Critically ill patients often require admission to the intensive care unit (ICU). When patients develop organ failures and end up on a ventilator, there are changes in the body's cell function that can increase the risk of poor outcomes. All cells, in order to function normally, have mitochondria, which help them generate energy and transfer vital messages between cells. However, during critical illness, the mitochondria in the cells can function less effectively and die prematurely, or their new synthesis and regeneration can be severely affected. This can result in continuous multi-organ failure with a lack of recovery and muscle wasting, causing severe weakness and an inability to function normally.

In this study, the investigators aim to assess mitochondrial capacity using three methods with varying levels of invasiveness. The investigators are planning to recruit 20 patients in the ICU who are on a ventilator for breathing support. The investigators plan to measure mitochondrial capacity from a breath test, blood cells, and muscle cells.

The investigators will collect breath samples after consuming an amino acid, which is a component of protein in our body and is commonly found in food. This amino acid is only broken down by the mitochondria. This safe test allows us to measure how much mitochondrial capacity remains in the body after the modified amino acid is broken down by the mitochondria. In comparison, the investigators will use standard methods which includes blood tests and muscle biopsy to examine the mitochondrial function of platelets (blood cells) and muscle cells. The investigators will also use non-invasive techniques (ultrasound and 'MyotonPRO') to assess muscle.

This study will help us determine the best way to assess mitochondrial function and capacity in critically ill patients and to understand strengths and weaknesses of different approaches.

When patients' mitochondrial function or capacity is impaired, the investigators can provide them with particular nutrition to improve mitochondrial activity. Because evaluating this at the bedside is challenging, it is impossible to tell which patients may benefit from specific therapies that improve mitochondrial function. If this breath test provides an assessment similar to the standard, sophisticated mitochondrial testing, the investigators could use it at the bedside in the future, which may improve patient outcomes and help design large clinical trials.

Critical illness is associated with significantly increased risk of morbidity and mortality (Morgan, 2021). Patients are often admitted to the intensive care unit (ICU) with single or multiple organ failure of diverse aetiology (e.g., severe infection, inflammation, trauma). Multi-organ failure is related to cellular and bioenergetic dysfunction caused by hypoxia, hyperoxia and increased oxidative stress and despite organ support measures, the anticipated mortality is high with ~20-50% (Zambon and Vincent, 2008). Despite these negative consequences, therapeutic strategies are limited with nearly all ICU based clinical studies failing to identify treatments that improve clinical outcomes. The heterogeneity within the ICU patients coupled with the lack of phenotypic characterisation has contributed to this poor progress, highlighting the need to comprehensively characterise the ICU phenotype to develop effective interventions that improve clinical outcomes.

Mitochondria have vital functions beyond cellular energy metabolism, including regulating cell death, calcium homeostasis and modulation of the cell cycle. All of these functions have been shown to impact outcomes during critical illness with mitochondria exhibiting structural changes within days of being admitted to ICU (Klawitter et al., 2023). Despite appropriate ICU management with antibiotics, fluids, oxygenation and nutrition, sepsis- associated mortality remains high and this is thought to be due to persistence of inflammation and impaired mitochondrial processes (Supinski et al, 2020). Stressed mitochondria produce higher levels of reactive oxygen species, activating caspases, and triggering cell death. Following mitochondrial death, subsequent lack in ATP production has been suggested to lead to poor clinical outcomes. Mitochondrial death itself leads to poor clinical outcomes and a study in 2013 directly linked higher levels of circulating mtDNA to increased ICU mortality at 28-days post admission (Jameson et al., 2023).

Physical inactivity is strongly associated with alterations in mitochondrial dysfunction and consequently prolonged inactivity in ICU may lead to adverse outcomes. Studies show downregulation of key mitochondrial transcription factors in skeletal muscle even in early stages of critical illness (Klawitter et al., 2023). A recent randomised control trial concluded that there was persistent intramuscular inflammation in critical illness and demonstrated that exercise alone is insufficient to restore muscle function (Jameson et al., 2023). A defining feature of ICU patients is the rapid and substantial loss of muscle (Puthucheary et al., 2023), which significantly increases morbidity and mortality risk (key clinical outcomes of ICU) (Lee et al., 2021).

Associated with ICU induced muscle decline is mitochondrial dysfunction, characterised by reduced mitochondrial content and function (Puthucheary et al., 2018). However, where mitochondrial deficiencies occur (e.g., which complex of the electron transport chain), and thus which element of the mitochondria are best to target therapeutically to improve mitochondrial health remains' elusive. These fundamental questions can be addressed using sophisticated omic-informatic techniques; however, this system biology approach requires interdisciplinary expertise, which has stunted progress. Bridging this gap, the investigators have interdisciplinary expertise in applying advanced computational analysis to human biological samples, permitting the detection of molecular targets to improve muscle mitochondrial health (Deane et al., 2019; Deane et al., 2023; Deane et al., 2021). Thus, using our pipeline, it is possible to identify molecular regulators of, and promising interventional avenues for, improving mitochondrial health in ICU patients.

