high-resolution-respirometry-in-a-small-volume-chamber

High-Resolution Respirometry in a Small-Volume Chamber

Investigating respiratory fluxes is decisive for understanding the complex interplay between metabolic processes. Studies of cells and tissues with ...

Oroboros Instruments

Medical university of innsbruck

Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling control and electron transfer pathway states. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The noncoupled state of maximum respiration is experimentally induced by titration of established uncouplers (CCCP, FCCP, DNP) to collapse the protonmotive force across the mitochondrial inner membrane and measure the electron transfer (ET) capacity (open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ET capacity. If OXPHOS capacity (maximally ADP-stimulated O flux) is less than ET capacity, the phosphorylation pathway contributes to flux control. Physiological substrate combinations supporting the NADH and succinate pathway are required to reconstitute tricarboxylic acid cycle function. This supports maximum ET and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. ET pathways with electron entry separately through NADH (pyruvate and malate or glutamate and malate) or succinate (succinate and rotenone) restrict ET capacity and artificially enhance flux control upstream of the Q-cycle, providing diagnostic information on specific ET-pathway branches. O concentration is maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental O limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background O flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in High-Resolution FluoRespirometry.

High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria.

This study aimed to investigate and compare the effects of repeated-sprint (RSH) and sprint interval training in hypoxia (SIH) on sea level running and cycling performance, and to elucidate potential common or divergent adaptations of muscle perfusion and -oxygenation as well as mitochondrial respiration of blood cells. Eleven team-sport athletes performed either RSH (3x5x10s, 20s and 5min recovery between repetitions and sets) or SIH (4x30s, 5min recovery) cycling training for 3weeks (3 times/week) at a simulated altitude of 2,200m. Before and three days after the training period, a Wingate and a repeated cycling sprint test (5x6s, 20s recovery) were performed with a 30min resting period between the tests. Four to five days after the training, participants performed a repeated running sprint test (RSA, 6x17m back and forth, 20s recovery) and a Yo-Yo intermittent recovery test (YYIR2) with 1 hour active recovery between tests. The order of the tests as well as the duration of the resting periods remained the same before and after the training period. During the cycling tests near-infrared spectroscopy was performed on the vastus lateralis. In four participants, mitochondrial respiration of peripheral blood mononuclear cells (PBMC) and platelets was measured before and after training. YYIR2 running distance increased by +96.7 ± 145.6 m after RSH and by +100.0 ± 51.6 m after SIH (p = 0.034, eta² = 0.449). RSA mean running time improved by -0.138 ± 0.14s and -0.107 ± 0.08s after RSH and SIH respectively (p = 0.012, eta² = 0.564). RSH compared to SIH improved re-oxygenation during repeated sprinting. Improvements in repeated cycling were associated with improvements in re-oxygenation (r = 0.707, p <0.05). Mitochondrial electron transfer capacity normalized per PBMC count was decreased in RSH only. This study showed that cycling RSH and SIH training improves sea-level running performance. Our preliminary results suggest that RSH and SIH training results in different patterns of muscular oxygen extraction and PBMC mitochondrial respiration, without effect on platelets respiration.

Exercise Performance, Muscle Oxygen Extraction and Blood Cell Mitochondrial Respiration after Repeated-Sprint and Sprint Interval Training in Hypoxia: A Pilot Study.

Human blood cells may offer a minimally invasive strategy to study systemic alterations of mitochondrial function. Here we tested the reliability of a protocol designed to study mitochondrial respiratory control in human platelets (PLTs) in field studies, using high-resolution respirometry (HRR). Several factors may trigger PLT aggregation during the assay, altering the homogeneity of the cell suspension and distorting the number of cells added to the two chambers (A, B) of the Oroboros Oxygraph-2k (O2k). Thus, inter-chamber variability (∆) was calculated by normalizing oxygen consumption to chamber volume () or to a specific respiratory control state (flux control ratio, ) as a reliable parameter of experimental quality. The method's reliability was tested by comparing the ∆ of laboratory-performed experiments (LAB,  = 9) to those of an ultramarathon field study (three sampling time-points: before competition (PRE,  = 7), immediately after (POST,  = 10) and 24 h after competition (REC;  = 10)). Our results show that ∆&nbsp; changed PRE-POST, but also for LAB-POST and LAB-REC, while all ∆ remained unchanged. Thus, we conclude that our method is reliable for assessing PLT mitochondrial function in LAB and field studies and after systemic stress conditions.

