At the end of 2019, patients with unexplained pneumonia appeared in Wuhan, China. At 21:00 on January 7, 2020, a new coronavirus was detected in the laboratory, and the detection of pathogenic nucleic acids was completed at 20:00 on January 10.
Subsequently, the World Health Organization officially named the new coronavirus that caused the pneumonia epidemic in Wuhan as 2019 new coronavirus (2019-nCoV), and the pneumonia was named severe acute respiratory infection (SARI).
There is a lack of effective targeted antiviral drugs, and symptomatic supportive treatment is still the current main treatment for SARI.
Vitamin C is significant to human body and plays a role in reducing inflammatory response and preventing common cold.
In addtion, a few studies have shown that vitamin C deficiency is related to the increased risk and severity of influenza infections.
Statistics of the 41 patients with SARI published in JAMA initially showed that 13 patients were transferred into the ICU, of which 11 (85%) had ARDS and 3 (23%) had shock.
Of these, 10 (77%) required mechanical ventilation support, and 2 (15%) required ECMO support.
Of the above 13 patients, 5 (38%) eventually died and 7 (38%) were transferred out of the ICU.
Viral pneumonia is a dangerous condition with a poor clinical prognosis.
For most viral infections, there is a lack of effective targeted antiviral drugs, and symptomatic supportive treatment is still the current main treatment.
Starting with a study in Zhongnan Hospital of Wuhan University (ZNOWU) , they hypothesized that Vitamin C infusion can help improve the prognosis of patients with SARI. [ZhiYong Peng, Zhongnan Hospital – Zhongnan Hospital of Wuhan University (ZNWU)]
Vitamin C, also known as ascorbic acid, has antioxidant properties.
When sepsis happens, the cytokine surge caused by sepsis is activated, and neutrophils in the lungs accumulate in the lungs, destroying alveolar capillaries.
Early clinical studies have shown that vitamin C can effectively prevent this process.
In addition, vitamin C can help to eliminate alveolar fluid by preventing the activation and accumulation of neutrophils, and reducing alveolar epithelial water channel damage.
At the same time, vitamin C can prevent the formation of neutrophil extracellular traps, which is a biological event of vascular injury caused by neutrophil activation.
A study on ARDS has shown the validity of using vitamin C as a powerful healing medium.
Viral diseases can produce the acute respiratory distress syndrome (ARDS)[1].
Pandemic viruses are the most common viruses that produce lung injury.
Influenza viruses and coronaviruses (e.g., H5N1, H1N1 2009, severe acute respiratory syndrome coronavirus, and middle east respiratory syndrome coronavirus) are potentially lethal pathogens known to produce lung injury and death from ARDS[2-5].
The exact pathogenesis of virus-induced ARDS is slowly becoming understood.
Unlike the “cytokine storm” occurring in bacterial sepsis that leads to up-regulation of pro-inflammatory cytokines [e.g., interleukin-1β (IL-1β), IL-8, IL-6] and generation of reactive nitrogen and oxygen species in the vascular space, viruses such as the influenza virus target alveolar epithelium, disabling sodium pump activity, damaging tight junctions, and inducing cell death in infected cells.
Cytokines produced by virally infected alveolar epithelial cells activate adjacent lung capillary endothelial cells which then leads to neutrophil infiltration.
Subsequent production of reactive oxygen and nitrogen species by infiltrating neutrophils further damages lung barrier function[1].
Apart from pandemic viruses other viruses, are increasingly reported to produce severe ARDS.
While most of the approximately 100 strains of enterovirus primarily infect the gastrointestinal tract, enterovirus-D68 (EV-D68) has tropism for the respiratory tract. EV-D68 produces acute respiratory disease ranging from mild upper respiratory tract symptoms to severe pneumonia and lung injury.
We report here the first application of high dose intravenous vitamin C employed as an interventional drug treatment for virus-induced ARDS.
Very few studies in critically ill patients with ARDS have reported the use of intravenous vitamin C.
The use of vitamin C to treat lung injury is still investigational.
- Nathens et al[15] infused ascorbic acid at 1 g every 8 h combined with oral vitamin E for 28 d in 594 surgically critically ill patients and found a significantly lower incidence of acute lung injury and multiple organ failure.
- Tanaka et al[16] infused ascorbic acid continuously at 66 mg/kg per hour for the first 24 h in patients with greater than 50% surface area burns and showed significantly reduced burn capillary permeability.
- A single report (published as abstract only) of a clinical study of large intravenous doses of ascorbic acid, and other antioxidants (tocopherol, N-acetyl-cysteine, selenium), in patients with established ARDS showed reduction in mortality of 50%[17].
- Clinical protocols currently in use for hospitalized septic patients fail to normalize ascorbic acid levels. Vitamin C dosages utilized in the treatment of the patient we describe in this case report arose from our previous human studies, infusing high dose intravenous vitamin C into critically ill patients with severe sepsis[18] and in our preclinical studies[19-21].