Monitoring mitochondrial function in patients is a bedside challenge, as it requires muscle biopsies followed by laboursome laboratory processing and analysis. Therefore, there is an unmet need to develop minimally or non-invasive methods to assess mitochondrial function to aid clinical decision-making processes. Addressing this research gap, the investigators have developed a novel isotope labelled non-invasive breath test to evaluate mitochondrial function rapidly and repeatedly, which has been validated in healthy subjects and in patients with non-alcoholic fatty liver disease (Afolabi et al., 2018).

Emerging pilot data from healthy and clinical (elective surgery) cohorts demonstrating our inhouse ability to perform mitochondrial functional assessment in muscle tissue and platelets using the Oroboros, and via the breath test using mass spectrometry. However, the correspondence between the measures and the applicability in ICU patients remains to be determined. Taken together and based on our pilot data, the investigators strongly believe it is feasible to take complementary mitochondria-focussed metabolic and molecular measurements in ICU patients, to better characterise ICU cohorts and identify future therapeutic avenues. By using a multitude of different strategies to assess mitochondrial capacity the investigators aim to better understand how it is affected in critical illness on both a systemic and tissue level.

One emerging and promising approach is the use of 13C-breath tests (13C-BTs) to characterise mitochondrial capacity. 13C-BTs involves the oral administration of a metabolic substrate labelled with a non-radioactive stable isotope of carbon (13C-atom), which is metabolised exclusively within the mitochondria and is followed by the recovery of the 13C-tracer on the breath as 13CO2. The 13C-substrate can be chosen to evaluate mitochondrial metabolism in the whole body. An example of a 13C-BT is the 13C-ketoisocaproate breath test (13C-KICA BT) which assesses mitochondrial function. It involves the oral ingestion of 13C-KICA, a metabolic intermediate of the branched chain amino acid leucine, which is metabolised in the mitochondria to 13C-labelled carbon dioxide (13CO2) which is recovered on the breath of the subject (Afolabi et al., 2018). This pathway is catalysed by the branched chain alpha-ketoacid dehydrogenase, which is in the mitochondria over a period of time. Thus, the generation of 13CO2 following the oral administration of 13C-KICA should reflect mitochondrial metabolism.

The ability to monitor changes in mitochondrial metabolism in patients with critical illness at an early stage is crucial, to develop effective approaches to treat patients early. This novel non-invasive method of assessing mitochondrial capacity has the potential to use at the bedside to inform treatment decisions.

There is an urgent need for practical, sensitive tests of metabolic function that can help to identify potential therapeutic targets in critical illness and quantify mitochondrial dysfunction.

Non-invasive 13C-breath tests such as the 13C-ketoisocaproate (13C-KICA) breath test can give a rapid quantitative measure of whole-body mitochondrial capacity providing invaluable information regarding the patients' metabolic function at any given point in time. Thus, allowing the evaluation of the response to targeted therapies.

Hypothesis of MitoICU: Mitochondrial capacity can be assessed in critically ill patients from multiple compartments (whole body and liver: 13C-KICA; cellular: platelets; and tissue: skeletal muscle) and the non-invasive measures (muscle ultrasound and MyotonPRO) may be used as surrogate for mitochondrial assessment in ventilated patients.

Inclusion Criteria:

• Adults ≥ 18 years
• Mechanically ventilated at time of recruitment
• Defined as critically ill by the responsible clinician
• Recruited within 48-hours of intubation
• Likely to remain intubated and ventilated for > 72-hours

Exclusion Criteria:

• Patients < 18 years
• Patient is being treated on an end-of-life pathway or active treatment is likely to be withdrawn within 24-hours
• Patient has significant liver dysfunction (Child-Pugh ≥ class 3)
• Patient is not absorbing enterally (defined as 2 x NG aspirates of > 500ml)
• Known pregnancy or positive urinary pregnancy test on testing
• Specific exclusion criteria for the muscle biopsy component of the study: Patients taking treatment dose anticoagulation, antiplatelet agents or with severe coagulopathy (or disease process leading to increased risk of bleeding) will be excluded from having a muscle biopsy taken as part of data collection.

Critically ill adult (>18 years) patients who have been mechanically ventilated will be recruited from the Intensive Care Unit at University Hospital Southampton.