*3.4. Summary of Benchmark Human Samples to Study Mitochondrial Bioenergetics*

There is growing evidence that impaired mitochondrial function contributes to aging and age-related diseases. Age-related impairments in mitochondrial function include decreased OXPHOS capacity and ATP synthesis and well as increased ROS generation and accumulation of mtDNA mutations and deletions [111–113]. Therefore, monitoring mitochondrial function could provide a useful clinical tool for both diagnosis and monitoring the effectiveness of treatments. To accomplish this, there is a need for a less invasive way to monitor mitochondrial function than testing primary tissues. While fibroblasts provide an important research model that can be used for disease diagnosis and pre-clinical research, this approach is not suitable for clinical testing and monitoring due to poor scalability. Muscle biopsies are optimal for bioenergetics measurements, nevertheless collecting these samples is often considered as a last resort due to availability and invasiveness [42]. Therefore, other minimally invasive samples are needed to provide a translational approach to monitoring mitochondrial respiratory function. There is growing evidence that respirometry measurements in circulating blood cells may be a systemic biomarker of mitochondrial bioenergetic function. Blood-based bioenergetics present a model that is minimally invasive to obtain and more feasible to test in clinical settings and on a larger scale in clinical trials.

#### **4. Blood-Based Bioenergetics—Respirometry as a Systemic Biomarker of Mitochondrial Function**

Mitochondrial content, substrate preference, protein composition, and mitochondrial morphology vary between tissues based on energetic and metabolic demands. Therefore, there are clear advantages to measuring mitochondrial function in the tissue of interest, which is the approach commonly used in basic research. However, primary tissue samples can be difficult to obtain in human subjects and the amount of sample can be limiting for downstream bioenergetic applications. Therefore, functional measurements of mitochondria cannot always be used in translational research or to identify impairment in

mitochondrial function clinically, even if it would be a useful readout. While there is differential mitochondrial function between tissues, there is also evidence that circulating cells can act as a biomarker for overall mitochondrial function. Using platelets and leukocytes as a less invasive biomarker of mitochondrial function has emerged as a potential approach for situations when testing primary tissue samples is not feasible. These cells represent an attractive systemic biomarker candidate and an alternative to invasive tissue biopsies in human studies, since blood can be easily obtained in sufficient quantities for respirometry assays and other bioenergetic measurements. Additionally, isolated blood cells can be cryopreserved for later testing if the cells need to be isolated at an off-site location and shipped for respirometry analysis. Cryopreservation allows for samples that are collected at different times to be stored and subsequently run together in the same experiment, which may cut down on variability. However, long-term cryopreservation can also decrease cell viability and impair mitochondrial bioenergetic parameters and should be considered when using this approach [114].

Protocols for measuring respirometry in circulating blood cells have been well established for both intact and permeabilized cells and previously described [115–117]. Oxygen consumption measurements in circulating blood cells are compatible with any respirometry platform. Given that platelets and leukocytes are non-adherent, they are well suited for measurements in oxygraph closed-chamber respirometers, but they can also be tested in plate-based respirometers by coating plates to promote cell adhesion. The primary advantage of using a plate-based respirometer, such as the Seahorse Extracellular Flux Analyzer, is minimizing the amount of sample required for measurements, which is about ten times less in the Seahorse XF96 compared with an oxygraph [118].

Tyrrell et al. have compared respirometry in permeabilized muscle fibers to intact platelets and monocytes in healthy vervet/African green monkeys [119]. Their data demonstrate that the maximal respiratory capacity of circulating blood cells correlates with the maximal OXPHOS capacity of permeabilized skeletal muscle from the same animal. Both platelet and monocyte maximal oxygen consumption displayed a significant correlation with (1) the combined CI and CII-linked OXPHOS (State 3 respiration) in permeabilized skeletal muscle fibers and (2) the respiratory control ratio in isolated skeletal muscle mitochondria. Comparisons of skeletal muscle bioenergetics and circulating blood cells have also been completed in human subjects [120]. Braganza et al. show significant correlations between bioenergetic parameters in platelets and permeabilized skeletal muscle fibers from the same subject. Maximal respiration in platelets significantly correlated with maximal respiration in muscle and platelet proton leak correlated with State 4 (leak) respiration in muscle. Furthermore, basal and ATP-linked respiration was decreased in older subjects compared with a younger cohort. However, not all studies demonstrate correlations between circulating blood cells and muscle fibers. In a study of 32 women, Rose et al. did not observe a correlation in intact respirometry in peripheral blood mononuclear cells (PBMCs) or platelets with permeabilized muscle fibers, though there were some correlations with permeabilized platelets and permeabilized muscle [118]. Taken together, these results suggest that bioenergetic profiling of platelets and leukocytes may be used as a biomarker of mitochondrial function and, in some cases, reflect the bioenergetic capacity of muscle tissues and potentially other highly metabolic primary tissues.

Pecina et al. could identify defects in CI in permeabilized lymphocytes isolated from pediatric patients, some with known mitochondrial disorders, using high-resolution respirometry [121]. The authors used a combined approach testing functional methods and protein analysis and were able to detect several types of isolated OXPHOS disorders. They suggest that lymphocytes are a suitable sample to detect isolated defects of CI, CIV, and ATP synthase and that repeated analysis of lymphocytes could allow for monitoring changes in mitochondrial function during disease progression. Additionally, changes in platelet and leukocyte mitochondrial respiratory function have been reported in several diseases including type 2 diabetes, human immunodeficiency virus (HIV), neurodegenerative diseases, as well as in aging [120,122–132]. Interestingly, there does not seem to be a

single respiratory parameter that demonstrates consistent differences in healthy controls compared with disease patients. Changes in respiration, both increased and decreased, have been found in basal, ATP-linked, and maximal respiration [133], can be variable, and sex-associated differences in oxygen consumption rates have been reported [130,134]. The wide range of mitochondrial changes observed in these studies could be due to different bioenergetic impairments and compensations between diseases. Additionally, there could be differences arising from the subpopulation of cells tested or potential confounding factors such as the presence of other, contaminating cell types, or inadvertent cell activation during isolation.
