Mechanisms of oxygen toxicity in the context of mitochondrial dysfunction

Background: Oxygen is one of the most important elements in the atmosphere, and is essential required for production of the body's main source of energy (adenosine triphosphate or ATP). ATP is produced by cellular structures called mitochondria through a process called oxidative phosphorylation (OXPHOS), which consumes oxygen. It has recently been observed that high oxygen levels can be acutely toxic in patients with faulty mitochondria. A similar situation has been observed in mouse models with mitochondrial dysfunction, but the mechanism involved is not yet understood. We propose that a 'block' in the OXPHOS pathway leads to a build-up of oxygen in the tissues, limiting the amount of energy that can be produced. Adding more oxygen can potentially exacerbate the situation and cause patients' symptoms to worsen. It is important for us to understand the mechanisms behind this oxygen toxicity to ensure that individual patients are treated appropriately without causing any further harm.

The aim of this project is to learn more about the underlying reasons for oxygen toxicity in two experimental groups:

Group 1: Patients with rare inherited mitochondrial disorders are known to have a specific defect in OXPHOS, which affects energy production in the brain and skeletal muscle. We predict that inhaling 55% oxygen will lead to reduced energy production in the patients with mitochondrial dysfunction, but not in healthy age-matched volunteers.

Group 2: Patients with Traumatic Brain Injury (TBI). TBI is often associated with impaired mitochondrial function. Studying the effects of oxygen in this group of patients will help the interpretation of our results in a broader medical context. We will compare TBI patients who have impaired mitochondrial function to patients who have TBI with no impairment of mitochondrial function.

Approach: We will test our theory in humans by comparing the effects of regular room air (roughly 20% oxygen) and high level oxygen (55%) inhaled for 1 hour. We will use the following measures:

1. Functional magnetic resonance (MR) scanning will let us measure energy production and mitochondrial function inside the body in real time in the brain and muscle.
2. A non-invasive probe will be used to measure blood oxygen levels and analyse how well oxygen is being taken up and utilised by the tissues (oximetry).
3. Brain and tissue oxygen and metabolism levels will be measured by a technique called microdialysis in the TBI patients. This group already have brain tissue oxygen sensors in place, which allows us to directly measure oxygen delivery and metabolism within the brain.
4. Blood samples from all participants before and after oxygen inhalation will be taken to analyse blood cells for molecules called biomarkers, which can give further clues about the mechanisms involved in oxygen toxicity, and link the two study groups.

Importance: Studying these parameters in both the body and the blood will guide the development of clinical biomarkers of oxygen toxicity, and will influence new approaches regarding the delivery of oxygen in the clinical setting. These findings will impact on the use of oxygen in rare mitochondrial diseases, and potentially influence the clinical management of common disorders including sepsis, critical illness, stroke, trauma and myocardial infarction.

Background: Recent pre-clinical studies indicate that high oxygen levels are toxic in the context of mitochondrial dysfunction, but the underlying mechanisms are not known. The prevailing hypothesis is that impaired mitochondrial oxidative phosphorylation (OXPHOS) reduces cellular oxygen consumption, leading to local tissue hyperoxia. In this context, further oxygen will compromise adenosine triphosphate (ATP) synthesis.

Approach: We aim to test this hypothesis in humans, comparing room air (20% oxygen) to 55% oxygen in two complementary disease groups: (1) Rare inherited mitochondrial disorders to provide proof-of-principle; and (2) Traumatic Brain Injury (TBI) to show broader medical relevance. Our prediction is that inhaling 55% oxygen for one hour will lead to decreased ATP synthesis in both contexts, but not in controls.

Our primary in vivo measure will be ATP synthesis measured in brain using 31-phosphorus magnetic resonance spectroscopy (31P-MRS). We will also measure brain N-acetyl aspartate (NAA) by 1H-MRS as an established marker of neuronal mitochondrial function; and tissue oxygen delivery and extraction using non-invasive oximetry. In the TBI group we will also perform in situ tissue microdialysis and brain tissue oxygen sensors allowing direct measurement of brain oxygen levels, oxidative and glycolytic metabolism. In both groups we will carry out linked to ex vivo exploratory measurements in lymphocytes and serum which we anticipate will provide a mechanistic link.

Importance: The parallel evaluation of ex vivo measures and non-invasive monitoring will guide the development of clinical biomarkers of oxygen toxicity, enabling the design of new management approaches matching oxygen delivery (tissue oxygenation) to local consumption (modulating mitochondrial function).

The results of this research will have impact in two inter-related areas - both clinical and academic. The primary impact will be delivered through healthcare services affecting treatment given to patients with both rare and common diseases. We anticipate this work will lay the foundations for future clinical guidelines, influencing public policy delivered by the NHS and health service sectors worldwide. This will be implemented through standardised protocols for oxygen use in the context of mitochondrial impairment, including the development of new approaches to evaluate the impairment of oxidative phosphorylation in specific tissues.

The primary potential impact is likely to be for clinical medicine, but will inevitably have academic benefit. Cross-disciplinary interaction between clinicians, basic scientists and radiography teams will encourage future collaboration; with any resulting publications benefitting the MRC-MBU and Cambridge Biomedical Research Centre as a whole (see academic beneficiaries section for full details). There is not likely to be any financial gain for the researchers involved as there is not any scope for commercialisation or intellectual property. This research is primarily focused on improving the outcome for patients in the long term.

Therefore, the main beneficiaries of this research will be:

1. Patients with rare inherited mitochondrial diseases. If our hypothesis is correct, we anticipate showing that high flow oxygen will be detrimental in patients with primary disorders of the mitochondrial respiratory chain. When combined with preclinical data and clinical case reports, this will immediately suggest that patients with these disorders should only receive oxygen therapy when absolutely necessary and, when required, this should be done in a carefully titrated manner. This research will have direct impact on the clinical management of patients receiving oxygen therapy, and offers the opportunity to improve patient outcomes through the careful balance of oxygen delivery.

2. Patients with common medical disorders. These findings will also have implications for the management of a diverse group of common medical disorders, including traumatic brain injury, sepsis, critical illness, stroke, trauma, myocardial infarction and cardiac arrest. If our hypothesis is correct, these findings will provide the evidence base for careful monitoring of oxygen delivery in traumatic brain injury, and open the door for similar mechanistic studies in several common disease states, including sepsis and ischaemia. The data provided by this study will underpin larger scale studies in all of the above contexts to refine the clinical guidance when using oxygen therapy.

3. Researchers investigating the fundamental biology of oxygen sensing in the context of respiratory chain impairment.
The linked mechanistic studies will cast light on the underlying biology of oxygen sensing in the context of respiratory chain impairment. This is a fundamental cellular pathway of broad relevance for cell homeostasis. Beneficiaries in this context are likely to be academic in the short-term, but could have therapeutic implications for other diseases known to be linked to oxygen sensing and metabolism, including cancer and ischaemic states.

We will complete a draft impact statement for each sub-study which will be submitted to the Cambridge University REF Impact Repository to allow efficient reporting in the future Research Evaluation Framework. As part of our ongoing pathways to impact we will continue to monitor for impact outside the academic sector over the next five years.