their chain reaction. It is also an O 2 − and hydroxyl (OH) sequestrant (64).
In summary, antioxidants bene t subjects with septic shock. Septic shock is triggered by bacterial stimuli, fungi or viruses. In this medical condition, it is necessary to regulate in ammation and other mechanisms that lead to OS (65).

Limitations
The absorption may be altered by the enteral route of administration. However, we found increments on Vit C levels in serum.
The present trial is underpowered to detect differences in mortality and in outcomes between groups because the sample size was calculated for differences of OS.

Conclusion
In patients with septic shock, adding antioxidants to standard therapy regulates in ammation. In pulmonary sepsis, replacement therapy with Vit C increases its serum levels, which is associated with decreased levels of CRP, PCT, and NO 3 − /NO 2 − . MT decreases LPO and SOFA score. NAC reduces LPO and improves antioxidant capacity. Vit E tends to decrease LPO. Each antioxidant has bene cial effect; thus, they might be combined in clinical trials in patients with septic shock. We suggest the use of antioxidants as an adjuvant to standard therapy in patients with COVID-19, adjusting for comorbidities and drug interaction. Availability of data and materials The data generated or analyzed during the current study are included in this manuscript and its additional les.
Ethics approval and consent to participate.
This study was approved by the Ethics Committees of American British Cowdray (ABC) Medical Center, I.A.P., and Instituto Nacional de Cardiologia Ignacio Chavez, informed consents were obtained from all patients Consent for publication Not applicable AAA: Analysis data collection patient care, laboratory work, protocol work in written master's program and article review. IPT: Conceptualization, design, laboratory elaboration, writing, review and approval for publication. GCA: Patient care, article review. JFG: Patients attention and review of the article. VGL: Participation in conceptualization, written laboratory work and review and approval for publication. EMR: Patient care, database management, laboratory work. RG, Laboratory work, review and approval of the writing. MES: Conceptualization, design, thesis tutor, statistical analysis, preparation of the protocol and manuscript revision and approval of the manuscript.All authors read and approved the nal manuscript