Validation of volatile organic compounds for the assessment of liver disease

Abstract

Chronic liver disease is one of the few conditions with increasing morbidity and mortality rates. Up to 75% of individuals with cirrhosis are diagnosed during a decompensation episode, at which point the prognosis is poor. Cirrhotic patients also have an annual risk of 2 to 4% of developing hepatocellular carcinoma (HCC). HCC is currently the fourth leading cause of cancer-related mortality worldwide, which is at least in part due to late diagnosis and inadequate screening. Gas chromatography-mass spectrometry (GC-MS) analysis of volatile organic compounds (VOCs) in breath has the potential to form the basis of a non-invasive diagnostic test for chronic liver disease and HCC. However, exhaled VOCs can be influenced by multiple confounding factors and the equipment used to collect and analyse breath can be cost prohibitive.

The aims of my PhD were four-fold. Firstly, to develop and validate a novel, cost-effective breath collection device and to formulate a standard operating procedure for its use in clinical studies. Secondly, to analyse the VOC profile of background room air within common clinical sampling locations and to assess their potential impact upon the collection of breath samples. Thirdly, to investigate a methodology for sample splitting using GC-MS as a way of facilitating sample analysis across multiple mass spectrometry platforms. With the information garnered from this methodology work, my final aim was to perform a clinical study to profile the VOCs in the exhaled breath of patients with cirrhosis, HCC, and normal liver parenchyma. Prior to this, I also performed a critical analysis of the pre-existing literature on VOCs for assessment of liver disease to help guide my study design.

Analysis of the novel breath collection device revealed acceptable repeatability for a wide range of VOCs and optimum settings for flow rates and volumes of breath were determined and included within a standard operating procedure. Profiling the background air volatiles in sampling locations identified specific VOC signatures for each location. Breath samples did not separate by location but monitoring of background volatiles in parallel to breath sampling remains important for identification of contaminant VOCs. Splitting of desorbed breath samples via GC-MS and recollection of two samples back on to one thermal desorption tube provides the best discrimination between samples. For my main clinical study, breath samples of 149 patients were analysed using GC-MS. Elevated levels of limonene and 2-pentanone were identified in those with hepatopathology, validating the results of previous studies. Additional VOCs were also discovered as candidate biomarkers and further studies are required to validate these findings.

The results of my clinical study have added to the existing literature that specific VOCs in exhaled breath have the potential to form a non-invasive diagnostic test for hepatopathology that could potentially help enhance earlier diagnosis of liver disease and reverse the trend in mortality rates.