key: cord-0993446-7o408jxr
authors: Wang, Jun; Mei, Fanghua; Bai, Lu; Zhou, Suhua; Liu, Di; Yao, Lulu; Ahluwalia, Amrita; Ghiladi, Reza A.; Su, Lei; Shu, Tong; Gong, Miaozi; Wang, Xiaofang; Zhu, Lijun; Cai, Kun; Zhang, Xueji
title: Serum nitrite and nitrate: A potential biomarker for post-covid-19 complications?
date: 2021-08-30
journal: Free Radic Biol Med
DOI: 10.1016/j.freeradbiomed.2021.08.237
sha: 0a355c9eb4f4b3549c28875ae37e40947e5e5f94
doc_id: 993446
cord_uid: 7o408jxr

Nitric oxide (NO) plays an important role in cardiovascular and immune systems. Quantification of blood nitrite and nitrate, two relatively stable metabolites of NO (generally as NO(x)), has been acknowledged representing NO bioactivity partially. Dysregulation of NO(x) had been reported in SARS-CoV-2 infected populations, but whether patients recovered from COVID-19 disease present with restored NO(x) is unknown. In this study, serum NO(2)(−) and NO(3)(−) were quantified and analyzed among 109 recovered adults in comparison to a control group of 166 uninfected adults. Nitrite or nitrate levels were not significantly different among mild-, common-, severe- and critical-type patients. However, these recovered patients had dramatically lower NO(2)(−) and NO(2)(−)/NO(3)(−) than the uninfected group (p < 0.0001), with significantly higher NO(3)(−) levels (p = 0.0023) than the uninfected group. Nitrate and nitrite/nitrate were positively and negatively correlated with patient age, respectively, with age 65 being a turning point among recovered patients. These results indicate that low NO(2)(−), low NO(2)(−)/NO(3)(−) and high NO(3)(−) may be a potential biomarker of long-term poor or irreversible outcomes after SARS-CoV-2 infection. It suggests that NO metabolites might serve as a predictor to track the health status of recovered COVID-19 patients, highlighting the need to elucidate the role of NO after SARS-CoV-2 infection.

COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused human life losses. As of August 22, 2021 , the total number of confirmed infected cases of COVID-19 stood at 211 million worldwide, with a death toll of 4.4 million (from the Johns Hopkins Center for Systems Science and Engineering) [1] . The possibility of developing a severe case of disease and the mortality rate are reported to be influenced substantially by age and the presence of comorbidities, as well as sex [2] [3] [4] . Estimates of the influence of age using data from 16 countries suggest that people aged 65 years and older were more susceptible to SARS-CoV-2 infection, with a higher admission rate to intensive care units (ICUs) and a higher fatality rate than younger individuals [5] . Multiple reasons for this age-related susceptibility have been proposed including a link to metabolic, respiratory, or cardiovascular comorbidities [6] [7] [8] [9] [10] [11] . For example, COVID-19 patients with existing hypertension have been reported to be more vulnerable to severe disease progression and mortality [7, 12] , with evidence supported by numerous further larger studies and recent meta-analyses of 24 studies from across the globe [13] . Moreover, substantial organ dysfunction among recovered COVID-19 patients has been increasingly reported, which is both unexpected and troubling [14] [15] [16] .

The mechanisms underlying these links are hotly debated. However, it is accepted that vascular dysfunction and impaired bioavailability of protective nitric oxide (NO) plays a key role in the progression of these co-morbidities including in patients with COPD, hypertension and other vascular diseases [17] . Besides its role as an important vasodilator, NO also functions as a vital immune mediator, including non-specific antiviral effects. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, has been reported to improve the host cell survival following infection in one study [18, 19] , and suppress the replication cycle of severe acute respiratory syndrome (SARS-CoV-1) at RNA and cellular levels and interfere with spike protein activity in another study [20] . More recently, NO has been shown to exert inhibitory effects on SARS-CoV-2 entry into cells and replication [21] .

Indeed, SNAP was found to delay or prevent viral cytopathic effects of SARS-CoV-2 in vitro via covalent binding and inhibition by released NO [22] . Currently, a few clinical trials of iNO are currently underway, for the purpose of prevention or therapy of COVID-19 disease [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] , but its precise role in COVID-19 pathogenesis has not been fully elucidated yet.

