key: cord-0977474-08ccmxkl
authors: Dominic, Paari; Ahmad, Javaria; Bhandari, Ruchi; Pardue, Sibile; Solorzano, Juan; Jaisingh, Keerthish; Watts, Megan; Bailey, Steven R.; Orr, A. Wayne; Kevil, Christopher G.; Kolluru, Gopi K.
title: Decreased availability of nitric oxide and hydrogen sulfide is a Hallmark of COVID-19
date: 2021-05-08
journal: Redox Biol
DOI: 10.1016/j.redox.2021.101982
sha: 64f4f32141244578567f4949c82ff62b1e513d0c
doc_id: 977474
cord_uid: 08ccmxkl

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in a global outbreak affecting millions of people who manifest a variety of symptoms. Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is increasingly associated with cardiovascular complications requiring hospitalizations; however, the mechanisms underlying these complications remain unknown. Nitric oxide (NO) and hydrogen sulfide (H(2)S) are gasotransmitters that regulate key cardiovascular functions. METHODS: Blood samples were obtained from 68 COVID-19 patients and 33 controls and NO and H(2)S metabolites were assessed. H(2)S and NO levels were compared between cases and controls in the entire study population and subgroups based on race. The availability of gasotransmitters was examined based on severity and outcome of COVID-19 infection. The performance of H(2)S and NO levels in predicting COVID-19 infection was also analyzed. Multivariable regression analysis was performed to identify the effects of traditional determinants of gasotransmitters on NO and H(2)S levels in the patients with COVID-19 infection. RESULTS: Significantly reduced NO and H(2)S levels were observed in both Caucasian and African American COVID-19 patients compared to healthy controls. COVID-19 patients who died had significantly higher NO and H(2)S levels compared to COVID-19 patients who survived. Receiver-operating characteristic analysis of NO and H(2)S metabolites in the study population showed free sulfide levels to be highly predictive of COVID-19 infection based on reduced availability. Traditional determinants of gasotransmitters, namely age, race, sex, diabetes, and hypertension had no effect on NO and H(2)S levels in COVID-19 patients. CONCLUSION: These observations provide the first insight into the role of NO and H(2)S in COVID-19 infection, where their low availability may be a result of reduced synthesis secondary to endotheliitis, or increased consumption from scavenging of reactive oxygen species.

Coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected over 77.8 million people in over 220 countries during the recent worldwide pandemic, approximately 18.5 millions of whom are in the United States.

Although COVID-19 causes significant morbidity and mortality when it manifests as 'viral pneumonia,' available evidence suggests that COVID-19 is associated with cardiovascular complications. These are rapidly emerging as a key threat, leading to increasing hospitalizations accompanied by a host of complications, including myocarditis, thrombo-embolism, acute coronary syndrome, and resultant cardiac arrhythmias, together referred to as Acute COVID-19

Cardiovascular Syndrome (ACovCS) 1, 2 . The complications of COVID-19 are significantly exacerbated due to preexisting comorbidities, including pulmonary and cardiovascular disease.

Studies of the SARS and SARS-CoV-2 viruses reveal a potential role for cytokine storm, altered blood pressure regulation, and thrombosis in the pathogenesis of COVID-19 [3] [4] [5] . Moreover, COVID-19 has been shown to directly target endothelial cells and cause endotheliitis, thus affecting downstream functions that may contribute to cardiovascular complications 6 . However, the link between cardiovascular complications and COVID-19, along with the underlying molecular mechanisms, remains poorly understood.

Nitric oxide (NO) and hydrogen sulfide (H 2 S) are ubiquitous signaling molecules popularly referred to as gasotransmitters that play protective roles in limiting the severity of cardiovascular disease 7, 8 . NO acts as a vasodilator and an antiviral agent in patients with SARS and can inhibit in vitro replication of SARS-CoV-2 9, 10 . While several recent reviews also suggest an association between H 2 S and SARS-CoV-1/2, they provide little evidence of any of such relationship [11] [12] [13] [14] . Consistent with these suppositions is the possibility that endothelial dysfunction concomitant with COVID-19 infection is likely to result in reduced NO and H 2 S metabolite availability. However, no studies have been reported to date evaluating specific J o u r n a l P r e -p r o o f levels of gasotransmitters in relation to In this study, we assessed the relationship between NO and H 2 S metabolite availability in patients with COVID-19 and further evaluated them as prognostic biomarkers in severely ill COVID-19 patients.

This was a case-control study approved by the Institutional Review Board (IRB) of Louisiana State University Health Sciences Center at Shreveport (LSUHSC-S) (STUDY00001501).

