## 3 The Metabolites of EB Volatiles
### Nitrogen Containing Compounds  
#### 3.3.1 Nitric Oxide (NO)  
NO is the most important nitrogen compound in terms of volatile EB biomarkers, being produced by different types of cells in the respiratory tract, such as inflammatory, epithelial, and vascular endothelial cells and airway nerves [205]. NO has one unpaired electron, which makes it highly reactive towards  
Figure 4: Metabolism of acetone and its precursors in liver and muscle. other molecules. It has a half-life of around 1-5 seconds and diffuses freely across membranes. These properties make NO an important transient signalling molecule working both within a single cell (autocrine) and between adjacent cells (paracrine) [206]. In fact, NO modulates the endothelium vasodilitation activity and for this reason is also termed as the endothelium-derived relaxing factor, \(\mathrm{EDRF}\). It is used by the cells to send signals to the surrounding smooth muscle to relax, thereby, causing vasodilation and blood flow increase. This physiological activity is influenced by several factors, as the levels and activity of the enzymes involved in NO production, its uptake rate by antioxidant molecules (glutathione and haemoglobin for instance), and the levels of oxidative stress [207]. Additionally, NO can be generated in the phagocytes (macrophages, neutrophils, and monocytes) that use it against pathogens (human immune response). Phagocytes express an inducible NO synthase (iNOS) that is regulated by interferon-gamma (IFN-\(\gamma\)) and tumour necrosis factor (TNF) (activators) [208, 209, 210], transforming growth factor-beta (TGF-\(\beta\)) (strong inhibitors) and the interleukin-4 (IL-4) and IL-10 (weak inhibition). This tight regulation of iNOS reflects the importance of NO in inflammation and immune responses [211]. In fact, NO is distinctively observed in the bronchial system and is reported to be a marker for airway inflammation [154]. This assumption results from extensive studies in different lung diseases, particularly those in which NO metabolism is altered. Regarding this, [PERSON] _et al._, for instance, suggested that high NO levels are present in the asthmatic airway environment, which is oxidative in nature. This further leads to an increase in the generation of reactive nitrogen species (RNS) and consequent protein nitration and oxidation. Ultimately, these modifications adversely affected protein functions, contributing to chronic inflammation [212]. Therefore, NO, through the measurement of its fractional concentration in exhaled breath (FENO), has become an important example of an exhaled biomarker that has reached clinical practice, particularly in the management of asthma [213, 214]. This pulmonary complication is characterised by recurrent and reversible airflow obstruction with different degrees of severity [215] and FENO helps not only in its diagnosis, as well as in the discrimination of its phenotypes and treatment monitoring, [216, 217], being a surrogate marker of eosinophilic airway inflammation and good predictor of corticosteroid response [218]. Overall, FENO constitutes a valuable diagnostic tool for a reliable clinical guidance in asthma treatment, particularly when its values are low [219]. Consequently, several devices using chemiluminescent and electrochemical sensors able to accurately measure FENO are already present in the clinical environment and other approaches, using quantum cascade lasers, for instance, are being developed [220, 221]. Chronic obstructive pulmonary disease (COPD), is characterised by a generalized inflammation that causes airways obstruction and, consequently, breath shortness [222]. In this case, NO has been correlated to the severity of COPD and higher levels are found in unstable COPD patients [223]. Similarly, promising FENO discriminative variations between patients and controls are observed in other complications besides COPD and asthma, including cystic fibrosis (CF), systemic sclerosis, hepatopulmonary syndrome and primary ciliary dyskinesia [224, 225].