Despite the promise of iNO therapy, systematic evaluations are still needed for safety and J o u r n a l P r e -p r o o f 3 efficacy of inhaled nitric oxide. Targeting NO is a double-edged sword, depending on where, how and how much of it is produced [33] . The effects described above of NO are evidenced with high concentrations of ~30-200 M. In a small study, detection of nitrite (NO2 -) and nitrate (NO3 -) (commonly referred as NOx) using photometric analysis or the Griess assay was shown to be elevated, suggesting that NO levels were increased, in 11 critically ill non-surviving COVID-19 patients compared to 45 survivors [34] . However, conversely, in several reports, decreased basal NO production or bioavailability (normally in the 100-400 nM range) has been suggested to underlie elevated apoptosis of endothelial cells induced by SARS-CoV-2 infection, increased inflammation, or excessive reactive oxygen species (ROS) [23, 24, 35] . One recent study reported decreased total NO, nitrite and SNO in COVID-19 patients in hospital [36] . Another study in Spain showed that nitric oxide diffusion was reduced in around 40% patients discharged from hospital, via respiratory function tests with a MasterScreen PFT equipment [37] . To date, the levels of NO in blood among patients discharged from hospitals, i.e. "recovered patients", remain unknown.

To determine whether alternations in NO metabolite levels might be associated with so-called "long COVID", we measured serum nitrite and nitrate of the COVID-19 recovered patients after being discharged from hospitals for at least 4 months. Since the clinical spectrum of SARS-CoV-2 infection is broad, 113 recruited recovered patients were categorized in this study, according to reported guidelines [38] . Another 175 adults who did not get infected with SARS-CoV-2 were recruited as a control uninfected group. Sex and age factors were evaluated in these two groups.

Since food is one source of nitrite and nitrate for human and it is extremely challenging to execute a clinical trial of diet on COVID-19 patients in hospital, an alternative way was used by comparing serum NOx between two small groups of healthy adults. Another 45 male adults were recruited as a diet-controlled group, who lived for two weeks with the same meal plan as hospitalized patients were, and whose serum NOx levels were compared with age-matched men in the uninfected group. After exclusion of unqualified volunteers, remaining 109 recovered patients, 166 control uninfected adults and 31 diet-controlled men were enrolled in this study, Patients were considered as recovered when they met the COVID-19 discharge criteria and were released from the hospital, according to established guidelines [38] . Blood samples were collected for nitrite and nitrate analyses. Each participant gave informed consent prior to participation in the study.

In order to evaluate the potential effect of diet on serum NOx, healthy male adults were fed the same dining menu for two weeks that COVID-19 patients received in hospitals before blood collection. Breakfast options included boiled eggs, milk, steamed buns, and noodles, which was delivered at 8:00-8:30 AM every morning. Lunch and dinner options included pork meat, chicken meat, cabbage, and green beans, and were delivered at 12:00-1:00 PM and 5:30-6:00 PM, respectively.

In order to minimize the interference of food and drink on endogenous serum nitrite and nitrate, blood was collected after overnight fasting, and no beverages were allowed overnight except for limited amount of water. Samples were obtained in the morning between 8:00 and 10:00 AM. The tube containing whole blood remained static on the bench for 50 min, after which serum was isolated from blood via centrifugation at 4000 g for 5 min at room temperature (RT). Serum was then put on dry ice and transported to the lab for immediate analysis, or stored at -80 ºC for next-step management.

Serum specimens were prepared via three different methods [39] : 1) precipitation with sodium hydroxide (NaOH) plus zinc sulfate (ZnSO4): a 0.1 mL serum sample was mixed with 0.2 mL NaOH (0.5 M) and 0.2 mL ZnSO4 (10% in water), the mixture vortexed for 30 s, sat for 15 min at RT and centrifuged at 13000 g for 5 min before analysis; 2) precipitation with ethanol: a 0.2 mL serum sample was mixed with 0.4 mL cold ethanol, the mixture was kept on ice for 30 min, and then centrifuged at 13000 g for 5 min before analysis; 3) omission of pre-treatment: serum samples were directly injected for analysis. 

