Assaying all of the nitrogen oxides in breath modifies the interpretation of exhaled nitric oxide
Introduction
Exhaled nitric oxide (NO) has received extensive attention in the literature as a marker of airway inflammation. However exhaled NO does not reliably correlate with inflammation in all settings. Indeed, in the highly inflamed lungs of some patients (such as those with cystic fibrosis), exhaled NO is commonly paroxysmally lower than in healthy controls (Grasemann et al., 1997). Despite this important limitation, exhaled NO has become a valuable clinical tool, primarily because our reticence to biopsy the lung during acute illness (which sharply contrasts to diseases of most organs) has left us with very little knowledge of lung disease. Non-invasive methodology, including measurements of exhaled gases and solutes in exhaled breath condensate therefore are increasingly relied upon to compensate for our unwillingness to biopsy the lung.
When exhaled NO is measured, what is actually being assessed is not NO production in the lung, but rather the net output of various NO production mechanisms minus NO consumption by numerous pathways in the entire airway. The NO production occurs not only by means of the three nitric oxide synthase (NOS) isoforms (Gaston et al., 1994), but also by nitrite (NO2−) protonation in acidic fluids with subsequent decomposition to NO (Hunt et al., 2000, Lundberg et al., 1994), as well as homolytic cleavage of S-nitrosothiols (SNO) (Gaston et al., 1998). Nitrate reduction by bacteria increases levels of the precursor NO2− (Gaston et al., 2002). Nitrite and SNO serve as storage pools of NO bioactivity (although the SNO more importantly are signaling molecules in their own right through nitrosonium (NO+) chemistry, unrelated to the NO radical) (Gaston et al., 1993).
The amount of exhaled NO is log orders lower than the amount of NO formed in the lungs. Before it is exhaled, the gas is reacting away within a complex nitrogen redox ecology. Nitric oxide consumptive processes include oxidation of NO to the higher nitrogen oxides (“HiNOx”) which include NO2−, nitrate (NO3−), peroxynitrite (OONO–), peroxynitrous acid (OONOH), and nitrotyrosine, as well as reaction with thiols to form SNO, and with iron centers such as in hemoglobin. Further, NO can be enzymatically reduced by prokaryotic and fungal enzymes in the airway, forming hydroxylamine (NH2OH) and ammonia (NH3) (Gaston et al., 2002).
The net gas phase NO that is exhaled will be increased by upregulation of the NOS isoforms or certain chemical conditions that lead to NO release from higher oxides (such as acidic conditions in the airway), or augmented bacterial NO3− reductive activity. The measured exhaled NO will be reduced by the consumptive processes, importantly reaction with oxidants (such as superoxide, hydroxyl radical and others).
To some extent, the NO that is oxidized and trapped as HiNOx can be assessed in the exhaled breath also. Although many compounds are difficult to identify in exhaled breath condensate, NO2− and NO3− are almost always readily within the range of the chemiluminescent assays (Hunt et al., 1995, Hunt et al., 2000, Balint et al., 2001, Corradi et al., 2003a, Loukides et al., 2001, Csoma et al., 2003, Gessner et al., 2003). Given the focus on exhaled NO as an inflammatory marker, it is intriguing to consider that a human being may exhale more HiNOx than they do NO, mole for mole. In this investigation, we examined HiNOx in the context of the exhaled breath to determine if the clinical testing of exhaled NO (gas phase) might be improved if the HiNOx were concurrently evaluated.
We therefore performed concurrent testing of exhaled NO and collection of exhaled breath condensate (EBC) for measurement of the HiNOx in both controls and in patients with asthma. We performed calculations of the NO and HiNOx production per hour.
We identified that, mole for mole, HiNOx contribute substantially to the total nitrogen oxides exhaled, and that disease states can substantially affect their ratios. The changes in ratios seem to reflect differences in oxidative processes in the airway.
Section snippets
Subjects
All subjects were non-smokers. Patients with asthma, currently clinically stable and not actively exacerbating, were enrolled from the outpatient clinics at the University of Virginia. Control subjects were enrolled from the same clinics, but presenting for non-respiratory, non-acute issues. All subjects read and signed informed consent prior to participating, and the study was approved by the University Human Investigation Committee.
Tests of lung health
Spirometry was performed with an SPG portable spirometer (SDI
Results
All patients tolerated all aspects of the study without adverse events of any kind. Subjects with asthma were 18.0±10.8 years old, with age ranging from 5 to 45 (n = 27, 17 female). Thirteen of these patients were taking inhaled steroids at the time of breath sampling. Two subjects were receiving a leukotriene antagonist (one was on both classes of medication). Control subjects were 19.9±8.4 years old, with age ranging from 6 to 39 (n = 17, 10 female), p = 0.62. There was a trend to lower mean %
Discussion
Exhaled NO certainly has achieved prominence as a simple non-invasive marker of lung disease. But it should be recognized that what one assesses when one measures NO in the exhaled breath is simply that: the NO in the exhaled breath. Although exhaled NO correlates with inflammation in certain population groups, one always needs to consider the possibility that it might not correlate in the patient sitting in front of the doctor. Consider patients with cystic fibrosis, who certainly have marked
Acknowledgement
This research was funded in part by the United States National Institutes of Health grants 1R01 HL69170-01 and R01 HL72429-01. JH notes that he is a co-founder of the company that manufactures the breath condensate collection equipment used in this study.
References (25)
- et al.
Nitrate in exhaled breath condensate of patients with different airway diseases
Nitric Oxide
(2003) - et al.
Nitric oxide metabolites are not reduced in exhaled breath condensate of patients with primary ciliary dyskinesia
Chest
(2003) - et al.
Bronchodilator S-nitrosothiol deficiency in asthmatic respiratory failure
Lancet
(1998) - et al.
Exhaled breath condensate nitrite and its relation to tidal volume in acute lung injury
Chest
(2003) - et al.
Condensed expirate nitrite as a home marker for acute asthma [letter]
Lancet
(1995) - et al.
Nitrotyrosine formation in the airways and lung parenchyma of patients with asthma
J. Allergy Clin. Immunol.
(1999) - et al.
Acid stress in the pathology of asthma
J. Allergy Clin. Immunol.
(2004) Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children—1999
Am. J. Respir. Crit. Care Med.
(1999)- et al.
Increased hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients
Eur. Respir. J.
(1997) - et al.
Increased nitric oxide metabolites in exhaled breath condensate after exposure to tobacco smoke
Thorax
(2001)