Our work thus far shows vitamin C to exert potent “pleotropic effects” when used as described in this report.
We showed that septic patients receiving high dose intravenous vitamin C exhibit significant reduction in multiple organ injury and reduced inflammatory biomarker levels[18].
Our preclinical work in septic lung-injured animals shows that vitamin C down-regulates pro-inflammatory genes that are driven by transcription factor NF-κB.
Furthermore, vitamin C significantly increases alveolar fluid clearance in septic lung-injured animals[21].
Finally, infused vitamin C’s capability to down-regulate liberated reactive oxygen and nitrogen species appears to be critical for attenuating lung injury[22].
CASE REPORT
A 20-year-old white female presented to urgent care with 24 h of increasing dyspnea after returning from a 7-d trip to Italy.
While in Italy she was exposed to several members of the family with whom she was visiting who had symptoms of upper tract respiratory infection. One family member had recently traveled to Morocco. While in Italy, the patient had visited a buffalo farm and ate unpasteurized cheese. There were no other unusual exposures. She noted cough and yellow sputum for 3 d with intermittent fever and night sweats.
DISCUSSION
A chest X-ray revealed diffuse bilateral opacities (Figure (Figure 1). Arterial blood gas testing revealed severe hypoxemia while receiving 100% oxygen by non-rebreather mask. Antibiotics were initiated and she was admitted to intensive care unit (ICU) with a diagnosis of community acquired pneumonia.
She denied GI symptoms, rash or arthralgia. She denied any history of thromboembolic disease, chest or leg pain or swelling. Her only medication was oral contraceptive for migraines associated with her menstrual cycle.
Non-invasive positive pressure ventilation failed to support hypoxemic respiratory failure and intubation was required on hospital day 3.
An echocardiogram revealed normal cardiac function. Respiratory cultures were negative, but a molecular detection viral respiratory panel was positive for enterovirus/rhinovirus (FilmArray, BioFire Diagnostics, LLC, Salt Lake City, Utah). Despite high PEEP and low tidal volume ventilation, hypoxemia (PaO2/FiO2 = 75) and hypercapnia remained severe. Chest imaging on hospital day 3 revealed dense bilateral opacities with central air bronchograms (Figure 2).
Due to failure of conventional ventilatory strategies, veno-venous extracorporeal membrane oxygenation (ECMO) was initiated on hospital day 3. Low tidal volume assist-control, pressure-control ventilatory strategy was continued. Vancomycin, piperacillin-tazobactam and levofloxacin started at ICU admission were continued.
High-dose intravenous vitamin C (200 mg/kg per 24 h) was initiated on ECMO day 1 with the total daily vitamin C dosage divided equally into four doses and infused every 6 h. AP chest X-ray imaging on ECMO day 2 following institution of vitamin C infusion revealed significant improvement in bilateral lung opacities (Figure (Figure 3).
Given the patient’s hemodynamic instability and vasopressor requirements, the critical care physician staff and nursing staff were very careful to keep the patient’s intake and output fluid balance even, being careful not to volume load a patient who was suffering from permeability pulmonary edema.
Bronchoscopy on ECMO day 3 was negative for bacterial or fungal respiratory pathogens. Histoplasma, Blastomyces, Aspergillus, and Legionella antigen studies were negative. Furosemide was used to achieve a daily negative fluid balance. Daily chest imaging with AP chest X-rays documented continued resolution of bilateral opacities.
Importantly, lung gas exchange significantly improved following institution of vitamin C infusions. Chest imaging on ECMO day 6 revealed significant further reduction in lung opacities. ECMO decannulation and extubation from ventilation occurred on ECMO day 7 (Figure (Figure 4). Vitamin C dosing was continued while the patient remained on ECMO.
Vitamin C dosing was reduced by half (100 mg/kg per 24 h) for one day following decannulation from ECMO then reduced by half again (50 mg/kg per 24 h) for an additional day.
Post-extubation the patient required 4 L/min nasal oxygen for 48 h and then was discharged home on room air. She was discharged home on hospital day 12.
Although we did not quantify the plasma ascorbic acid levels in the patient we report here, we have previously reported that critically ill patients with severe sepsis treated with the identical vitamin C infusion protocol achieved plasma ascorbic acid levels of 3.2 mmol, values which are 60 fold higher than normal plasma ascorbic acid levels[18].
In conclusion, we report here the first use of vitamin C as an interventional drug to attenuate lung injury produced by viral infection.
The patient described here was discharged home 12 d following hospitalization, requiring no oxygen therapy. Follow-up exam at 1 mo following the patient’s initial hospitalization revealed her to have completely recovered.