Human Platelet Mitochondrial Function Reflects Systemic Mitochondrial Alterations: A Protocol for Application in Field Studies.

Multiple non-aggregatory functions of human platelets (PLT) are widely acknowledged, yet their functional examination is limited mainly due to a lack of standardized isolation and analytic methods. Platelet apheresis (PA) is an established clinical method for PLT isolation aiming at the treatment of bleeding diathesis in severe thrombocytopenia. On the other hand, density gradient centrifugation (DC) is an isolation method applied in research for the analysis of the mitochondrial metabolic profile of oxidative phosphorylation (OXPHOS) in PLT obtained from small samples of human blood. We studied PLT obtained from 29 healthy donors by high-resolution respirometry for comparison of PA and DC isolates. ROUTINE respiration and electron transfer capacity of living PLT isolated by PA were significantly higher than in the DC group, whereas plasma membrane permeabilization resulted in a 57% decrease of succinate oxidation in PA compared to DC. These differences were eliminated after washing the PA platelets with phosphate buffer containing 10 mmol·L ethylene glycol-bis (2-aminoethyl ether)-,,','-tetra-acetic acid, suggesting that several components, particularly Ca and fuel substrates, were carried over into the respiratory assay from the serum in PA. A simple washing step was sufficient to enable functional mitochondrial analysis in subsamples obtained from PA. The combination of the standard clinical PA isolation procedure with PLT quality control and routine mitochondrial OXPHOS diagnostics meets an acute clinical demand in biomedical research of patients suffering from thrombocytopenia and metabolic diseases.

Mitochondrial Respiration of Platelets: Comparison of Isolation Methods.

Iron is an essential component for metabolic processes, including oxygen transport within hemoglobin, tricarboxylic acid (TCA) cycle activity, and mitochondrial energy transformation. Iron deficiency can thus lead to metabolic dysfunction and eventually result in iron deficiency anemia (IDA), which affects approximately 1.5 billion people worldwide. Using a rat model of IDA induced by phlebotomy, we studied the effects of IDA on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) and the liver. Furthermore, we evaluated whether the mitochondrial function evaluated by high-resolution respirometry in PBMCs reflects corresponding alterations in the liver. Surprisingly, mitochondrial respiratory capacity was increased in PBMCs from rats with IDA compared to the controls. In contrast, mitochondrial respiration remained unaffected in livers from IDA rats. Of note, citrate synthase activity indicated an increased mitochondrial density in PBMCs, whereas it remained unchanged in the liver, partly explaining the different responses of mitochondrial respiration in PBMCs and the liver. Taken together, these results indicate that mitochondrial function determined in PBMCs cannot serve as a valid surrogate for respiration in the liver. Metabolic adaptions to iron deficiency resulted in different metabolic reprogramming in the blood cells and liver tissue.

Article	Year
High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29850993	2018
Exercise Performance, Muscle Oxygen Extraction and Blood Cell Mitochondrial Respiration after Repeated-Sprint and Sprint Interval Training in Hypoxia: A Pilot Study. Journal of sports science & medicine\| PubMed ID: 30116106	2018
Human Platelet Mitochondrial Function Reflects Systemic Mitochondrial Alterations: A Protocol for Application in Field Studies. Cells\| PubMed ID: 34440857	2021
Mitochondrial Respiration of Platelets: Comparison of Isolation Methods. Biomedicines\| PubMed ID: 34944675	2021
Mitochondrial Respiration in Response to Iron Deficiency Anemia: Comparison of Peripheral Blood Mononuclear Cells and Liver. Metabolites\| PubMed ID: 35323713	2022

Luiz F. Garcia-Souza

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