Towards standardisation in breathomics

Exhaled breath VOCs analysis is safe and non-invasive method of monitoring for human metabolic profiles and has the potential to become diagnostic tool in clinical practise. This thesis first describe in detail the different aspects of exhaled breath VOCs and its use as diagnostic tool in respiratory diseases. The current exhaled breath analysis work-flow including breath sampling, analysis and data processing is also described. A single exhaled breath sample can contain in excess of 500 different chemical species. There is a wide range of factors that can cause the variability to individual breath profiles. In order to detect small changes in breath profiles, a standardised and reproducible approach to exhaled breath analysis methodology is required. The long term storage of exhaled breath samples using multi-sorbent tubes is investigated, the optimum storage protocol and condition is discussed. A portable breath sampling system was also developed for remote sampling. The introduction of this new feature enables breath sampling to be carried out outside the designated laboratory with no location restriction. This feature combined with the easy to use and non-invasive original sampling unit designed for subjects with impaired lung function minimise participant stress level and discomfort. It also utilises the custom developed air supply filtration assembly to create a standardised purified breathable air that can minimise the method variability and improve standardisation to breath samples collected. This methodology is tested in an excise induced bronchoconstriction (EIB) study where two groups of participants: healthy and excise induced bronchoconstriction (EIB) positive undergo high intensity cardiopulmonary exercise testing (CPET). The data from two groups of participants is analysed and three markers which shown correlation with EIB positive participants are determined

Influence of cancer associated microbiome on volatile organic compound production in oesophago-gastric adenocarcinoma

Oesophago-gastric cancer is a significant health problem with poor prognosis in Western countries. This is due to a paucity of alarm symptoms in early stages of the disease resulting in late clinical presentation and associated delays in initiation of treatment. The development of non-invasive breath tests using exhaled Volatile Organic compounds (VOCs) to determine oesophago-gastric cancer risk would help facilitate earlier diagnosis and potentially improve patient survival. Whilst many of the biochemical pathways relating to the origin of these VOCs within humans are as yet unknown, it is postulated that that specific VOCs are produced directly by cancer tissues. Contributions from other endogenous sources including the intestinal microbiome and healthy tissues within the intestinal tract as well as other organ systems. The aim of this thesis was to understand the interaction between the upper gastrointestinal microbiome and VOC production in patients with oesophago-gastric cancer and to explore how this onco-microbial axis can be exploited to augment VOC production. The production of cancer associated VOCs (fatty acids and phenol) were investigated by analysing the ex vivo headspace above un-derivatised tissue samples as well as in vivo mixed breath, isolated bronchial breath and gastric endoluminal air. Increased concentrations of these VOCs were detected in the headspace of cancer tissue samples as well as isolated endoluminal air adjacent to tumours. Findings therefore implicate that the tumour and its local environment are the likely source of upregulated VOCs in oesophago-gastric cancer. The relative contribution of the tumour associated microbiome remains unknown. 16S RNA sequencing analysis for 185 oesophago-gastric tissue samples from cancer and control subjects were performed in order to assess the microbial diversity. Results revealed higher abundance of Firmicutes (e.g. Streptococcus salivarius, Escherichia coli and Streptococcus anginosus) in oesophago-gastric cancer samples compared to controls. The headspace of in vitro and patient derived (ex vivo) cultures of specific targeted bacteria was subsequently found to contain similar VOCs as those previously detected in oesophago-gastric cancer. To increase the sensitivity of breath testing, further work was performed to augment the diagnostic response using simple metabolic substrates (sugars, proteins, lipids). When added to in vitro cultures of cancer-associated bacteria, these nutrients resulted in upregulated VOC production. Oesophago-gastric cancer patients who were given the same substrates orally were found to have a transient rise in the same VOCs that was greater than observed in healthy controls. This thesis provides new insight into the biological origin of VOC production in oesophago-gastric cancer. Experiments linking the cancer-associated microbiome, exogenous substrates to upregulated VOC production in cancer patients offers the potential for a future augmented breath test for this disease. The augmented breath test is expected to increase earlier cancer detection leading to improvement in overall survival.Open Acces