Chemiluminescence analysis has been widely recognized as a reliable method for nitrite and nitrate quantification in biological fluids [40] [41] [42] . Based on the highly sensitive ozone-chemiluminescence method, its sensitivity reaches 1 picomole (1 nM for a 1 mL injection) for liquid samples. A Nitric Oxide Analyzer 280i (NOA 280i, GE) was applied to measure the levels of nitrite and nitrate in this study. Reducing reagents employed were the triiodide ion for nitrite detection, and vanadium trichloride (VCl3) solution containing 1 M HCl for nitrate quantification.

Duplicate injections of standard solutions into the NOA purge vessel were performed. The NOA instrument passed daily calibration when calibration curves were obtained with R 2 > 0.999.

Comparison among the three pretreatment methods was performed via injection of each sample six times in order to evaluate the recovery ratio and RSD. Detection of each serum sample was immediately carried out after pretreatment, while samples stayed on ice during measurement. Assays for every group sample were repeated in triplicate.

Comparisons among groups and subgroups were performed using a two-tailed unpaired Student's t-test with OriginPro 9.1 (OriginLab) and Prism 7 (GraphPad). Correlation analysis, multivariate analysis of variance (MANOVA) and independent t-test were performed with SPSS Statistics V22.0 (IBM). Values for all measurements were expressed as mean±SD for parametric distributions. A p-value of <0.05 was considered statistically significant. A p-value between 0.05 and 0.1was considered borderline significant. All experiments were performed at least three times.

All subjects recruited provided informed consent for participation in this research study. Among the recruited 175 adults (18-83 years old) who never got infected by SARS-CoV-2 before the blood collection, 71 were men and 104 were women. Among them, nine people were excluded due to hypertension, diabetes or being under medication, with the remaining 166 volunteers for blood collection to measure nitrite and nitrate. Among the recruited 113 recovered patients (18-83 years old) from COVID-19, there were 3 mild-type, 90 common-type, 9 severe-type, 5 critical-type, 5 unclassified and 1 asymptomatic subjects, according to the established guidelines [38] for clinical J o u r n a l P r e -p r o o f 6 types of COVID-19 disease. Four patients out of these 107 classified subjects were excluded due to hypertension or being under medication. The remaining 103 classified, 5 unclassified and 1 symptomatic volunteers were enrolled for blood collection ( Figure S1 , Table S1 ).

The hospital discharge criteria for the COVID-19 subjects were: 1) a return to normal body temperature for more than 3 days; 2) significant improvements in respiratory symptoms; 3) lung imaging showing significant improvement in acute exudative lesions; and 4) two consecutive respiratory tract samples (at least 24 hours apart) that returned negative nucleic acid tests for SARS-CoV-2. All these recruited patients were under treatment, and subsequently discharged from hospital for at least four months prior to study participation, who were within the recovered group in this study.

Of the diet-controlled group, only male adults were recruited in order to simplify data analysis by removing factors attributable to female menopause and menstruation. 45 men were recruited, with 14 were excluded due to hypertension, diabetes or being under medication. The remaining 31 men were age-matched with 31 men from the uninfected group ( Figure S1 , Table S1 ). These 31 uninfected adults were not under controlling diet, in order to clarify whether diet casts a difference for serum NO between these two small groups of male participants. For two weeks prior to blood collection, this diet-controlled population followed a meal plan for COVID-19 patients that have been adopted by hospitals in Hubei Province. Volunteers in these two male populations were 28-60 years old.

Analyte detection in serum is complicated due to the presence of the serum matrix that includes proteins, lipids and other contents. An efficient pretreatment method is required to reduce matrix interference and achieve high accuracy of NOx levels. In this study, three pretreatment methods were investigated to select the most efficient one for analysis of serum nitrite and nitrate, i.e. the stable metabolites of NO. The results are shown in Figure 1 , Table S2 .

We carefully compared the foaming intensity following injection of serum samples after pretreatment. When serum was mixed with NaOH and ZnSO4 in a volumetric ratio of 1:2:2 (serum:NaOH:ZnSO4), a greater amount of precipitation was observed compared to the ethanol pretreatment (serum:ethanol=1:2), with very little precipitation observed in the absence of the pretreatment step ( Figure 1A) . No matter whether serum nitrite or nitrate was detected, samples with J o u r n a l P r e -p r o o f 7 the NaOH/ZnSO4 pretreatment had less foaming than those with ethanol pretreatment (Figure 1B) .