Consecutive patients admitted with COVID-19 viral pneumonia to Ochsner-LSU hospital in Shreveport were approached for inclusion in the study. Patients who tested positive for COVID-19 by rapid testing or by PCR within 14 days were included. Pregnant women, prisoners, and patients younger than 18 years of age or older than 89 years of age were excluded from the study. Among those who met the inclusion criteria, a total of 73 patients were consented; two patients withdrew their consent, we could not obtain blood samples from two other patients, and one sample was inadequate for performing analysis. Volunteers were invited to enroll in the study using flyers and by word of mouth. Blood samples from healthy race-and sex-matched volunteers with no prior history of COVID-19 infection were also obtained in the cardiology clinic at Ochsner-LSU Hospital in Shreveport after the volunteers provided an informed consent.

After obtaining an informed consent, blood samples were collected from human healthy subjects and COVID-19 patients into 6 mL BD vacutainer tubes with lithium heparin. Samples were transported to the lab within 15 min on ice and were centrifuged at 1500 RCF for 4 min at 4 °C; plasma was collected and snap frozen for further analyses. Medical record data pertaining to baseline characteristics and comorbidities of healthy subjects and COVID-19 patients were collected and compared (Tables 1 and 2 ). J o u r n a l P r e -p r o o f 2.3 NO metabolite measurements. NO metabolites (NOx) were measured using an ozonebased chemiluminescence assay (Sievers Nitric Oxide Analyzer 280i, Weddington, NC) as described previously 15 . Plasma samples were collected in NO stabilization buffer (1.25 mol/L potassium ferricyanide, 56.9 mmol/L N-ethylmaleimide, 6% Nonidet P-40 substitute in PBS), or free nitrite and S-nitrosothiol (SNO) preservation buffers (Zysense, Weddington, NC), respectively. Aliquots of samples were injected into the analyzer and tested for total NO and for individual NO metabolites.

Plasma samples were analyzed for free sulfide, acid-labile sulfide (ALS), bound sulfane sulfur (BSS), and total sulfide levels using the monobromobimane (MBB) method reported previously 16 . Free sulfide was measured using 30 μL of plasma with MBB; for detection of ALS and BSS, 50 μL of plasma was processed separately in two 4 mL BD vacutainer tubes with 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA) for the ALS reaction, and 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA) containing 1 mM TCEP for the total sulfide reaction. Following a 30minute incubation on a nutator mixer, to trap the evolved sulfide gas and incubated with 100 mM Tris-HCl buffer (pH 9.5, 0.1 mM DTPA) for 30 min on a nutator mixer. The trapped sulfide was then measured using the MBB method and calculations performed to determine total sulfide and its pools as previously described 16 .

Quantitative determination of nitrotyrosine in the plasma of control subjects and COVID-19 patients was performed by a competitive ELISA kit (Cell biolabs, Inc.) as per manufacturer's instructions.

J o u r n a l P r e -p r o o f 2.6 Statistical analyses. Levels of NO and sulfide metabolites were assessed by group means and standard deviations with subsequent pairwise comparison using analysis of variance (ANOVA). Receiver-operating characteristic analysis (ROC) was conducted to assess the predictive accuracy in correlating NO and sulfide levels with COVID-19 infection. Cutoff values for positive classification were included in the curve, with a nonparametric distribution assumption and a confidence level of 95%. These statistical analyses were performed using GraphPad Prism 5.0. We also conducted multivariable regression analyses to estimate the effect of various predictor variables on NO and H 2 S in separate models with 95% confidence intervals. A descriptive analysis of study variables was performed using SPSS Version 26.0 (IBM Corp., Armonk, NY). A Chi-square test of independence was used to determine associations between categorical variables. For continuous variables, means of two independent groups were compared using the independent samples Student's t-test. For all analyses, a p-value of <0.05 was considered statistically significant. We assumed equal variance for the independent samples Student's t-test result when Levene's test had a p-value <0.05. Otherwise, we used the results from equal variance not assumed.

A total of 68 COVID-19 cases and 33 controls were included in the study. Plasma NO availability was measured and compared between control subjects and COVID-19 patients (Fig.   1 ). We found a significant reduction in the total NO levels in the plasma of COVID-19 patients 

We next examined the impact of COVID-19 infection on sulfide metabolites. Figure 2 illustrates free, acid labile, bound sulfide, and total sulfide pools that were quantified in plasma samples from healthy controls and COVID-19 patients. Sulfide levels, including free (0.310.14 M vs 0.180.05 M, p<0.0001; Fig 

The association of plasma NO levels were compared between COVID-19 patients and control subjects based on race. Analysis by race revealed a significant reduction in plasma total p=0.275) was seen in AA COVID-19 patients compared to AA controls, no statistical J o u r n a l P r e -p r o o f significance was observed (Fig. 3E) . Moreover, no race-based differences were observed when NO levels were compared between control and/or COVID-19 groups in Caucasians vs AA.

