Measuring Mitochondrial Dysfunction in Humans
Mitochondria are essential organelles found in almost every cell in the human body. They host a number of important metabolic pathways and carry out essential biological functions such as ATP synthesis and regulating cell death. There is a slow decline in mitochondrial function associated with ageing and mitochondrial dysfunction is proposed to act causally in a number of diseases. Previously it has been difficult to measure the health of human mitochondria as tests have required tissue from invasive muscle biopsies. The Seahorse XF Analyser is a recent technological advance that enables researchers to test mitochondrial function in small numbers of live cells. Recently, using the Seahorse analyser, peripheral blood cells such as platelets, monocytes and lymphocytes have been shown to display individually distinct bioenergetic profiles. During circulation, these cells are exposed to metabolic or environmental stressors throughout the body, potentially allowing them to act as biomarkers of bioenergetic health and ageing. Different cell preparations were trialled to purify and isolate platelets, monocytes and lymphocytes from freshly drawn whole blood. These protocols succeeded in preparing platelet and T-lymphocyte samples for XF analysis, however inconsistent results indicated that the protocols need further development. The Seahorse XF analyser was used to measure bioenergetic function in human platelets and T-lymphocytes from healthy donors ranging from 21 to 56 years of age. Each cell type required optimisation experiments to determine the optimal inhibitor and substrate concentrations to generate a meaningful bioenergetic profile. Similarly, seeding densities were determined to ensure oxygen consumption values that were suitable to the instruments sensitivity. These cell types have elastic metabolic phenotypes, and appeared sensitive to metabolic switching during early stages of the XF assay. Platelets were particularly difficult to work with because of their inclination to cease using oxidative phosphorylation and switch metabolism to using purely glycolytic pathways. Platelet susceptibility for metabolic switching is undetermined at this point. Lymphocyte optimization experiments also indicated possible premature activation during the assay. Further work is needed to fine tune this protocol to ensure consistent and uniform measurements before accurate BHI values are calculated for donors of different ages.
Advisor: Pearson, Andree; Hampton, Mark
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: CFFR - Centre for Free Radical Research
Publisher: University of Otago
Keywords: Seahorse; Mitochondrial; BHI; Bioenergetic; Platelets; PBMCs
Research Type: Thesis