Mitochondrial Assessment in Critical Ill Patients in Intensive Care

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.