We next compared subjects based on race for sulfide metabolites (Fig. 4A-D) . M, p<0.0001), while no significant changes were seen in the levels of BSS (Fig. 4G ). When sulfide levels were compared between Caucasian and AA controls, there was a significant reduction in free sulfide levels (0.310.08 M vs 0.250.08 M; p=0.04) in AA subjects. No significance was seen in other pools of sulfide in comparisons between these races in either the control or COVID-19 groups.

To determine NO-derived oxidants, we measured systemic levels of nitrotyrosine in the plasma from healthy controls and COVID-19 patients ( 

C-reactive protein (CRP) levels have been shown to be an early prognosticator in COVID-19 pneumonia and can indicate disease severity, whereas the gasotransmitters NO and H 2 S are known for their anti-inflammatory properties 17 . We measured nitrotyrosine, CRP as well as NO and H 2 S levels in a single subject who was initially a control subject, but 9 days later contracted a COVID-19 infection (Fig. 6 ). The subject's CRP levels, which were significantly elevated with 

We next performed ROC with sulfide and its metabolites by analyzing the AUC in healthy controls and COVID-19 patients. Free sulfide with an AUC of 0.8697 (95% CI-0.7878-0.9517, p < 0.0001) was a strong predictor of COVID-19 in the overall study population (Fig. 8A) . A free sulfide of 0.30 M or below had a sensitivity of 96% and a specificity of 33% of predicting COVID-19 infection and a level of 0.24 M or below had a sensitivity of 91% with a specificity of 67%. Total sulfide was also fairly able to predict COVID-19 infection with an AUC of 0.753(p<0.0001, Fig. 8B ). We further analyzed the accuracy of reduced sulfide levels as a predictor of COVID-19 based on race in Caucasian and AA subjects. We found that free sulfide was a powerful predictor of COVID-19 infection in Caucasians with an AUC of 0.915 (p<0.0001, (Fig. 8E, F ). With a free sulfide level of 0.30 M or below, the sulfide levels were able to predict COVID-19 with 95% sensitivity and 14 % specificity in AA.

Independent sample Student's t-tests were performed to study the association between biomarkers (cardiac injury, thrombosis, and inflammatory) and NO and H 2 S levels in the COVID-19 cases ( 96±60.40nM, p=0.11 ). The patients who had higher levels of cardiac, inflammatory, and thrombosis biomarkers had higher NO and H 2 S levels although most were non-significant (Table 3) .

We performed multivariable regression analysis to identify any association between comorbidities and total NO and sulfide levels ( Table 4 ). It is worth noting that we did not find any further association between NO and H 2 S levels and cardiovascular risk factors, including age, race, sex, diabetes, and hypertension (Table 4 ).

The gasotransmitters NO and H 2 S have overlapping pathophysiological roles with significant influence in regulating cardio-and vaso-protective functions and possessing anti-inflammatory, anti-thrombotic, and antiviral properties 7, 18, 19 20, 21 . While researchers have pondered the possible use of NO and H 2 S in the treatment of COVID-19, studies exploring the availability of these two gasotransmitters in COVID-19 patients are limited 11, 13, 14, [22] [23] [24] . For the first time, our study analyzed and compared both NO and sulfide metabolites in healthy subjects and COVID-19 patients and observed a significant and parallel reduction in both NO and sulfide metabolites in the COVID-19 patients compared to controls (Figs 1 and 2) . compared to non-infected controls 28 but did not include data from mildly ill COVID-19 patients.

In contrast, our study found significantly lower NO metabolites in patients with COVID-19

infections of different severities compared to controls. H 2 S is another gasotransmitter with antiviral properties that is cardioprotective, antiinflammatory, and antioxidant 8, 29, 30 . Considering its varied functions, it has been contemplated as a possible therapy in COVID-19 infection 11, 22, 31 . We have previously reported H 2 S availability as a predictive biomarker for cardiovascular disease in a race-and sex-based manner 16 . A recent study has suggested a correlation between the severity of SARS-CoV-2 infection, cytokine production, and H 2 S plasma levels 13 . H 2 S levels were significantly reduced in deceased patients compared to those who survived following COVID-19 infection, suggesting a possible role of H 2 S in the outcome of pneumonia caused by SARS-CoV-2 13 . However, that study was limited to COVID-19 patients with viral pneumonia and did not include non-infected controls. In a biological system, H 2 S can be present in various forms including free, acid labile, and bound sulfane sulfur that regulate and contribute to the total amount of bioavailable sulfide 16 . For the first time, we demonstrate that all of these sulfide biochemical forms are significantly reduced in COVID-19 patients compared to healthy controls (Fig. 2) . The interaction between H 2 S and NO can be complex and could range from synergism, based on evidence from the cardiovascular disease models 32, 33 to antagonistic regulation of each other found in inflammatory cells, 34 especially in pulmonary infections 35 . Our finding that both H 2 S and NO are reduced in COVID-19

infection simultaneously hints at a more synergistic role for these two gasotransmitters in this context.

