Previous investigations have shown that aCOHb levels may be elevated in trauma and surgical patients [2,3], and in patients with inflammatory lung disease [4,5] and critical illness [6,7,8]. The presence and role of COHb is still debated in COVID-19 patients . The majority of ICU hospitalized COVID-19 patients experienced ARDS, vascular inflammation, thrombosis, and ultimately multi-organ damage. The triggering factors are likely multiple. Among them, excess of free heme is a potential offending agent as it has been shown to exacerbate and contribute to the pathogenesis of numerous inflammatory diseases . Therefore, it may be postulated that accumulated free heme and hemoglobin could be involved in the mechanisms increasing pulmonary permeability and inflammation in COVID-19 patients . Protective mechanisms against free heme include the heme oxygenases, which break down heme into equimolar parts of biliverdin/bilirubin, CO, and iron/ferritin. Neither admission nor peak aCOHb levels were predictive of outcome. The increase in aCOHb occurred with some delay and was significantly greater in the patients who were mechanically ventilated. Regarding the interpretation of [a-v]COHb, we noted this did not change over time and was similar among survivors and non-survivors. On the pooled data for [a-v]COHb, we found lower values (mean 0.2%) than those documented in a series of patients with inflammatory diseases who were tested while breathing air room (mean 0.5% [a-v]COHb) . This later series had suggested that the measurement of [a-v]COHb concentration differences could be a valuable marker to define the site of inflammation as this difference was more pronounced in patients with inflammatory pulmonary diseases in comparison with extrapulmonary inflammatory diseases .
The percentage of smokers was low in the present study but consistent with other COVID-19 studies. There was no difference at baseline following ICU admission in aCOHb levels among smokers and non-smokers. Therefore, it was assumed that exogenous CO did not play a significant role. It is also documented that aCOHb may vary following hemorrhage or hemolysis but these conditions were not encountered in our patients.
The main source of endogenous CO results from the metabolism of heme by heme oxygenase. There is a direct relationship between the aCOHb and the endogenous production of CO either in healthy volunteers or in clinical studies [6,13,14]. Changes in ventilator variables may also affect aCOHb concentration . Several factors may affect the elimination of the endogenously produced CO: CO lung diffusing capacity, alveolar ventilation, lung capillary oxygen pressure, aCOHb concentration, endogenous CO production and CO catabolism. The increase of the inspired fraction of oxygen will logically result in a transient increase in CO lung elimination as a result of a competition of CO and O2 for the same binding sites. In addition, an experiment conducted on healthy volunteers showed that inhalation of FiO2 1.0 increase endogenous exhaled levels by a factor of four . In this experiment, the most likely mechanism was CO displacement from hemoglobin, according to the very short duration of exposure to FiO2 1.0. This could be different in a setting a prolonged exposure to FiO2 1.0 following hypoxic conditions, with a possible over-expression of HO-1.
In the present study, changes in inspired oxygen fraction or ventilator settings occurred in almost all of the patients, particularly at the acute initial stage. As aCOHb was a mean of 4 to 8 daily values for each patient, we can estimate that these changes would have a minimal impact on CO lung excretion. On the other hand, aCOHb represents a balance between endogenous CO production and CO elimination from blood to lungs and extravascular tissues. It remains difficult to establish if the increase in aCOHb has to be ascribed to an increase of CO production or to an impaired CO elimination through the alveolo-capillary membrane. On the whole dataset, there was a correlation between aCOHb and PaCO2 in both survivors and non-survivors, but a strict parallelism between CO and CO2 pulmonary elimination is not expected. The present study confirms that the respective kinetic profile of aCOHb and PaCO2 varied over time, with a parallel increase in both aCOHb and PaCO2 for the patients who were mechanically ventilated < 20 days, and a discordant evolution over the subsequent period. This was particularly evident for some patients with a very long period of mechanical ventilation and in whom recovery of PaCO2 occurred while high aCOHb values were maintained for a longer period. The reasons for this discrepancy are not precisely known. Release of COHb from peripheral tissues to venous blood was not documented by vCOHb. Difference in diffusion capacity between CO and CO2 appears unlikely. A third hypothesis should be that induction of heme catabolism mediated by HO-1 could be prolonged following a sustained hypoxic or inflammatory stress.
Indeed, the heme-catabolizing enzyme heme oxygenase (HO)-1 is highly inducible in oxidative stress. Patients with acute respiratory distress syndrome are reported to have an increased expression of (HO)-1 . Arterial carboxyhemoglobin level was measured in a cohort of 1267 ICU patients mainly admitted after cardiovascular surgery. Both low minimum and high maximum levels of aCOHb were associated with increased intensive care mortality . Arterial carboxyhemoglobin levels also correlated with biomarkers of the inflammatory response. These data suggested that the failure to up-regulate the activity of the HO system in the presence of a pro-inflammatory stress may be associated with a worse prognosis, while excessive (HO)-1 induction may also affect negatively the outcome. In patients from a medical ICU, survivors had slightly higher minimal and marginally higher average aCOHb levels when compared to non-survivors .
The administration of exogenous CO has been proposed as therapeutic intervention in various conditions including acute lung injury [20-22]. Contrasting results have been published as some in vivo studies suggested that the endogenous production of CO or its exogenous administration was protective, while other studies were negative . In a human model of sepsis-related ARDS, inhalation of low doses of CO was associated with an increase in aCOHb ranging from 3.48 to 4.9%. No serious adverse events occurred in the CO-treated group, while circulating mitochondrial DNA levels were reduced .
Carbon monoxide can confer anti-inflammatory protection in rodent models of ventilator-induced lung injury (VILI). This modulation could be partly due to an increased expression of caveolin-1 . Among the drugs recently proposed to treat COVID-19 infection, the effect of dexamethasone remains debatable as it may induce HO-1 in macrophages in some experimental conditions . Induction of HO-1 can also been achieved by a large variety of agents, including aspirin, statins, probucol, valsartan, niacin, resveratrol, and curcumin. The possible role of inhaled nitric oxide (iNO) therapy on carboxyhemoglobin formation is poorly documented in humans [26,27]. While NO has been reported to induce HO-1 expression in various cell types, iNO is usually considered has having minimal systemic dissemination . In the present series, the patients who received iNO therapy should not be excluded from the analysis as it may be assumed that iNO therapy was applied to the most severe patients who were more prone to develop high aCOHb levels.