Eugenio Aprea1, Filomena Morisco2,3, Paola Vitaglione3, Luca Cappellin1, Vincenzo Lembo2, Flavia Gasperi1, Giuseppe D’Argenio2, Vincenzo Fogliano3, Nicola Caporaso2, Franco Biasioli1
1Research and Innovation Centre, Fondazione Edmund Mach, via Mach 1, San
Michele all’Adige, Italy, franco.biasioli@fmach.it
2Gastroenterology Unit, Department of Clinical and Experimental Medicine,
University of Naples ‘‘Federico II’’ Naples, Italy.
3Department of Food Science, University of Naples ‘‘Federico II’’ Portici, Italy.
Abstract
The upgrade of PTR-MS from Quadrupole to Time of Flight mass spectrometer [1] represents a great advance in the analytical capability of PTR-MS technology. Due to higher mass range and higher mass and time resolution [2-3], ToF mass analyzers appears to be an ideal tool for breath monitoring. On the other hand the larger and more complex data generated requires great efforts to handle and extract the analytical information. We recently developed a full methodology from basic mass spectra handling to the application of up-to date data mining methods [4] which allows for extracting highly relevant features from large amounts of complex spectral data.
Here we present our recent experiences in breath monitoring using PTR-ToF-MS for non-invasive physiology monitoring and disease diagnosis. The first application is the monitoring of the effects of the diet on the physiology state in awake rats affected by non-alcoholic steatohepatitis (NASH) [5]. The second application is the implementation of a procedure for the diagnosis of liver cirrhosis and the assessment of disease severity in humans.
Application 1.
Rat suffering of NASH were subjected to 2 different diet regimes (standard or high fat). Since in a recent study was demonstrated that decaffeinated coffee reduces liver inflammation [6], water or decaffeinated coffee was administrated to the rats to verify if through breath analysis was possible to see also such effect. In the rats’ exhaled breath we found several spectrometric peaks that are reliable markers both for diet fat content or coffee supplementation. The high resolution and accuracy of PTR-ToF-MS allows the identification of related compounds such as methanol, dimethyl sulphide, dimethyl sulphone and ammonia most of them related to the metabolism of liver inflammation.
In conclusion the rapid and minimally invasive breath analysis of awake rats allows the identification of markers related to the pathologic conditions and the influence of the diet on it. The use of animal models for disease studies has the advantage to reduce the confounding factors induced by non standardized dietary regimens and life style. The proposed method can be used to monitor some parameters over a treatment period, and it can be applied for a wide range of pathologies and for studies on broader populations.
The exhaled breath of 14 healthy subjects (M/F 5/9, mean age 52.3, range 35-77 years) and of 12 patients (M/F 8/4, mean age 70.5, range 42-80 years) with liver cirrhosis of different class severity (The Child-Pugh class was A in 6, B in 3, and C in 3 patients) was analyzed. Real time breath analysis was performed using a buffered end-tidal (BET) on-line sampler coupled to a PTR-ToF-MS. Spectra were acquired using the data acquisition software TOF-DAQ (Tofwerk AG, Switzerland) with a mass/charge range of 10–400 Th. The data were analyzed by non- parametric ANOVA (Kruskal-Wallis test) using the Statistica 9.1 (StatSoft, USA) software. Eight compounds (2-pentanone, C8-ketone, 2 monoterpenes and 4 sulfur compounds) resulted significantly different in cirrhotic patients compared to healthy controls. Furthermore, a C8- ketone, a monoterpene and a NS-compound permitted the discrimination between Child-Pugh A and Child-Pugh B+C cirrhotic patients.
In conclusion, breath analysis allows to distinguish cirrhotic from healthy subjects and well compensated liver disease from more advanced liver stage. The proposed method can be used to identify the stage and severity of liver disease in real time with a safe and non-invasive procedure.
Final remarks and perspectives
With these two examples of applications of breath monitoring by PTR-ToF-MS we demonstrated the feasibility of an easy and fast on-line procedure for breath analysis applicable both in animals and humans. The PTR-ToF-MS allows the screening and monitoring of virtually hundreds of compounds, being capable of acquire a full spectrum in a split second, and thus not limited to few pre-selected trace ions. The analytical information entangled in the recorded spectra and extracted with ad hoc procedure brought to the identification of compounds related to liver disease and to the physiological state of subject as influenced by the diet. We believe that the new PTR-MS configuration will boost the research in the field of breath analysis for the identification of markers of diseases and will provide a useful tool for nutrigenomic investigations.
References
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[5] E. Aprea, F. Morisco, F. Biasioli, P. Vitaglione, L. Cappellin, C. Soukoulis, et al., Analysis of breath by proton transfer reaction time of flight mass spectrometry in rats with steatohepatitis induced by high-fat diet, Journal of Mass Spectrometry. 47 (2012) 1098–1103.
[6] P. Vitaglione, F. Morisco, G. Mazzone, D.C. Amoruso, M.T. Ribecco, A. Romano, et al., Coffee reduces liver damage in a rat model of steatohepatitis: The underlying mechanisms and the role of polyphenols and melanoidins, Hepatology. 52 (2010) 1652–1661.