There are known variations in NO and H 2 S levels based on race in vascular disease patients 16, 36 . ROC analyses with NO showed a significantly predictable relationship between COVID-19 and NO levels, including total NO, free nitrite, and SNO metabolites in all of the COVID-19 subjects, irrespective of race ( Fig. 6 A-C) . Interestingly, sulfide metabolites, especially total sulfide and free sulfide, were more predictive of COVID-19 infection than NO metabolites. ROC analysis of free sulfide showed that a free sulfide level of 0.30 M was 96% sensitive and 33% specific in predicting COVID-19 infection in the general population; >94% sensitive and 58% specific in the Caucasian population; and 95% sensitive and 14% specific in the AA population. Assuming a roughly 10% prevalence of COVID-19 infection in the United States, free sulfide levels of 0.30 M predicted COVID-19 infection with a positive predictive value (PPV) of 14% but a negative predictive value (NPV) of 99% in the general population and a PPV of 20% and a NPV of 99% in Caucasians, suggesting that higher free sulfide levels can rule out COVID-19 infection with certainty. The majority of the control population in this study was healthy and did not have significant comorbidities, while 25% of COVID-19 cases had CVD.

Previously, we have shown that while the levels of other sulfide metabolites in the plasma are decreased with cardiovascular disease, free sulfide levels are elevated in these patients 37, 38 Therefore, the finding that free sulfide levels are significantly reduced in and are the best J o u r n a l P r e -p r o o f predictors of COVID-19 infection in the COVID-19 cases with 25% CVD prevalence assumes prominence.

procalcitonin were associated with poor outcomes in COVID-19 infection 39 . We therefore analyzed the effects of various inflammatory and cardiovascular biomarkers on NO and H 2 S in COVID-19 patients (Table 3) . We saw a significant association between LDH and NO levels in COVID-19 infected subjects (Table 3) . Surprisingly, patients with LDH levels >260 U/L had higher total NO levels compared to patients with LDH levels 260 U/L. NO also showed a significant association with mortality, with increased NO levels in expired COVID-19 subjects compared to patients who survived. This is in agreement with the findings in the study by Alamdari et al. 28 Similarly, COVID-19 patients who were severely ill or expired had a significantly higher plasma H 2 S levels compared to patients who were mild-to-moderately ill or survived. Although the gasotransmitter levels were significantly reduced in COVID-19 patients compared to controls, it is unclear why sicker COVID-19 patients had relatively elevated levels compared to less sick patients. One possible explanation is that the elevated NO and H 2 S levels in sicker COVID-19 patients is a last-ditch compensatory response to the severely noxious effects of the COVID-19 infection. Another reason could be a hypothetical inability to utilize or underutilization of NO and H 2 S to reduce oxidative stress leading to poor outcomes. NO-derived oxidant generation can also reduce NO availability, thereby reducing its levels. Peroxynitrite is one such oxidant that promotes nitration of protein tyrosine residues such as nitrotyrosine. 40 We observed a significant increase in nitrotyrosine levels in the plasma of COVID-19 patients Comorbidities in COVID-19 patients may be associated with increased hospitalizations, complications, and mortality [43] [44] [45] . Therefore, we used multivariable regression analyses to find the association between the gasotransmitters NO and H 2 S and other risk factors (Table 3) in COVID-19 positive cases. Remarkably, there were no further differences in either NO or sulfide metabolites with patient demographics or cardiovascular comorbidities known to affect their levels, including age, race, sex, diabetes, and hypertension ( Table 4 ), suggesting that the effect of COVID-19 on these gasotransmitters was overwhelming, leaving no room for variations.

In summary, our findings reveal that the availability NO and sulfide metabolites is significantly reduced in individuals with COVID-19 infection but is not affected by comorbidities. In addition, reduced free sulfide levels have a high sensitivity in predicting COVID-19 infection in the study population regardless of race. Based on a case study within the cohort, inflammatory and oxidative stress markers CRP and nitrotyrosine, were inversely related to and NO/H 2 S availability with the onset of COVID-19 infection, which should be studied in a wider population. Tables   Table 1 : Demographics of COVID-19 cases and healthy controls included in the study. 

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Supplementary figure 1. Receiver-operating characteristic analysis of free nitrite by race

ROC curves with area under the curve of free nitrite -altogether (A)

Caucasian (B) and African American (C) populations respectively