The measurement of serum without any pretreatment was significantly more challenging, as foaming was too vigorous to perform multiple replicates using a single triiodide or vanadium solution, and the reductive reagents had to be frequently replaced.

As shown in Figures 1C and 1D and tabulated in Table S2 (see Supplementary Information), the relative recoveries employing the NaOH/ZnSO4 pretreatment were in the range of 85.2 -111.0% with a relative standard deviation (RSD) of no more than 4.2%. The relative recoveries from ethanol pretreatment were in the range of 73.4 -110.4% with an RSD in the range of 1.3 -11.7%. In the absence of a pretreatment step, the relative recoveries were in the range of 40.1 -76.9% with an RSD in the range of 3.9 -8.8%. These results confirmed the reliability of NaOH/ZnSO4 pretreatment as our preferred method for quantification of serum nitrate and nitrate in the uninfected and COVID-19 recovered patients.

We also carefully evaluated the association between injected sample volume and area under curve (AUC) with NaOH/ZnSO4 pretreatment. This procedure was performed to minimize systematic errors during detection ( Figure S2) . The results showed that the chemiluminescent signal was linear with increasing sample volume in the range of 10 -100 μL (R 2 > 0.999), which covered the injected volumes used for nitrite or nitrate measurement.

In order to investigate whether NOx levels in the recovered population were different from uninfected adults, blood was collected from 166 uninfected adults and 109 recovered patients without hypertension, diabetes or being under medication after overnight fasting.

Nitrite levels (Figure 2A) The ratio of nitrite versus nitrate in the uninfected group (0.0214±0.0189; Figure 2C ) was statistically higher (p < 0.0001) than in the recovered group (0.0057±0.0041) ( Table S3) 

Since vascular tone, as well as immune responses, may be regulated by sex, age and pathophysiological status [43] , the data were further analyzed to see if serum nitrite and nitrate levels got impacted by the factors of sex, age, and clinical severity before recovery. Between the two groups, recovered men had lower nitrite levels than men in the uninfected group (p < 0.0001), a similar finding to that observed for women (p < 0.0001) (Figure 2D) . Inside the recovered group, no difference was found between male and female patients, but male nitrite levels were lower than those of females inside the uninfected group. As for serum nitrate, recovered men had higher nitrate levels than men in the uninfected group (p = 0.0066), a similar trend to that observed for women with borderline significance (p = 0.0728) ( Figure 2E ). No significant difference of nitrate levels was observed between men and women in the same group. As for the ratio of nitrite versus nitrate, the same sex in two groups had statistically different values, but the two sexes in the recovered group had similar levels and women had higher nitrite/nitrate levels than men in the uninfected group ( Figure 2F, Table S3 ). It may suggest that infection attenuates the sex-related nitrite difference among healthy subjects, as reported in literature [44] .

Age is another factor influencing viral infection and disease progression during the COVID-19 pandemic [3] . Therefore, the association between nitrite and nitrate (NOx) and age were analyzed in these two groups (Figure 3, Figure S3 ). In the recovered group, there existed positive correlation between nitrate and age, and negative correlation between the ratio of nitrite/nitrate and age ( Figure   3D -F). Such correlations were not observed in the uninfected group (Figure 3A-C) . Since an age of 65 years or older is considered as a high-risk factor for COVID-19 [5] , we further split each group into 18-64 and 65-83 years old subgroups and performed multivariate analysis of variance (MANOVA) and a simple effect analysis. As for nitrite and nitrite/nitrate, recovered adults in the same-age ranges still had dramatically lower levels than the uninfected peers ( Figure 3G, 3I) , with the opposite trend of nitrate levels ( Figure 3H ). In addition, nitrate and nitrite/nitrate levels were significantly higher and lower in the recovered (65-83) than in the recovered (18-64) subgroup ( Figure 3H-I) , respectively. The results indicate that high NO3and low NO2 -/NO3may serve a promising marker for long-term consequences among aged people infected by SARS-CoV-2.

