Bronchiolitis obliterans syndrome (BOS) is the main complication that occurs after lung transplantation, affecting up to 60% of recipients within 5 years after surgery and leading to death. Patients undergo routine bronchoscopies to monitor their conditions through the analysis of bronchoalveolar lavage fluid (BALf), retrieved from lungs. Since this biofluid is still poorly characterized from the point-of-view of its content in metabolites, its metabolomic profiling could permit to recognize promising candidates for effective therapy of BOS. For this work, thirty samples were selected from lung transplant recipients, some of them being stable and others suffering from BOS, and the remaining samples from patients with other respiratory diseases. To carry out a high throughout analysis of BALf, two different complementary techniques have been applied. The rationale for this decision was that the use of complementary methodologies would offer the advantage of obtaining further confirmed and therefore reliable data. While the GC-MS system is more suitable for the detection of volatile and semi-volatile compounds, on the other hand NMR spectroscopy is able to accurately identify polar analytes, preventing sample destruction and can be easily repeated. The results of each analysis would have been compared, identifying the most promising metabolites. Unfortunately, the analysis through GC-MS did not achieve reliable results and, for this reason, only those obtained through 1H NMR spectroscopy have been considered in my work. 38 polar metabolites, mainly including branched amino acids (isoleucine, leucine, and valine), alanine, threonine, tryptophan, lactate, pyruvate, acetone, acetate, ethanolamine, taurine, and urea have been identified. As expected, some were strongly involved in cellular energy processes, such as pyruvate, lactate, and acetone, while others were more surprising, such as taurine/hypotaurine. We try to explain those metabolic alterations, in the attempt to describe a complex picture where many players could be still unknown to our knowledge. The choice to use both GC-MS and 1H NMR spectroscopy represents a novelty in the effort to characterize a biofluid still largely unknown, moreover in the context of a specific pathology such as BOS, restricted only to patients who undergo lung transplant.
La sindrome nota come Sindrome da Bronchiolite Obliterante (BOS) è la principale complicanza riscontrata in seguito al trapianto di polmone e colpisce fino al 60% dei pazienti nei primi 5 anni dopo l'operazione, provocando danni irreversibili all'organo che portano alla morte. Per monitorare le condizioni di salute, i pazienti vengono sottoposti frequentemente al lavaggio broncoalveolare per ottenere campioni da analizzare per verificare l'insorgenza di eventuali infezioni. L'analisi metabolomica del fluido da lavaggio broncoalveolare potrebbe fornire importanti indicazioni riguardo l'identificazione di markers potenzialmente utili per migliorare le prospettive di vita dei pazienti e per instaurare una terapia. Il lavoro descritto nella mia tesi riguarda trenta pazienti sottoposti a trapianto, alcuni stabili ed altri affetti da BOS ed altri campioni provenienti da pazienti affetti da altre patologie respiratorie che sono serviti per confrontare i diversi profili metabolomici. Per questa analisi sono state utilizzate due differenti tecniche, seppur complementari, in modo da ottenere risultati affidabili. La gas cromatografia seguita dalla spettrometria di massa (GC-MS) consente di individuare composti volatili e semivolatili; la spettroscopia di risonanza magnetica nucleare (NMR) è una tecnica che identifica principalmente composti di natura polare. I dati ottenuti in seguito all'analisi con la GC-MS non sono risultati tuttavia conclusivi e, per questa ragione, il mio lavoro si è concentrato su quelli ottenuti dalla NMR. Sono stati identificati 38 metaboliti polari, di cui i principali sono aminoacidi ramificati (isoleucina, leucina e valina), alanina, treonina, triptofano, lattato, piruvato, acetone, acetato, etanolammina, taurina e urea. Se alcuni sono coinvolti in processi energetici della cellula, altri invece, come la taurina, sono inaspettati. La scelta di utilizzare due diverse tecniche per caratterizzare il fluido da lavaggio broncoalveolare dal punto di vista del suo metaboloma rappresenta una novità che offre la possibilità di identificare metaboliti che possono essere considerati potenziali candidati per capire i meccanismi della malattia in modo più profondo e, forse, per migliorare le terapie disponibili nel trattamento della BOS.
Analisi metabolomica del fluido da lavaggio broncoalveolare di pazienti affetti da Sindrome da Bronchiolite Obliterante (BOS) per identificare potenziali marker della patologia
GALAVOTTI, LUDOVICA
2019/2020
Abstract
Bronchiolitis obliterans syndrome (BOS) is the main complication that occurs after lung transplantation, affecting up to 60% of recipients within 5 years after surgery and leading to death. Patients undergo routine bronchoscopies to monitor their conditions through the analysis of bronchoalveolar lavage fluid (BALf), retrieved from lungs. Since this biofluid is still poorly characterized from the point-of-view of its content in metabolites, its metabolomic profiling could permit to recognize promising candidates for effective therapy of BOS. For this work, thirty samples were selected from lung transplant recipients, some of them being stable and others suffering from BOS, and the remaining samples from patients with other respiratory diseases. To carry out a high throughout analysis of BALf, two different complementary techniques have been applied. The rationale for this decision was that the use of complementary methodologies would offer the advantage of obtaining further confirmed and therefore reliable data. While the GC-MS system is more suitable for the detection of volatile and semi-volatile compounds, on the other hand NMR spectroscopy is able to accurately identify polar analytes, preventing sample destruction and can be easily repeated. The results of each analysis would have been compared, identifying the most promising metabolites. Unfortunately, the analysis through GC-MS did not achieve reliable results and, for this reason, only those obtained through 1H NMR spectroscopy have been considered in my work. 38 polar metabolites, mainly including branched amino acids (isoleucine, leucine, and valine), alanine, threonine, tryptophan, lactate, pyruvate, acetone, acetate, ethanolamine, taurine, and urea have been identified. As expected, some were strongly involved in cellular energy processes, such as pyruvate, lactate, and acetone, while others were more surprising, such as taurine/hypotaurine. We try to explain those metabolic alterations, in the attempt to describe a complex picture where many players could be still unknown to our knowledge. The choice to use both GC-MS and 1H NMR spectroscopy represents a novelty in the effort to characterize a biofluid still largely unknown, moreover in the context of a specific pathology such as BOS, restricted only to patients who undergo lung transplant.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/13491