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Title: Oxidative stress and metal homeostasis at the air-lung interface in Chronic Obstructive Pulmonary Disease
Author: Leong-Smith, Phe
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2012
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Abstract:
Background: Chronic obstructive pulmonary disease (COPD) represents a spectrum of disorders encompassing chronic bronchitis and emphysema, associated with cough, excess mucus and exercise-related dyspnoea and characterized by a progressive reduction in airflow. Development of these symptoms is associated with chronic exposure to noxious particles or gas, most commonly from tobacco smoking, which triggers abnormal inflammation in the lung. Consistent with this increased inflammatory burden, oxidative stress has been demonstrated in COPD patients, largely through determination of antioxidant and oxidative damage marker concentrations in exhaled breath condensate and induced sputum. Whilst these samples are easier to obtain than bronchoscopy-based lavage, there remains contention concerning how well they reflect the distal airway lining fluids and hence the actual disease state. In the present study, I therefore investigated the oxidative status of bronchoalveolar lavage fluids (BAL) from well defined groups of COPD patients; current and ex-smoker, as well as aged and smoking matched controls. In addition, as COPD has been argued to be a disease of accelerated ageing a group of young controls was included to examine the extent to which age influences the endpoints under consideration. Chapter 2: In the first experimental chapter I investigated the evidence for oxidative stress in the airways of subjects with COPD (smokers and ex-smoking) relative to age and smoking matched controls. In addition, young healthy non-smokers and mild asthmatics were included to investigate the impact of age on the examined parameters, as well as to compare antioxidant defences in the context of acute and chronic inflammation. In this chapter, I examined respiratory tract lining fluid antioxidants sampled in bronchoalveolar lavage fluid, focusing on low molecular weight antioxidants (glutathione, urate and ascorbate) and their oxidation products (glutathione disulphide and dehydroaspresence) of COPD, but observed smoking-related increases in glutathione and ferritin, and age-related increases in dehydroascorbate and 4-hydroxy-2-nonenal. Chapter 3: In the second experimental chapter I attempted to understand the age-related increase in oxidation markers observed in the lavage samples from chapter 2. Pro-oxidant metal (Fe and Cu) concentrations were determined in the lavage samples from each of the groups used in chapter 2, by inductively coupled plasma mass spectrometry and a novel assay was developed based on metal catalysed oxidation of ascorbate, to provide a functional measure of the catalytically active metal pools at the surface of the lung. Through the use of selective chelators, the relative contribution of labile Fe and Cu pools was assessed. In this chapter I was able to demonstrate that respiratory tract lining fluid Cu concentrations increased with age, in parallel to an increased pro-oxidant status in the RTLF and evidence of a non-transferrin bound Fe pool. These indices appeared related to the concentration of oxidation markers reported in chapter 2. Chapter 4: Given the failure to detect increases in gross measures of oxidative damage in COPD patients, it was decided that the focus should shift toward a more refined focus on specific oxidations to proteins functionally related to the pathogenesis of the disease. I decided based on the pre-existing literature, that there was merit in focusing on 4-hydroxy-2-nonenal adduction of proteins. To achieve this, I attempted to develop a mass spectrometry-based method for the identification of adducted proteins and protein sequences. This was an ambitious undertaking and unfortunately I was only able to take these experiments so far, but at least was able to demonstrate the feasibility of the method. The methodology developed was sufficiently promising to warrant further investigation after the completion of my PhD. Short conclusion: In this study I was unable to demonstrate any evidence of oxidative stress in the airways of patients with COPD, either ex- or current smokers. I did however, observe evidence of increased oxidative damage, catalytic metal (Cu) concentrations and pro-oxidant metal activities (Cu and non-transferrin bound Fe) with age. At the end of this thesis, despite the attempt to investigate markers of oxidative stress in a relevant compartment (the distal lung) and in carefully controlled groups (aged and smoking matched), the role of oxidative processes in COPD remains oblique.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.628205  DOI: Not available
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