Non-invasive strategy in assessing asthma through biofluids metabolomics exploration: exhaled breath and urine potentialities

Asthma is a significant health issue in the pediatric population with a noteworthy growth over the years. The proposed challenge for this PhD thesis was the development of advanced methodologies to establish metabolomic patterns in urine and exhaled breath associated with asthma whose applicability was subsequently exploited to evaluate the disease state, the therapy adhesion and effect and for diagnostic purposes. The volatile composition of exhaled breath was studied combining headspace solid phase microextraction (HS-SPME) with gas chromatography coupled to mass spectrometry or with comprehensive two-dimensional gas chromatography coupled to mass spectrometry with a high resolution time of flight analyzer (GC×GC–ToFMS). These methodologies allowed the identification of several hundred compounds from different chemical families. Multivariate analysis (MVA) led to the conclusion that the metabolomic profile of asthma individuals is characterized by higher levels of compounds associated with lipid peroxidation, possibly linked to oxidative stress and inflammation (alkanes and aldehydes) known to play an important role in asthma. For future applications in clinical settings a set of nine compounds was defined and the clinical applicability was proven in monitoring the disease status and in the evaluation of the effect and / or adherence to therapy. The global volatile metabolome of urine was also explored using an HSSPME/GC×GC–ToFMS method and c.a. 200 compounds were identified. A targeted analysis was performed, with 78 compounds related with lipid peroxidation and consequently to oxidative stress levels and inflammation. The urinary non-volatile metabolomic pattern of asthma was established using proton nuclear magnetic resonance (1H NMR). This analysis allowed identifying central metabolic pathways such as oxidative stress, amino acid and lipid metabolism, gut microflora alterations, alterations in the tricarboxylic acid (TCA) cycle, histidine metabolism, lactic acidosis, and modification of free tyrosine residues after eosinophil stimulation. The obtained results allowed exploring and demonstrating the potential of analyzing the metabolomic profile of exhaled air and urine in asthma. Besides the successful development of analysis methodologies, it was possible to explore through exhaled air and urine biochemical pathways affected by asthma, observing complementarity between matrices, as well as, verify the clinical applicability.

Allergic asthma exhaled breath metabolome: a challenge for comprehensive two-dimensional gas chromatography

Allergic asthma represents an important public health issue, most common in the paediatric population, characterized by airway inflammation that may lead to changes in volatiles secreted via the lungs. Thus, exhaled breath has potential to be a matrix with relevant metabolomic information to characterize this disease. Progress in biochemistry, health sciences and related areas depends on instrumental advances, and a high throughput and sensitive equipment such as comprehensive two-dimensional gas chromatography–time of flight mass spectrometry (GC × GC–ToFMS) was considered. GC × GC–ToFMS application in the analysis of the exhaled breath of 32 children with allergic asthma, from which 10 had also allergic rhinitis, and 27 control children allowed the identification of several hundreds of compounds belonging to different chemical families. Multivariate analysis, using Partial Least Squares-Discriminant Analysis in tandem with Monte Carlo Cross Validation was performed to assess the predictive power and to help the interpretation of recovered compounds possibly linked to oxidative stress, inflammation processes or other cellular processes that may characterize asthma. The results suggest that the model is robust, considering the high classification rate, sensitivity, and specificity. A pattern of six compounds belonging to the alkanes characterized the asthmatic population: nonane, 2,2,4,6,6-pentamethylheptane, decane, 3,6-dimethyldecane, dodecane, and tetradecane. To explore future clinical applications, and considering the future role of molecular-based methodologies, a compound set was established to rapid access of information from exhaled breath, reducing the time of data processing, and thus, becoming more expedite method for the clinical purposes.