Exhaled nitric oxide as a marker of lung injury in coronary artery bypass surgery

N. Marczin1, T. Kövesis1, D. Royston1, B. H. Cuthbertson2, S. A. Stott2 and N. R. Webster2

1 Harefield, UK 2 Aberdeen, UK

Editor—We read with interest the publication by Cuthbertson and colleagues1 concerning exhaled nitric oxide as a marker of lung injury in coronary artery bypass surgery. We are particularly interested in this work.

We have been pursuing a similar area of research and published a series of observations in this field regarding exhaled nitric oxide and lung injury in the setting of heart surgery and transplantation during the last 5 yr.29 In addition, the work of Cuthbertson and colleagues appears to support our opinions expressed in the editorial: nitric oxide as mediator, marker and modulator of lung injury.10

We find that the potentially important article by the Aberdeen group contains intriguing data that require further clarification. This is related largely to the methodology, which appears to have important effects on the data obtained and its interpretation. As this is largely attributable to influence of ventilation parameters on exhaled nitric oxide concentrations, we feel it important to point out and initiate discussions within the anaesthetic community on this important aspect of exhaled nitric oxide research.

At the heart of the matter is the reported baseline value of exhaled nitric oxide levels (in parts per billion, p.p.b.) in patients undergoing coronary surgery after induction of anaesthesia. The median of baseline exhaled nitric oxide—obtained by a methodology thought to allow sampling of end-expiratory alveolar gas—appears to be close to 60 p.p.b. However, we believe that this value is nearly 10-fold higher than that which has been obtained in normal human conducting airways, and 50–100 times greater than the scientific community believes are alveolar concentrations of gas phase nitric oxide.

Several groups have confirmed the presence of nitric oxide in the lower airways by direct and indirect methods. Perhaps the most convincing evidence comes from studies utilizing bronchoscopy for detection of gaseous nitric oxide concentrations in the main and lower airways. These studies confirm the concept that, because of its high diffusion coefficient, nitric oxide released to the gas phase from its production site in the fluid phase equilibrates quickly along the airways, leading to relatively uniform nitric oxide concentrations in the gas phase from the trachea to the segmental bronchi.11 12 As alveolar gases cannot be sampled by this direct method, it is estimated from end-expiratory gas concentrations. Such studies employing various ventilation manoeuvres surprisingly suggest that alveolar gases appear to contain near zero concentrations of nitric oxide.12

One of the early observations in the exhaled nitric oxide field was the extreme flow dependency of measured concentrations of exhaled nitric oxide.13 This recognition precipitated important research to explain the flow dependency. It led to recommendations by both the European Respiratory Society (ERS) and the American Thoracic Society (ATS) to standardize this measurement in both adults and children.14 15 These recommendations take into consideration nitric oxide contamination from upper airways and take account of the flow dependency by performing the measurement at a constant expiratory flow rate in spontaneously breathing and cooperative patients. With these single exhalation profiles, normal and pathological levels of exhaled nitric oxide measured at the mouth have been established, which correlate well with nitric oxide concentrations measured by bronchoscopy at the level of bronchi. These data suggest that normal levels are below 15 p.p.b. and higher levels in patients with respiratory symptoms represent airway inflammation with a 90% specificity and a 95% positive predictive value for asthma.16

The main problem with measuring exhaled nitric oxide in artificially ventilated patients, is that standard expiratory flow cannot be guaranteed and thus, because of the flow dependency of nitric oxide concentrations, the changing expiratory flow rate produces a continuously variable concentration of nitric oxide in the gas phase. We, and Brett and Evans, independently recognized this problem in ventilated patients and overcame this by calculating mean, peak and area under curve nitric oxide values using standardized ventilation.29 17 Figure 1, utilizing real-time measurement of gas phase nitric oxide, shows that this is absolutely crucial, not only because contamination of inspired air with nitric oxide might confound exhaled nitric oxide values, but more importantly, because ventilatory frequency, tidal volume, inspiratory:expiratory (I:E) ratio, and PEEP have significant influence on measured nitric oxide concentrations. In other words, as we have pointed out; exhaled nitric oxide values in ventilated patients are meaningless without a report of the exact ventilator settings and measurement conditions.7



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Fig 1 Influence of ventilation variables on exhaled nitric oxide and carbon dioxide concentrations. (A) The influence of increased minute volume by increasing tidal volume (VT) at a constant ventilatory frequency of 10 bpm. (B) The influence of increased minute volume by increasing ventilatory frequency at constant tidal volume (5 ml kg–1). (C) The effects of altered I:E ratio on flow patterns (top traces), exhaled nitric oxide and carbon dioxide.