Whether the clinical classification during infection impacts NOx levels in blood stays unknown.

In this study, we performed a comparison of serum NOx among patients from four categories. The J o u r n a l P r e -p r o o f 9 clinical types (mild, common, severe, and critical) of COVID-19 patients were categorized based on their symptoms according to established guidelines [38] . As the name implies, mild-type COVID-19 patients presented with mild clinical symptoms and no evidence of pneumonia in imaging. Patients categorized as common-type COVID-19 presented with fever and respiratory symptoms, with pneumonia visible on imaging. Severe type criteria in adults included one of the following symptoms:

shortness of breath with a respiratory rate (RR) 30 times/min; upon air inhalation, oxygen saturation was 93% at rest; arterial partial pressure of oxygen (PaO2)/inhaled oxygen concentration (FiO2) 300 mmHg (1 mmHg = 0.133 kPa); or progressively worsening of clinical symptoms, with >50% significant progress of lesions within 24-48 hours by pulmonary imaging. The criteria for critical-type COVID-19 disease include one of the following: respiratory failure occurs and mechanical ventilation is required; shock occurs; respiratory failure is combined with other organ failure, and ICU monitoring and treatment are required.

Among the 103 classified recovered patients, there were 3 mild type, 86 common type, 9 severe type and 5 critical type patients when they were in hospital. As shown in Figure 4 and Table S3 , Table S3 ).

In short, nitrite and nitrite/nitrate were significantly lower in the recovered patients than in the uninfected control group. In the recovered population, high nitrate seemed a marker for poor long-term effects for patients aged 65 or older, as well as for COVID-19 patients in general. Sex appeared to only impact serum nitrite levels in the uninfected group.

Nitrite and nitrate from diet has been suggested a source of endogenous NO in humans [45] .

Since the role of sex in NO circulation and metabolism has not yet been fully elucidated, and female statistically significant difference was found between these two groups of healthy men, suggesting that hospital meals did not influence serum NOx level in uninfected adults, at least for a short-term period of two weeks. Whether diet affects NOx in COVID-19 patients in hospital and recovered patients, a well-designed study is highly needed.

The pathogenesis of COVID-19 disease has been linked to oxidative stress or redox imbalance, including higher nitrate and cytokine storm in patients, and the use of antioxidants has been proposed as a potential therapy [46] . Taking advantage of the sensitive and precise properties of the chemiluminescent technique, this study systematically measured serum nitrite and nitrate in recovered COVID-19 patients discharged from hospital for more than four months for the first time, in order to clarify whether different metabolites of NO may serve a potential biomarker for long-term effects of SARS-CoV-2 infection.

There was no parallel comparison to achieve the optional method for chemiluminescence measurements of NOx in previous literarures, even though multiple serum preparation methods were noted [47] [48] [49] . After evaluating the recovery ratio and foaming conditions during sample preparation, we thereafter established a NaOH/ZnSO4 pretreatment method for the reproducible and quantifiable determination of nitrite and nitrate levels in serum. In this study, the opposite changes were observed for serum nitrite and nitrate in the recovered group compared to the uninfected group. It suggests that nitrite and nitrate may get produced and function differently in vivo. In one study by Joshi et al. in 2002 , NO was majorly and irreversibly consumed by oxy-hemoglobin to form nitrate under normoxia, instead of being conserved as nitrite or S-nitrosohemoglobin (one type of S-nitrosothiol, SNO) [50] . Less than 1% of homogenously delivered NO from its donor was converted into nitrite or SNO [50] . As reported for recovered COVID-19 patients [51] , they usually encounter shortness of breath or difficulty breathing, which might suggest that their blood hemoglobin needs to work more diligently in order to deliver enough oxygen and therefore consume more NO. This speculation of a compensatory effect of hemoglobin certainly needs experimental confirmation.

