Metabolomics of equine Exhaled Breath Condensate (EBC) in healthy horses and horses affected by equine asthma

Objectives - Metabolomics can be defined as the analysis of the small molecules, called metabolites, representing the end products of interactions between genes and the environment. This rapidly developing approach shows considerable potential in veterinary pharmacology, toxicology, and clinical diagnostics and therapeutics1 . The most common biofluids used in metabolomic analysis applied to clinical and nutritional studies are blood and urine, however other biofluids like exhaled breath are gaining popularity. Exhaled breath condensate (EBC) can be obtained without adverse effects in children, patients with serious respiratory diseases2 and animals3-5. Although some studies have been performed on EBC in horses [3-5] this is the first report on EBC metabolomics performed in equine species. Because of the increasing interest towards these kind of specimens in people with respiratory disease6 , we aimed to investigate the metabolomic profile of EBC in healthy horses and horses affected by equine asthma. Materials and methods - Six clinically healthy horses (1 male, 3 geldings, 2 females), mean age 19±3 years, mean body weight 423±17 Kg, with no history of respiratory disease were included in Group H. Six horses (1 male, 2 geldings, 3 females) showing different degrees of respiratory symptoms (cough, dyspnea), having a known history of equine asthma, mean age 13±4 years, mean body weight 449±30 Kg, were included in Group A. EBC samples were collected by means of a face mask, with two non-rebreathing valves, connected via tubing to a condensation chamber, having a one-way valve on the top, immersed in an ice block at 15±5 °C. EBC samples were collected and stored at -80°C until metabolomic analysis, performed by 1 H-NMR7 . Endoscopic examination of the upper airways and cytological examination of tracheal wash samples (TW) were also performed to validate clinical findings in both groups. The Wilcoxon test was applied to look for changes in metabolites concentrations between groups, with a significance limit of p<0.05. Results - Endoscopic and cytological findings highlighted the presence of airways inflammation in Group A compared to Group H. The metabolomic analysis of equine EBC allowed the quantification of 8 molecules, namely methanol, ethanol, formate, trimethylamine, acetone, acetate, lactate and butanone. Higher methanol concentrations were found in group A compared to Group H. Conclusions - Little is still known about the genesis of exhaled breath volatile organic compounds (VOCs), even though some are thought to be endogenous, produced by the body as end-products of metabolic pathways [8]. Notwithstanding the differences between human and equine physiology, we could find some of the most common molecules observed in human EBC6, 8, 9. In particular, we found higher methanol concentrations in horses affected by asthma. Similar increase in methanol concentrations were observed by Maniscalco et al.9 in EBC of humans with chronic obstructive pulmonary disease (COPD). Interestingly, methanol is metabolized to formaldehyde, which shows a pro-inflammatory action and exacerbates airways inflammation in alveolar and bronchial cells and in animal models9 . Although presented in a pilot study, our results showed that metabolomic analysis of EBC could represent a potential diagnostic tool in horses with asthma.