 
While mean exhaled nitric oxide concentrations in ventilated control patients seem to be in the normal range established by the single exhalation technique in spontaneously breathing healthy volunteers, several groups of investigators, including Cuthbertson and colleagues, utilized different approaches and gained information about exhaled nitric oxide in the absence of ventilation. Thus, the question arises as to what the characteristics and determinants of exhaled nitric oxide are in the absence of ventilation (i.e. breath-holding).

Figure 1 suggests that reducing either tidal volume or ventilatory frequency appears to increase measured concentrations of nitric oxide. We have extended these measurements to an effective end-expiratory and end-inspiratory breath hold. Figure 2 demonstrates representative traces of more than ten patients. It suggests that nitric oxide continues to accumulate in the main airways during both types of breath-holding, reaching about 10-fold higher concentrations than those obtained with normal ventilation at end-expiration. This observation might explain why the Aberdeen group obtained similarly high concentrations of gaseous nitric oxide, 15 s after temporary disruption of ventilation. However, two questions remain. First, what are the kinetics of this increase in exhaled nitric oxide, and does this represent, at any given time, alveolar concentrations of nitric oxide?



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Fig 2 Representative traces exhibiting (A) end-expiratory and (B) end-inspiratory breath-holding manoeuvres to evaluate main airway and alveolar concentrations of gaseous nitric oxide and carbon dioxide in patients undergoing open heart surgery. The flow pattern is included for reference (top of panel): downward traces show inspiratory flow rate whereas the upward trace depicts expiratory flow.

 
In similar breath-holding studies in spontaneously breathing patients, Dweik and colleagues12 found that there was an increase in nitric oxide that could be described with exponential curve fitting. These studies and our data suggest that a plateau is reached 20–30 s after initiation of breath hold. This highlights the importance of timing of gas sampling in studies like the Aberdeen study, where instead of real-time data, manual sampling is performed. The plateau phase can be explained by a steady state between nitric oxide production/release to the gas phase, and consumption/removal from this phase. Many studies suggest that, in the normal lung, the major mechanism of nitric oxide production/release is the contribution of bronchiolar epithelium, whereas the most obvious explanation for removal of nitric oxide is by pulmonary blood flow via the alveoli.4 5 12 In this case, however, alveolar concentrations may be much less than main airway concentrations during breath-holding. True alveolar concentrations can be estimated in these experiments of end-inspiratory breath-holding by releasing the inspiratory pause. The first ventilator manoeuvre will be a spontaneous expiration by the patient (see flow trace) whereby true alveolar air will mix with gases accumulated in the conducting airways during breath-holding. Figure 2 shows that this true alveolar gas delivers additional carbon dioxide to the airways causing a further increase in measured carbon dioxide concentrations. In contrast, measured nitric oxide concentrations rapidly decline to near zero, suggesting that airway gases are now diluted with alveolar gas that must contain negligible concentrations of nitric oxide. Thus, the kinetics of nitric oxide are similar to end-expiratory breath-holding experiments, where the first manoeuvre after breath-holding is inspiration by the ventilator delivering oxygen 100%, free of nitric oxide, causing dilution of both nitric oxide and carbon dioxide, thus rapidly reducing measured concentrations of both of these gases (Fig. 2, top panel). These data, obtained in ventilated patients, fully support the observations of Dweik and colleagues regarding alveolar concentrations of nitric oxide.12

Thus, we have explained the huge discrepancy among the reported levels of exhaled nitric oxide by the Aberdeen group and us, and others using standardized ventilation methods. We have also provided strong evidence that these levels are unlikely to bear a relationship to alveolar nitric oxide content, the site which is implicated in acute lung injury. Where do we go from here? Can exhaled nitric oxide be utilized to monitor acute lung injury?