In 2001, Lauer et al. reported that plasma nitrite alone, instead of nitrate or the sum of nitrite and nitrate, was reflecting regional endothelial nitric oxide synthase (eNOS) activity in one study of 24 volunteers, which was positively correlated with forearm blood flow [52] . In this study, decreased nitrite might be linked to eNOS dysfunction in impaired endothelium during SARS-CoV-2 infection [23] , as shown in Scheme 1. In addition, dysfunction of iron metalism was reported among COVID-19 patients [53] . Hyperferritinemia was associated with severe COVID-19 disease, which also existed in recovered patients, especially those with severe or critical COVID-19 [54] . Whether and how the dysregulation of iron metabolism, such as release of free toxic circulating heme, is linked to the pathogenesis and NO in COVID-19 stays unclear [53] . Apparently, this opposite trends of NO2and NO3in recovered patients need furter investigation.

Furthermore, the positive association between nitrate and age in the recovered COVID-19 patients was observed. Aging has been linked to detrimental NO production and bioavailability [55] , which seems in accordance with lower NO2 -/NO3and higher NO3in patients 65 years or older in this study. One potential cause for the decreased nitrite levels in recovered group may lie in perturbed gut microbiota of COVID-19 patients [56] . In one study of 100 patients after clearance of SARS-CoV-2, the level of a gut bacterium with immunomodulatory potential, Faecalibacterium prausnitzii, was reported to be diminished when compared to a healthy control group. A summary of the above factors that influence NOx serum levels as they relate to COVID-19 disease are presented in Scheme 1. The commensal microbe F. prausnitzii has been shown to affect NO production via J o u r n a l P r e -p r o o f 12 iNOS induction [57] . Commensal gut bacteria also modulate nitrate reduction to nitrite [58] , and might be depleted in COVID-19 patients. These additive effects may then cause lower NO availability, which triggers more nitrite reduction to NO in order to remedy the loss of NO among patients.

On the other hand, endothelial cells are damaged during SARS-CoV-2 entry and progression, which either inactivates endothelial nitric oxide synthase (eNOS) or uncouples eNOS to release reactive oxygen species (ROS) (Scheme 1) [59] . Excessive ROS can consume NO to form peroxynitrite (ONOO -). In the presence of protons, i.e. neutral or acidic pH, the stability of peroxynitrite is compromised and peroxynitrous acid (ONOOH) will decomposed to form NO3 -.

Either in blood under physiological condition (~pH 7. 35-7.45) or blood from COVID-19 patients (~pH 7.40-7.51), the degradation of peroxynitrous acid and formation of nitrate seems natural. A similar phenomenon was observed in older people, who usually had slower or dysfunctional vascular function and decreased NO bioavailability [60] . It is highly possible that the combined effect of aging and viral infection and leads to low nitrite, low nitrite/nitrate and high nitrate among subjects recovered from COVID-19.

However, there also exists other pathways to inactivate NO, such as s-nitrosylation, nitration, As is a covalent post-translational oxidative modification, S-nitrosylation, also called S-nitrosation (SNO), has been known as an important modification in physiology and diseases [61, J o u r n a l P r e -p r o o f 13 

(LMW-SNO, such as S-nitrosolglutathione, S-nitroso-coenzyme A) and SNO-cysteine-proteins [61, 62] , as shown in Equilibrium 1. Quite a few literatures have suggested that NO exerts its pathophysiological function mainly via S-nitrosylation, instead of via other pathways as shown in Scheme 2. In one recent study by Dominic et al, S-nitrosothiol was significantly lower in the COVID-19 patients than in the control group [36] . It indicated that blood SNO levels changed during SARS-CoV-2 infection with uncovered mechanism yet.

R1SNO + R2SH → R1SH + R2SNO

LMW-SNO Protein1-SNO Protein2-SNO Equilibrium 1

On the other hand, S-nitrosylation may serve as a defense to viral attack during this pandemic [22] .

Using a NO donor SNAP, Akaberi et al identified that reduced activity of SARS-CoV-2 main protease was consistent with S-nitrosylation of cysteine in the active site of this protease [22] , though the mechanism needs to be clarified. This is not the first time that S-nitrosylation was suggested as the molecular base of NO's antiviral effects [20, 63] . Whether the decreased level of NO (in this case, the level of S-nitrosolthiol) is due to cellular defense against SARS-CoV-2 certainly needs further comprehensive investigation from both analytic measurement and mechanistic studies.