Validation of volatile organic compounds for the assessment of liver disease

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.Open Acces

Prediction and Detection of Hypoglycaemia in Type 1 Diabetes

Patients with Type 1 Diabetes still suffer from recurrent and troublesome hypoglycaemia despite improvements in glucose monitoring, insulin delivery and structured education. The key objectives of my thesis were to explore ways to identify patients at greater risk to develop hypoglycaemia and to investigate novel ways to prevent hypoglycaemic episodes by detecting this early and with ease. This might ultimately translate into reduction in overall risk of hypoglycaemia occurrence in those with Type 1 Diabetes. I investigated potential factors that could predict persistent presence of severe hypoglycaemia in the HypoCOMPaSS study, a multi-centre UK study examining clinical strategies to reduce burden of hypoglycaemia. Looking at a subset of these participants who underwent detailed hypoglycaemic clamp studies, I found no obvious parameters predicting persistent risk of severe hypoglycaemia. I then examined whether individual genetic factors might contribute to risk of persistent severe hypoglycaemia, exploring the association between polymorphisms in Angiotensin Converting Enzyme ACE gene and severe hypoglycaemia in 77 participants in HypoCOMPaSS study. Interestingly, I found that the homozygous DD ACE gene polymorphism was associated with a significantly increased risk of severe hypoglycaemia. Considering then practical approaches to trying to minimise hypoglycaemia risk, I examined the efficacy and safety of a novel implantable Continuous Glucose Monitoring Senseonics CGM System in 10 subjects with Type 1 Diabetes from the Cambridge cohort of the pivotal European PRECISE 1 study. In keeping with the global data, I found in Cambridge participants that this novel system was safe and its efficacy was comparable to commercially available Continuous Glucose Monitoring. This device was useful in detecting hypoglycaemia early with high device satisfaction among the users. I also explored alternative non-invasive methods to measure blood glucose and detect hypoglycaemia easily using breath sample in Type 1 Diabetes participants during experimental hypoglycaemia. The concentration of Isoprene exhaled in breath was significantly raised during hypoglycaemia. Finally, I looked at the stability of ‘diluted insulin aspart (NovoRapid®)’ in ambient temperature and CSII over 30 days. Both neat and diluted insulin aspart were stable beyond 30 days and could potentially be used by patients with T1D requiring very low insulin doses to avoid hypoglycaemia.NIHR Cambridge Biomedical research centre, Diabetes UK, University of East Anglia Senseonics Inc. Dr Frank Waldron-Lync

Effect of abdominal binding on cardiorespiratory function in paralympic athletes with cervical spinal cord injury

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University on 4 July 2011.Spinal cord injury (SCI) causes a lesion-dependent impairment in cardiorespiratory function that may limit exercise capacity. The aims of this thesis were to describe cardiorespiratory function in highly-trained athletes with low-cervical SCI, and to investigate whether abdominal binding enhances cardiorespiratory function at rest and during exercise in this population. Using body plethysmography, bilateral phrenic nerve stimulation and transthoracic ultrasound, it was demonstrated that Paralympic athletes with cervical SCI exhibit a restrictive pulmonary defect, impaired diaphragm and expiratory muscle function, and low left ventricular mass and ejection fraction compared to able-bodied controls. Using the same methods, it was shown that abdominal binding improves resting cardiorespiratory function by reducing operating lung volumes, and increasing vital capacity, twitch transdiaphragmatic pressure, expiratory muscle strength and cardiac output. A further finding was a positive relationship between binder tightness and cardiorespiratory function. During a field-based assessment of fitness, abdominal binding reduced the time taken to complete an acceleration/deceleration test and increased the distance covered during a repeated maximal 4-min push test. During laboratory-based incremental wheelchair propulsion, abdominal binding altered breathing mechanics by reducing operating lung volumes and attenuating the rise in the pressure-time index of the diaphragm. Furthermore, abdominal binding increased peak oxygen uptake and reduced peak blood lactate concentration, despite no change in peak work rate. Peak oxygen uptake in the laboratory was related to the distance covered during the maximal 4-min push, suggesting that the improvement in field-based performance with binding was due to an improvement in aerobic capacity. In conclusion, this thesis demonstrates that abdominal binding significantly enhances cardiorespiratory function at rest, improves exercise performance in the field, and improves operating lung volumes, breathing mechanics and peak oxygen uptake during incremental treadmill exercise. Thus, abdominal binding provides a simple, easy-to-use tool that can be used to enhance cardiorespiratory function at rest and during exercise in highly-trained athletes with cervical SCI

A validation and feasibility study of the non-invasive measurement of oxygen delivery and consumption after elective major abdominal surgery.

Major surgery triggers a physiological stress response that results in an increase in post-operative metabolic demand and oxygen consumption (V̇O2), which must be met by an increased oxygen delivery (DO2). Historical studies described the increase in V̇O2 in patients after major surgery and presented evidence that the inability to meet this increase oxygen demand and the temporal pattern of this oxygen deficit appears to differ according to whether patients survive, or develop complications or not. The survival and complication profile of patients in modern practice is different from that previously described, And the methods employed in these historical studies were invasive and inconsistent with contemporary practice. V̇O2 can be measured non-invasively with indirect calorimetery, and DO2 calculated from non-invasive cardiac output monitors, and haemoglobin and oxygen saturation measurement devices. This thesis describes two prospective observational studies which1) validate and 2) assess the feasibility of non-invasive measurements of V̇O2 and DO2 and explore their temporal patterns after contemporary abdominal surgery. These techniques demonstrate moderate to good trending ability when measuring changes in V̇O2 and DO2. The non-invasive measurement of V̇O2 and DO2 is feasible in patients after major abdominal surgery. There appear to be distinct patterns of V̇O2 and DO2 after contemporary abdominal surgery in those who develop complications or not. Contemporary patterns of net cumulative oxygen debt appear to differ from those previously described.Plymouth Hospitals NHS Trus