A variety of models, ranging from cell culture to lung transplants, have demonstrated consumption of nitric oxide in lung cells and in human studies, providing a clear answer half a decade ago.2 3 5 6 10 17 However, the more interesting question remains regarding the mechanisms of these changes, and whether exhaled nitric oxide could also be used to detect subclinical and developing lung injury. We have suggested that the reason why exhaled nitric oxide was decreased in lung injury, was epithelial injury or increased oxidative stress in the vicinity of bronchiolar epithelial cells. However, this can be complicated by increased production of nitric oxide via cytokine induced upregulation of nitric oxide synthases in certain stages of inflammation. Thus, additional measurements are needed to investigate the role of fluid-phase reactions, various nitric oxide metabolites, and different compartments of the lung. In this regard, we have suggested use of the microvascular metabolism of nitric oxide donor drugs such as nitroglycerine, which produce increases in exhaled nitric oxide, as a potential means of utilizing exhaled nitric oxide to detect microvascular changes in lung injury.7 8

Nevertheless, the study by Cuthbertson and colleagues is potentially significant because it suggests that basal exhaled nitric oxide measurement can detect developing lung injury at the bedside. However, this notion again should be interpreted with caution. First, although a weak relationship was found between the oxygenation index and exhaled nitric oxide, this relationship was not true for all patients involved in the study. Second, although not reported with clarity, it appears that only a small percentage of the oxygenation data would fulfil the criteria of acute lung injury. Third, none of these patients went on to develop clinically significant lung injury. In fact, this would argue against the predictive value of exhaled nitric oxide in this setting. Finally, the time course in their paper suggests that the greatest decrease in exhaled nitric oxide occurred in the period before cardiopulmonary bypass, which raises important questions, and is not easily explained by taking into consideration the pathophysiology of bypass surgery.

It is our view that the international research community should be encouraged to establish a consensus regarding non-invasive methods such as exhaled nitric oxide, carbon monoxide, volatile compounds, and breath condensate measurement to determine the value of these promising techniques in evaluating established and developing lung injury, and pulmonary involvement of systemic disease in ventilated patients. Tracheal intubation offers unique opportunities to sample the lower airways and alveolar compartment with appropriate methods, repeatedly, and with only minimal or no compromise to critically ill patients. While this represents an extraordinary potential to learn more about the pathophysiology and actual status of disease progression, mechanical ventilation also represents unique challenges in data collection and interpretation. These efforts should lead to convincing studies that can form the basis of an international consensus, and which should lead to recommendations by the ERS and ATS regarding these techniques in ventilated patients. Hopefully, our recent efforts to assemble the ERS/ATS Task Force on this topic will be successful in fulfilling this goal.18 In the mean time, the Aberdeen group should be congratulated for making a considerable contribution to this endeavour.

N. Marczin

T. Kövesi

D. Royston

Harefield, UK

Editor—Thank you for the opportunity to reply to the letter from the Harefield group. Marked published differences between the magnitude and direction of exhaled nitric oxide values in various models of lung injury make this a fascinating and slightly confusing field. The reasons for the high exhaled nitric oxide concentrations found in our study seem to be explained, in part, by methodological issues related to sampling and measurement of exhaled gas, as raised in this letter. What we had attempted to achieve was an easily obtainable and relatively easily quantifiable measurement technique, which would be clinically useful in the early stages of acute lung injury and acute respiratory distress syndrome.

As the authors suggest, there are clearly some issues which require further study. We would agree that consensus and standardization of measurement techniques for nitric oxide estimation in expired gases is required. Efforts to assemble an ERS/ATS task force to tackle these important issues are to be commended and encouraged.

B. H. Cuthbertson

S. A. Stott

N. R. Webster

Aberdeen, UK

References

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2 Marczin N. NO and oxidant stress in human lung ischemia–reperfusion (I/R). In: Boros, ed. Proceedings of the 37th Congress of the European Society for Surgical Research. Bologna: Monduzzi Editore, 69–74

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