The objective of the present study was to evaluate platelet mitochondrial oxygen consumption using high-resolution respirometry (HRR) and metabolic flux analysis (MFA) and to verify the effect of advanced age on these parameters. HRR was used to analyze permeabilized and intact platelets, MFA to measure oxygen consumption rates (OCR), extracellular acidification rates (ECAR) and ATP production rate in intact fixed platelets. Two groups of healthy volunteers were included in the study: YOUNG (20-42 years, n=44) and older adults (OLD; 70-89 years; n=15). Compared to YOUNG donors, platelets from group OLD participants displayed significantly lower values of oxygen consumption in the Complex II-linked phosphorylating and uncoupled states and the Complex IV activity in HRR protocols for permeabilized cells and significantly lower resting and uncoupled respirations in intact cells when analyzed by both methods. In addition, mitochondrial ATP production rate was also significantly lower in platelets isolated from older adults. Variables measured by both methods from the same bloods correlated significantly, nevertheless those acquired by MFA were higher than those measured using HRR. In conclusion, the study verifies compromised mitochondrial respiration and oxidative ATP production in the platelets of aged persons and documents good compatibility of the two most widely used methods for determining the global performance of the electron-transporting system, i.e. HRR and MFA
Hyperbaric oxygen (HBO) therapy, i.e. breathing pure oxygen
under increased environmental pressures serves as a treatment
for diverse medical conditions. However, elevated oxygen
concentration can be detrimental to central nervous system or
lungs. Our study aimed to evaluate the effects of repeated
exposure to HBO on mitochondrial respiration assessed by highresolution respirometry (HRR), cell viability estimated by
PrestoBlue® reaction, morphology analyzed by routine phase
contrast and fluorescent microscopy, and superoxide dismutase
(SOD) and citrate synthase (CS) activities using human lung
fibroblasts. The cells were exposed to HBO for 2 h per day for
5 consecutive days. One day after the last exposure, HBO cells
displayed significantly smaller area and perimeter, compromised
viability and elevated SOD activity. No changes were detected in
CS activity or quality of mitochondrial network. HRR revealed
impaired mitochondrial oxygen consumption manifested by
increased leak respiration, decreased activity of complex II and
compromised ATP-related oxygen consumption when fatty acids
were oxidized. Our findings document that in conditions
mimicking chronic intermittent exposure to HBO, lung fibroblasts
suffer from compromised mitochondrial respiration linked to
complex II and impaired cellular growth in spite of increased
antioxidant defense. Underlying mechanism of this HBO-induced
mitochondrial dysfunction should be further explored.