Measuring exhaled nitric oxide: not only a matter of how--but also why--should we do it?

The biological functions of nitric oxide are so diverse and complex that it is now becoming increasingly difficult to delineate briefly the physiological roles and pathophysiological implications of this seemingly simple messenger molecule [1]. In respiratory medicine, NO can either be viewed as a paracrine factor (derived from endothelium, epithelium, nerves, inflammatory cells, etc), a therapeutic gas or a marker of inflammation [2]. Amongst other paracrine factors, the endothelium-derived NO has, in its own right, a central role to play in the modulation of pulmonary vascular tone [3]. As a gaseous molecule, NO has been extensively investigated in clinical settings and used as inhalational therapy to relieve pulmonary hypertension and/or refractory hypoxaemia in adults and infants [4]. Although there are many questions that still remain to be properly answered [5], the use of inhaled NO has undoubtedly revived interest in molecules that can selectively reduce both pulmonary vascular resistance and intrapulmonary shunt. As a radical molecule, NO is highly reactive and readily combines with an array of biological molecules, ranging from reactive oxygen species to haeme moiety containing proteins [6]. This explains why measurement of NO in biological systems was often fraught with difficulties in the early days [7]. Since 1991, however, measurement of in vivo NO production in humans have been proven to be technically feasible by means of ex vivo manoeuvres, i.e. by sampling the exhaled breath and analysing it for NO content using a chemiluminescent NO analyser [8]. As the technique is noninvasive, it was immediately applied to patients, especially those with bronchial asthma, to assess endogenous production of NO by the lung [9–11]. Soon, the accumulating evidence suggested that measurement of exhaled NO could be viewed as a new lung function test [12] to monitor airway inflammation in asthma [13] and other conditions associated with inflammation of the respiratory tract [14]. It is still difficult to know the actual source of the endogenous NO that is detected in the exhaled air [15]. As NO is synthesized by many lung cells, it could originate from virtually anywhere in the respiratory tract, from alveolar space to the nose. Several recent and carefully conducted studies have clearly shown how the techniques of measurement are likely to affect the amount and origin of exhaled NO [16– 20]. This prompted the European Respiratory Society, in 1997, to issue specific recommendations for the measurement of exhaled and nasal NO [21], an initiative which was followed in 1998 by the American Thoracic Society. In contrast with the ongoing technical debate on how to measure exhaled NO, it seems that a consensus has been reached on the diagnostic value of NO measurement in asthma. Compelling evidence clearly demonstrates that asthmatic subjects who are not treated with inhaled glucocorticoids have on average higher amounts of exhaled NO as compared with healthy controls [9–14, 18, 21]. The strength of the evidence, and hence the validity of the finding, is based on both practical and theoretical grounds. Higher levels of exhaled NO in asthma have been consistently found by several independent investigators using different techniques of measurement [18, 22, 23]. Theoretical considerations are also consistent with the raw data. NO is synthesized by a group of three enzymes, called NO synthases (NOS). Each isoform is differentially distributed in organs and tissues, with a preferential expression of the constitutive NOS I in neurons, the inducible NOS II in inflammatory cells and the constitutive NOS III in vascular endothelium [1]. All three isoforms have been detected in lung cells. In particular, the inducible NOS II is markedly expressed in asthmatic airways [24]. Unlike the constitutive neuronal NOS I and endothelial NOS III which synthesize NO only in minute amounts to meet physiological demands, expression of inducible NOS II by asthmatic epithelial cells leads to a massive synthesis of NO, thus explaining high levels of exhaled NO in asthma. Moreover, the inhibitory effect of glucocorticoid hormones on NO production [25–28] is supported by the molecular links between inducible NOS II, nuclear factorκB (NF-κB) (a key transcription factor in asthma [29]) and the genomic effect of the glucocorticoids [30]. Briefly, binding of the hormone to its intracellular receptor leads to the transcription of the inhibitory subunit (IκB) which normally impedes NF-κB binding to the promoter regions of inflammatory genes, including the gene encoding inducible NOS II [30]. It is probably true that exhaled NO is increased in asthma as a result of airway inflammation. There are several practical issues that can be inferred from this observation. Because NO production is increased in inflammatory diseases, measuring exhaled NO can therefore be viewed as a noninvasive, though indirect, means of detecting inflammation in the respiratory tract. Because NOS II is induced by several inflammatory cytokines (e.g. interleukin-1β, tumour necrosis factor-α, interferon-γ), monitoring exhaled NO is also an elegant way to assess the efficacy of antiinflammatory agents, assuming that the more potent the Service de Physiologie-Explorations Fonctionnelles, CHU Cochin, APHP & Université Paris V-René Descartes, Paris, France.