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Digital archive of theses discussed at the University of Pisa


Thesis etd-03092017-131018

Thesis type
Tesi di dottorato di ricerca
Thesis title
Chemical biomarkers in human breath and oral fluid: Method development and applications in non-invasive clinical analyses
Academic discipline
Course of study
tutor Prof. Fuoco, Roger
correlatore Dott. Di Francesco, Fabio
  • Analytical methods
  • Biomarkers of heart failure
  • Exhaled breath
  • Monitoring of diseases
  • Oral fluid
Graduation session start date
Release date
Early detection plays a crucial role for the planning of treatment and the prognosis of diseases. The capability to evaluate the physiological conditions of a patient, trace the progression of a disease and monitor the efficacy of post-treatment therapies is a primary objective in healthcare research.
The analysis of blood, tissues or urine is a common approach to obtain clinical information, but this procedure entails drawbacks. In particular, the collection of the first two specimens is invasive and brings a significant stress for the patient, especially if repeated sampling is needed, whereas urine sampling is simple but with less controllable in frequency. Alternative specimens such as exhaled breath and oral fluid have gained importance as potential sources of clinical information because they reflect the biological activity as well as the health status, and they can be easily and unobtrusively collected. The possibility to obtain clinical information in a non-invasive way and without risk for the patients represents one of the most important goal of health monitoring. The analysis of these alternative matrices thus represents an interesting approach, especially for the screening of a large population.
The aim of this work was to develop and validate analytical methods for the quantification of specific biomarkers in human breath and oral fluid, and to apply them for monitoring of disease such as heart failure and psoriasis and the environmental exposure to toxic substances.
In a first study, an analytical method was optimized for the chemical characterization of exhaled breath based on two-stage thermal desorption capillary gas chromatography coupled to mass spectrometry. The method was used to monitor the exposure of hospital personnel to sevoflurane, a widely used anaesthetic, and isopropanol, a disinfectant.
In addition to these molecules, the determination of eighteen volatile compounds belonging to different chemical classes of environmental concern (i.e. hydrocarbons, ketones, aldehydes, aromatics, tio-compounds and esters) was also possible, which suggests the possible use of exhaled breath analysis for the monitoring of subjects exposed to environmental contaminants or patients suffering from specific disease.
In fact, in a second study, several analytical methods were developed and validated to quantify salivary biomarkers related to the clinical status of patients suffering from heart failure. Heart failure is a rapidly diffusing chronic cardiovascular disease and the main cause of mortality and poor quality of life in western societies. Aldosterone, cortisol and 8-iso-Prostaglandin F2 (8-isoPGF2) were determined in oral fluid by liquid chromatography coupled with tandem mass spectrometry, tumor necrosis factor- and interleukin-10 by enzyme-linked immunoassays, uric acid by liquid chromatography with ultraviolet detection, lactate by liquid chromatography with fluorescence detection, and salivary -amylase enzymatic activity by a spectrophotometric method. Each biomarker is representative of a pathological condition occurring with the heart failure syndrome, so that abnormal levels of these molecules could be associated with the severity and progression of the disease. The methods were then used in the framework of the H2020 HEARTEN Project and to support the development of specific biosensors able to anticipate critical exacerbation episodes by rising an alarm in hospital information systems remotely connected by an mHealth application.
The third research was focused on the development and validation of suitable analytical method for the determination of stress-related biomarkers in oral fluid, to investigate the response of the two primary neuroendocrine systems, namely the autonomic nervous system and the hypothalamic-pituitary-adrenal axis, whose main biomarkers of activation are salivary -amylase and cortisol, respectively. In this case, a spectrometric method was used for the determination of salivary -amylase enzymatic activity and a Reversed-Phase High-Performance Liquid Chromatography coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry method for cortisol. These methods were then applied to investigate the responsiveness of psoriatic patients to stressors. Psoriasis is a chronic and inflammatory skin disease characterised by a significant psychological distress and psychiatric morbidity, experiences of stigmatization, and decreased quality of life. The aetiology of psoriasis has not been fully understood yet, but it appears to be multifactorial, involving both genetic and environmental influences. Among these factors, emotional stress is considered to play an important role in the onset and exacerbation of the disease. The clinical application of the methods, even if in a limited number of psoriatic volunteers, showed potential to clarify the role of cortisol and salivary -amylase enzymatic activity and the mechanisms of activation of the two biological systems in response to a stressor. Moreover, information was achieved on their play in these mechanisms. The results suggested that the quantification of salivary stress-related biomarkers could be used to assess the effectiveness of stress reduction strategies in the management of psoriasis.