To date, mounting evidence suggests that COVID-19 survivors may encounter long-term health issues [64, 65] . Chronic unexplained medical symptoms of so called "long COVID" had been reported in both adults and children [66] [67] [68] [69] [70] , even among initially mild-infected patients, which included fatigue, headache, insomnia, dizziness, lung damage, erectile dysfunction, as well as other psychiatric and neurological symptoms. In a recent cohort study of COVID-19 patients discharged from hospitals for 6 months, most survivors were still troubled by a number of symptoms, including fatigue or muscle weakness, sleep difficulties, anxiety or depression [15] . These findings are consistent with another follow-up study performed 3 months after hospital discharge [71, 72] , and are similar to what was observed for SARS patients, suggesting long-term sequelae due to coronavirus infections [73] . In addition, as endothelial dysfunction diminishes NO bioavailability, it also causes erectile dysfunction, which was observed with significantly higher occurrence among men with SARS-CoV-2 infection [70, 74] .Unfortunately, validated biomarkers are still missing to J o u r n a l P r e -p r o o f 14 quantify these abnormalities for most of these post-infectious symptoms [75] .

As a key immune, cardiovascular and neurological mediator, circulating NO levels may represent some unknown long-term aftermath of SARS-CoV-2 infection. There appears from the results presented in this study that an association between levels of NOx, SARS-CoV-2 infection and age together suggest that NO is involved in the COVID-19 pathogenesis, possibly via cardiovascular and immune modulation. Comparison of serum NOx between two healthy adult groups in this study is certainly not an ideal method to investigate the effect of food-source NOx on SARS-CoV-2 infected patients in hospital. More strictly designed trials are necessary to elucidate the puzzle of diet, which contains both antioxidants and oxidants. One recent study in in primary human lung cells and intestinal organoids showed that SARS-CoV-2 dysregulated cellular metabolism and reduced autophagy [69] , which might indirectly modulate NO pathways due to interaction between them [76] .

There is an urgent need to understand how pro-oxidants and antioxidants function in patients with COVID-19 disease. Combined with chest imaging and pathological diagnosis, potential biomarkers or predictor like NOx may help elucidate persisting symptoms and substantial organ dysfunction [65] .

This is the first time that NO levels have been reported among patients recovered from COVID-19 disease after discharge from hospitals for at least 4 months. Lower nitrite and nitrite/nitrate, and higher nitrate levels were found among these patients. In addition, nitrate and nitrite/nitrate were positively and negatively correlated with patient age, respectively, with age 65 being a turning point.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 

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Nitrate reduction to nitrite, nitric oxide and ammonia by gut bacteria under physiological conditions

Endothelial activation and dysfunction in COVID-19: from basic mechanisms to potential therapeutic approaches

Endothelial dysfunction and aging: an update

Protein S-Nitrosylation: Determinants of Specificity and Enzymatic Regulation of S-Nitrosothiol-Based Signaling

S-nitrosylation: specificity, occupancy, and interaction with other post-translational modifications

S-nitrosylation of viral proteins: molecular bases for antiviral effect of nitric oxide

Reinfection with SARS-CoV-2: Implications for Vaccines

Protection of healthcare workers against SARS-CoV-2 reinfection

The lasting misery of coronavirus long-haulers

How COVID-19 can damage the brain

Persistent symptoms in Swedish children after hospitalisation due to COVID-19

SARS-CoV-2autophagy uncovers host-targeting antivirals

Mask up to keep it up": Preliminary evidence of the association between erectile dysfunction and COVID-19

Impact of coronavirus disease 2019 on pulmonary function in early convalescence phase

Follow-up study of the pulmonary function and related physiological characteristics of COVID-19 survivors three months after recovery

Long-term bone and lung consequences associated with hospital-acquired severe acute respiratory syndrome: a 15-year follow-up from a prospective cohort study

Addressing male sexual and reproductive health in the wake of COVID-19 outbreak

Long COVID has exposed medicine's blind-spot

Complex inhibitory effects of nitric oxide on autophagy

· Decreased nitrite, nitrite/nitrate and increased nitrate levels were observed among recovered COVID-19 patients discharged from hospital for more than four months. · Nitrate and nitrite/nitrate were positively and negatively correlated with patient age, respectively. · Serum NOx was not significantly different among recovered COVID-19 patients with varied clinical severity in hospital.