This thesis aims to introduce and describe the importance of small molecule volatiles as biomarkers, and both their present and future roles in disease diagnostics and basic research. A few examples of these molecules are introduced in more detail, including the medical conditions they are related to, as well as the advantages and challenges concerning their analysis. The biomarkers discussed in this thesis are hydrogen cyanide (HCN), ammonia (NH3), nitric oxide (NO), hydrogen sulfide (H2S), acetone (C3H6O) and methane (CH4). The volatility of these molecules makes them suitable for breath analysis, which is described thoroughly in this thesis. The diagnostic potential of breath analysis, as well as its comparability to traditional analysis techniques, such as blood and urine analysis, are also discussed. Measurement of small-molecule volatiles from the headspace of bacterial cultures is also introduced and described in detail, and its importance considering the understanding of bacterial activity and metabolism discussed further. Volatile biomarkers in human breath and produced by bacteria are thus the main themes of this work.
This thesis also describes a laser based, cavity-enhanced absorption technique that can be used to measure volatile, small-molecule biomarkers, such as those mentioned above. The measurement technique in question is cavity ring-down spectroscopy (CRDS), and its main features, as well as its advantages and limitations are discussed in detail, with special emphasis on features advantageous in breath analysis and bacterial headspace measurements. Sampling of the bacterial headspace and breath gas are introduced thoroughly. Special emphasis is given to the custom-built sampling line constructed in our laboratory, and it is used as a descriptive example when discussing the handling of gaseous samples. The measurement setups developed in our laboratory for the headspace measurements of both aerobic and anaerobic bacteria are also introduced in more detail.
The experimental part of this thesis describes the bacterial headspace measurements done with the custom built CRDS instrument and sampling setup constructed in our laboratory. The aim of this experimental demonstration was to investigate, whether some oral bacteria are able to produce detectable amounts of HCN in vitro, identify these bacteria, and monitor their HCN production as thoroughly as possible. Oral bacteria capable of producing HCN could possibly affect the concentrations measured from human exhaled breath, and this should be taken into account in the HCN breath analysis. In the future, HCN could also be considered a possible biomarker for oral pathogenic bacteria responsible for periodontal diseases, if these bacteria are able to produce HCN in detectable amounts. Another aim of this experimental demonstration was to prove that our suggested measurement setup is suitable for both anaerobic and aerobic bacterial headspace measurement, and that it offers reliable, consistent and reproducible results. We were able to prove that certain oral anaerobes from Porphyromonas, Prevotella and Fusobacterium genera can produce detectable levels of HCN in vitro, which is a novel finding. We also observed a strong correlation between the HCN and carbon dioxide (CO2) productions of Porphyromonas gingivalis, which indicates a connection between bacterial metabolic activity and HCN production. Measurements done for the positive control produced similar results to those observed in previous studies, which demonstrates that our proposed system can be applied in the screening and evaluation of HCN production by both aerobic and anaerobic bacteria. In addition, results from duplicate and triplicate measurements were consistent with each other, further indicating that our proposed measurement and sampling setup are valid.