Figure 5 displays her follow-up chest X-ray film. Importantly, it should be noted that this is a single case report.
The role of Vitamin C in this patient’s recovery is not certain, and clearly additional investigation will be required before this can be recommended as a therapy for ARDS.
Let’s try to understand why the vitamin is important for the immune defenses of our organism.
Mitochondrial Dynamics is Essential in Antiviral Immunity
Viruses, including coronaviruses, have the ability to alter cellular functions to increase proliferation. The ability to evade immune responses is perhaps the most important aspect of viral persistence and proliferation.
Recent discoveries have shown that mitochondria is the central regulator of our immune system, controlling innate immune signaling and cell fate of immune cells [42].
Mitochondrial outer membranes have emerged as a major platform for important signaling molecules, and mitochondrial dynamics involving fusion and fission play critical roles in immune-cell activation [43].
Immunity and mitochondria are now accepted to be tightly interlinked, as mitochondria can regulate the activation, differentiation and survival of immune cells [44].
During viral infections, mitochondrial dynamics is altered as viruses manipulate mitochondrial dynamics to influence infection progression.
The disruption of mitochondrial dynamics caused by viruses, including coronaviruses, can severely increase viral pathogenesis [45]. This is the reason why coronavirus SARS-CoV encoded 3a, 3b proteins target mitochondria to inflict damage and injury, causing apoptosis in order to deregulate the host immune system [37, 41].
During infection, damaged mitochondria would normally be cleared quickly via combined processes in mitochondrial dynamics and mitophagy.
Fission is a process that facilitates the segregation of damaged mitochondria, which is subsequently removed by mitophagy. The remaining healthy mitochondria would be fused with existing mitochondrial network through fusion processes.
In this way, mitochondria can maintain energy production to sustain cellular homeostasis [46].
However, if mitochondria become highly depolarized and therefore irreversibly damaged, these mitochondrial will be eliminated permanently with no possibility of being reincorporated via fusion events. The mitochondrial membrane potential (∆Ψm) becomes the determining factor in sorting out which mitochondria can be repaired and restored, and which must be segregated and permanently eliminated [45].
Membrane Permeabilization and Depolarization
The onset of mitochondrial depolarization is always coupled with depolarization of the plasma membrane potential. Studies have also shown that permeabilization of the outer mitochondrial membrane is necessary for the depolarization of membrane potential during apoptosis [47].
Mitochondrial membrane permeabilization and the loss of mitochondrial transmembrane potential, or ΔΨm depolarization are often used as biomarkers of apoptosis [53]
During mitochondrial depolarization, release of large quantities of cytochrome c slows down electron flow, interrupting the production of ATP that eventually leads to the formation of increased free radicals that will disturb cellular homeostasis [47].
The mitochondrial membrane potential (ΔΨm) is the main source of chemical energy that is responsible for driving proton re-entry from the intermembrane space through the ATP synthase back into the mitochondrial matrix [48].
The maintenance of appropriate ΔΨm is critical for mitochondrial energy production as the energy available for ATP synthesis is directly derived from mitochondrial membrane potential (ΔΨm). Depolarization translates into decreased energy available for ATP synthesis.
It is now understood that mitochondria contain individual interconnected powerhouses called cristae [49]. Individual crista is able to maintain different membrane potential along the inner mitochondrial membrane. During transient depolarization events, some cristae can maintain polarity despite the collapse of ΔΨm in adjacent cristae [50].
This means that during depolarization, the cristae that can sustain membrane potential can be rescued and fused into existing healthy mitochondria. However, fusion must happen before the cristae loses polarity permanently because reduced membrane potential can decrease the level of fusion protein OPA1, generating non-fusing mitochondria as a result [51].
Simultaneous tracking of fission and Δψm revealed that depolarized mitochondria produced during fission events are SIX times less likely to be fused within the next 10 minutes [52].
What can facilitate the rescue of depolarized mitochondria?
Ascorbic Acid, Mitochondria & Depolarization – the Plasma Membrane Redox System Revisited
Recent discoveries showed that ascorbic acid has the ability to return mitochondrial fusion rates to normal in a model for Parkinson’s disease [54].
Many earlier studies already demonstrated that ascorbic acid, vitamin C, can prevent loss of mitochondrial membrane potential. When cells were treated with uncoupling agents that can inhibit oxidative phosphorylation like CCCP (carbonyl cyanide m-chlorophenyl hydrazone), membrane potential was lowered together with the induction of membrane permeabilization, while cytochrome c proteins were released to initiate apoptosis.
The addition of ascorbic acid to these cells prevented depolarization and ensuing apoptotic cascade events [55].
The ability of ascorbic acid to prevent depolarization in mitochondrial membrane confers anti-cytotoxic effects resulting in dose-dependent decrease in apoptosis in cells during in vitro experiments [56] Ionizing radiation induce mitochondrial membrane depolarization in cells and causes apoptosis [57].
