Happy Hypoxia: Higher NO in red blood cells of COVID-19 patients

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) has spread to almost 100 countries, infected over 10M patients and resulted in 505K deaths worldwide as of 30 th June 2020. The major clinical feature of severe COVID-19 requiring ventilation is acute Respiratory Distress Syndrome (ARDS) with multi-functional failure as a result of a cytokine storm with increased serum levels of cytokines. The pathogenesis of the respiratory failure in COVID-19 is yet unknown, but diffuse alveolar damage with interstitial thickening leading to compromised gas exchange is a plausible mechanism. Hypoxia has been seen in the COVID-19 patients however, patients present with a distinct phenotype. Intracellular levels of NO playing important role in the vasodilation of small vessels. Objectives: To elucidate the intracellular levels of NO inside of RBCs in COVID-19 patients compared with that of healthy control subjects. Methods: We recruited 14 COVID-19 infected cases who had pulmonary involvement of their disease, 4 non-COVID-19 healthy controls (without pulmonary involvement and were not hypoxic) and 2 hypoxic non-COVID-19 patients subjects who presented at the Masih Daneshvari Hospital of Tehran, Iran between March-May 2020. Whole blood samples were harvested from patients and intracellular levels of NO in 1 million red blood cells (RBC) was measured by DAF staining using ow cytometry (FACS Calibour, BD, CA, USA). Results: The Mean orescent of intensity for NO was signicantly enhanced in COVID-19 patients compared with healthy control subjects (P ≤ 0.05). As a further control for whether hypoxia induced this higher intracellular NO, we evaluated the levels of NO inside RBC of hypoxic patients. No signicant differences in NO levels were seen between the hypoxic and non-hypoxic control group. Conclusions: This pilot study demonstrates increased levels of intracellular NO in RBCs from COVID-19 patients. Future studies should examine whether intracellular NO would be increased in large number of COVID-19 patients for usage of possible NO therapy in


Introduction
The coronavirus SARS-CoV-19 that causes coronavirus disease 2019 (COVID-19) has spread to almost 100 countries, infected over 10 million patients and resulted in 505K deaths worldwide as of 30 th June 2020 (1). The major clinical feature is acute Respiratory Distress Syndrome (ARDS) with a key complication being heart and multi-functional failure abnormal blood oxygen saturation is at least 95% in most lung diseases, such as pneumonia. Whilst decreasing oxygen saturation accompanies other change, such as stiff or oedematous lungs, increasing levels of carbon dioxide are usually seen in COVID-19 patients with pneumonia (2). Thus, many COVID-19-infected patients with pulmonary involvement have hypoxia and dyspnea as important hallmarks of disease. In COVID-19 patients, despite the respiratory system insu ciently oxygenating the blood, these patients are often alert and feeling relatively well and can easily talk (3).
Red blood cells (RBCs) are highly adapted cells for blood gas transport. At the high oxygen tensions (PO2) prevailing in the pulmonary system, the blood is normally completely saturated with oxygen and hemoglobin (Hb) will formed an R structure. When the blood enters the microcirculation, the PO2 is attenuated promoting oxygen dissociation from hemoglobin and a shift to the T form (4).
Clinical examination of severe cases of COVID-19 patients shows a decreased ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO 2 :FiO 2 ratio) with concomitant hypoxia and tachypnea in most cases (5). Nitric oxide (NO) plays a key role in controlling the vascular system by regulating vascular tone and blood ow following activation of soluble guanylate cyclase (sGC) within the vascular smooth muscle. NO also controls mitochondrial oxygen consumption by inhibiting cytochrome c oxidase (6). RBCs have long been considered as powerful scavengers of endothelial cell-derived NO, participating in systemic NO metabolism mainly by limiting NO bioavailability (7). RBCs passing through the microcirculation sense tissue oxygen conditions via their degree of deoxygenation and couple this information to the release of vasodilatory compounds including ATP and NO to enhance blood ow to hypoxic tissues (8). NO is a free radical and has a critical pathophysiological role in infectious diseases.
RBC intracellular NO is derived from three sources: a) entry from the cell exterior by binding to the highly conserved β-globin chain cysteine 93 residue to form bioactive S-nitrosohemoglobin (SNO-Hb) (9), b) formation from nitrite entering RBC due to the reductive potential of deoxyhemoglobin (10) and c) intracellular production of NO by RBC derived from an active and functional eNOS-like enzyme (RBC NOS). This is localized in the RBC membrane and cytoplasm and has similar properties to eNOS in terms of phosphorylation sites controlling enzymatic activity and its activity dependence on intracellular calcium and L-arginine concentrations (11).
Transfer of NO from SNO-Hb to the membrane-bound anion exchanger (AE1) is required for transfer of NO out of the RBC and is dependent on both the SNO-Hb state (T or R) and the SNO-Hb concentration. Therefore, the ability of SNO-Hb to transfer NO to AE1 or other proteins (e.g., glutathione) are limiting factors in respiratory e ciency. The kinetics and allosteric regulation of Hb nitrosylation by oxygen and pH are consistent with the physiologic mechanisms that modulate tissue blood ow, namely acidosis, hypoxemia and tissue hypoxia lead to NO generation by the RBC via SNO-protein transfer of NO activity (12). In addition, insults such as cellular stress activates RBC NOS, leading to NO release and vasodilation of vessel segments under hypoxic conditions. Together, this supports a prominent role of RBC-derived NO in the regulation of local blood ow (13).
Therefore, the erythrocrine function of RBCs i.e. the release of bioactive molecules including NO, NO metabolites, and ATP are likely to be important in tissue protection and regulation of cardiovascular homeostasis by RBCs. Despite this clear role of NO in vasodilation, there is little evidence regarding the role of NO in COVID-19 particularly in 'happy hypoxic' patients. To examine the hypothesis that NO is important in regulating vasodilation during hypoxia in these subjects we studied intracellular levels of NO in COVID-19 patients.