Pretreating cells with ascorbic acid protected cells against ionizing radiation induced apoptosis [58].
Coronavirus & Membrane Depolarization – The Ascorbic Acid Connection
The N-protein of SARS-CoV has been demonstrated to cause apoptosis via the mitochondrial apoptotic pathway under starvation of serum. Zhang et al. in 2009 found that the N-protein could cause increased generation of reactive oxygen species, leading to loss of membrane potential, increased membrane permeability, cytochrome C release and ultimately cell death after bovine serum was withdrawn for 24 hours [59].
Why is bovine serum important? Bovine serum contains various micronutrients [60] but most of all, it contains ascorbic acid [61].
There is no doubt that coronaviruses like 2019-nCoV, SARS-CoV, and MERS-CoV target mitochondria to induce apoptosis in order to disrupt host immune system in order to facilitate proliferation.
Protecting mitochondrial dynamics in fusion and fission events allow depolarized mitochondria to regain functional control. How does ascorbic acid maintain membrane potential to protect mitochondrial dynamics?
CYB5R3 & VDAC1 – A Tale of Membrane Potential & Ascorbic Acid
There is an extensive, dynamic and influential network of plasma membrane enzymes that regulate redox balance in the cellular environment. These enzymes were not formally classified until the early 2000’s because they were primarily NADH or NADPH oxidases that had been known under various other names based upon their physiological electron acceptors. Most of these plasma membrane redox enzymes that have been identified so far use ascorbic acid almost EXCLUSIVELY as their electron acceptor and donor, due to the unique characteristics of ascorbic acid [62].
The CYB5R plasma membrane enzyme is encoded by four genes CYB5R1, CYB5R2, CYB5R3 and CYB5R4. The isoform CYB5R3 has ubiquitous cytoplasmic expression and its membrane-bound form is present in mitochondria, nucleus, endoplasmic reticulum and plasma membrane [64].
The CYB5R3 (membrane-bound NADH:cytochrome b5 oxidoreductase 3) enzyme catalyzes rapid exchange of electrons between ascorbate and its one electron oxidation metabolite, ascorbyl free radical (AFR), also known as semidehydroascorbate.
The CYB5R3 enzyme uses electrons from NADH to convert AFR back into ascorbate [64].
CYB5R3 is located on the outer mitochondrial membrane and is functionally connected to VDAC1 (voltage-dependent anion channel 1). VDAC1 is the most abundant protein found on the outer mitochondrial membrane (OMM) [64].
The Cyb5R3/VDAC1 system is responsible for the conversion of AFR into ascorbate. VDAC1 has been shown to preserve mitochondrial membrane integrity, keeping cells intact when exposed to carcinogens that could induce depolarization and apoptosis [65, 66].
Mice bred without CYB5R3 show a loss of mitochondrial biogenesis, accompanied by 30% loss of total ATP, 50% loss of Complex IV activity, and 25% loss of Complex IV protein quality [67].
Why is CYB5R3 essential for maintaining normal functioning of mitochondria, including activity of mitochondrial ETC, oxygen consumption, ATP production and resistance to oxidative stress? CYB5R3/VDAC1 is actually an additional energy generating pathway that utilizes ascorbate/AFR as electron donors and acceptors [68].
When oxidative phosphorylation activity is not supported by the respiratory chain proteins due to collapsed membrane potential (depolarization), the activity of the CYB5R3/VDAC1 system is able to generate an electrochemical membrane potential catalyzed by electron transfers [69, 70, 71]. CYB5R3/VDAC1 transfer electrons from cytosolic NADH into mitochondria, utilizing Complex IV [69, 72].
Most surprisingly, when ascorbic acid, instead of NADH is supplied to intact mitochondria, the same non-enzymatic induction of this alternative energy production pathway was observed, where there was oxygen uptake, cytochrome c reduction and ascorbate oxidation [69,72].
The rapid conversion of AFR back to ascorbate using NADH as electron donor restores the ascorbate pool and maintains a high NAD+/NADH ratio in cells [68]. However, if there is a lack of NADH, then it may be necessary to maintain a constant supply of fresh ascorbate so that CYB5R3/VDAC1 can utilize electrons from ascorbate to generate membrane potential to rescue depolarizing mitochondria.
During severe infections, the ability of mitochondria to utilize plasma redox system CYB5R3/VDAC1 to generate transient supply of alternate energy would provide mitochondria the opportunity to maintain fusion and fission events to clear damaged mitochondria. Ascorbic acid provides electrons to CYB5R3/VDAC1 when NADH is inadequate as a result of disrupted oxidative phosphorylation.
The importance of a continuous supply of adequate ascorbic acid during cytokine storms induced by coronavirus infections cannot be underestimated.
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