Materials And Methods
We examined the 14 COVID-19 infected cases who had lung involvement of their disease , 4 non-COVID-19 healthy control (without lungs involvement and not hypoxic) and 2 hypoxic-nonCOVID-19 patients subjects who presented at the Masih Daneshvari Hospital of Tehran, Iran between March-May 2020. All COVID-19 infected cases were diagnosed based on the World Health Organization (WHO) interim guidance. Patients were con rmed positive for COVID-19 nucleic acid in the respiratory samples via realtime reverse-transcriptase polymerase-chain-reaction (RT-PCR) or serum speci c antibodies and chest imaging including chest X ray and CT sacn. Demographic data of all participants presented in Table 1.

Results
Chest X ray and CT imaging of the lungs shows signi cant changes in the lungs with bilateral alveolar diffusion ( Fig. 1 A and B).
After isolation of RBC, they were stained with DAF and intracellular NO determined. COVID-19 patients had a greater shift re ecting higher intracellular levels of NO (Fig. 1C). The mean orescent intensity (MFI) was calculated for each subject and plotted as a histogram (Fig. 1D). This showed a signi cant increase in intracellular NO levels in RBCs from COVID-19 subjects (P≤0.05).
To determine whether hypoxia may be responsible for the increased levels of intracellular NO in COVID-19 patient's RBC, we examined intracellular NO levels of RBC from hypoxic patients without COVID-19 (due to COPD and emphysema. As depicted Fig. 1E, no signi cant increase of intracellular NO was seen in hypoxic patients compared to non-hypoxic controls.

Discussion
We demonstrate increased levels of intracellular NO in RBC from COVOD-19 subjects. This is not due to the presence of hypoxia per se but may afford protection against the hypoxia seen in COVID-19 patients. During health, constitutive NO production in RBCs is largely NOS-dependent, whereas in hypoxic conditions NO production may involve nitrite reduction by deoxyhemoglobin carbonic anydrase and/or eNOS itself (14).
RBC-derived NO causes the vasodilation of small vessels in tissues allowing oxygen to be readily released to tissues. In our study, intracellular RBC NO of COVID-19 patients is signi cantly higher than in healthy controls and this may enable the release of oxygen to tissues resulting in the clinical manifestation of happy hypoxia in these patients. Since NO is a pulmonary vasodilator and also as antiviral activity against coronavirus strains it is likely that NO treatment may be effective in COVOD-19 subjects. There is no evidence that direct oxygen therapy is bene cial in the management of breathlessness in severe COVID-19 patients but our data suggests that NO therapy may be bene cial in COVID-19 patients with hypoxia (15).
In summary, COVID-19 patients show higher levels of NO inside RBC compared to non-COVID-19 hypoxemic patients. Whether higher levels of intracellular NO inside RBC of COVID-19 infected patients drive the unexpected silent hypoxia phenotype needs to be examined in future clinical studies using NO donors in hypoxemic COVID-19 patients.

Conclusions
This pilot study shows that elevated levels of intracellular NO may mask the effects of hypoxia in COVID-19 patients which present as a 'happy' hypoxic state. Further studies are required to con rm this but the data suggests that trials of NO therapy or NO donors may be useful in treating severe COVID-19 patients with hypoxia.