Only a few studies have evaluated changes in mitochondrial function and oxidative stress associated with ultramarathon running. Invasive biopsies are needed to assess mitochondrial function of skeletal muscle, which may not be well tolerated by some individuals. Platelets (PLTs) as a metabolically highly active and homogenous cell population were suggested as a potentially valuable surrogate to investigate mitochondrial function. Thus, this study was aimed to evaluate mitochondrial function of PLTs and its association with individual race performance and markers of oxidative stress, muscle damage and renal dysfunction. Race performance and mitochondrial function (high-resolution respirometry, HRR) of PLTs using different substrates inducing ROUTINE, LEAK, N-pathway control state (Complex I linked oxidative phosphorylation; CI, OXPHOS), NS-pathway control state (CI + II linked OXPHOS and electron transfer pathway; ET), S-pathway control state (CII linked ET) as well as parameters of oxidative stress and antioxidant capacity, and markers of muscle and renal injury were assessed in eight male ultramarathon runners (26–45 years) before, immediately after and 24 h after an ultramarathon race (PRE, POST, and REC). Ultramarathon running induced an increase in LEAK O 2 flux of PLT mitochondria and slight, largely non-significant changes in the oxidant/antioxidant balance. Levels of creatine kinase (CK), lactate dehydrogenase (LDH), blood urea nitrogen, and creatinine were all significantly elevated POST and remained high in REC. There were inverse correlations between race time and N-linked substrate state PRE-POST, and changes in CK and LDH levels were significantly related to PLT mitochondrial LEAK and N-linked respiration PRE. Although race-related changes in respirometry parameters of PLT mitochondria were rather small, a somewhat more pronounced increase in the relative N-linked respiration in faster runners might suggest PLT CI as indicator of physical fitness. The higher PLT LEAK PRE and diminished increase of CK during the race may represent a prophylactic preconditioning and the slight but non-significant elevation of the antioxidant potential post-race as a protective consequence of the race-related oxidative stress and potential threat to the kidney. Our findings point toward an interrelationship between mitochondrial function of PLTs, individual fitness levels and extreme physical and metal stresses, which stimulates further research.

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 (∆ab) was calculated by normalizing oxygen consumption to chamber volume (JO2) or to a specific respiratory control state (flux control ratio, FCR) as a reliable parameter of experimental quality. The method’s reliability was tested by comparing the ∆ab of laboratory-performed experiments (LAB, N = 9) to those of an ultramarathon field study (three sampling time-points: before competition (PRE, N = 7), immediately after (POST, N = 10) and 24 h after competition (REC; N = 10)). Our results show that ∆ab JO2 changed PRE-POST, but also for LAB-POST and LAB-REC, while all ∆ab FCR 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.

Executive summary – Box 1: In brief: Mitochondria and bioblasts .......................................... 2 1. Introduction ............................................................................................................................................................................. 8 2. Coupling states and rates in mitochondrial preparations ................................................................................... 8 2.1. Cellular and mitochondrial respiration ................................................................................................................. 8 2.1.1. Aerobic and anaerobic catabolism and ATP turnover Consortium Communication 2 of 44 Gnaiger E et al ― MitoEAGLE Task Group (2020) Bioenerg Commun 2020.1 2.1.2. Specification of biochemical dose and exposure 2.2. Mitochondrial preparations .................................................................................................................................... 10 2.3. Electron transfer pathways ..................................................................................................................................... 11 2.4. Respiratory coupling control .................................................................................................................................. 12 2.4.1. Coupling 2.4.2. Phosphorylation P» and P»/O2 ratio 2.4.3. Uncoupling 2.5. Coupling states and respiratory rates ................................................................................................................. 13 2.5.1. LEAK state 2.5.2. OXPHOS state 2.5.3. Electron transfer state 2.5.4. ROX state 2.5.5. Quantitative relations 2.5.6. The steady state 2.6. Classical terminology for isolated mitochondria ............................................................................................ 19 2.6.1. – 2.6.5. State 1 – State 5 2.7. Control and regulation .............................................................................................................................................. 21 3. What is a rate? – Box 2: Metabolic flows and fluxes: vectoral, vectorial, and scalar ............................. 21 4. Normalization of rate per sample................................................................................................................................. 23 4.1. Flow: per object ............................................................................................................................................................ 23 4.1.1. Count concentration 4.1.2. Flow per single object 4.2. Size-specific flux: per sample size .......................................................................................................................... 25 4.2.1. Mass concentration 4.2.2. Size-specific flux 4.3. Marker-specific flux: per mitochondrial content ............................................................................................. 26 4.3.1. Mitochondrial concentration and mitochondrial density 4.3.2. mt-Marker-specific flux 5. Normalization of rate per system ................................................................................................................................ 28 5.1. Flow: per chamber ...................................................................................................................................................... 28 5.2. Flux: per chamber volume ....................................................................................................................................... 28 5.2.1. System-specific flux 5.2.2. Advancement per volume 6. Conversion of units ............................................................................................................................................................ 30 7. Conclusions – Box 3: Recommendations for studies with mitochondrial preparations ...................... 31 References ............................................................................................................................................................................. 36 Authors (MitoEAGLE Task Group) – Author contributions .............................................................................. 41 Acknowledgements – Competing financial interests – Correspondence

Ultramarathon running represents a major physical challenge even for elite athletes. Runners wellbeing may be challenged by fluid and electrolyte disturbances, hemolysis and skeletal muscle damage, decline in hepatic function and kidney injury. We hypothesized that these effects may even be exacerbated in non-elite runners. Physiological, hematological and biochemical parameters of ten males (26-45 years, weekly training time 8.5 h), participating in a mountain ultramarathon (67 km; approximately 4,500 m of total ascent), were determined before (PRE), immediately after finishing the ultramarathon (POST), and 24 h after the individual finish (REC). Race times of the 8 finishers (2 drop-outs due to hot ambient temperature) varied between 10.4 and 16.1 h, which almost represents the range of the entire starter field (8.82 h-17.47 h). The following changes in mean values of selected markers for skeletal muscle damage and kidney injury were observed from PRE to POST: creatine kinase (CK) + 1289%, lactate dehydrogenase (LDH) + 87%, serum creatinine (CR) + 72%, blood urea nitrogen (BUN) + 96%, and estimated glomerular filtration rate (eGFR) - 45%. Values of CK + 1447%, LDH + 56%, and BUN + 71% remained elevated at REC. White blood cells were increased (+ 137%) only POST. In conclusion, CK and LDH levels and leucocytosis may be considered to be relatively harmless "side-effects" of prolonged running in this group of male subjects with rather moderate ultramarathon experience and training status. However, acute kidney injury may become clinically relevant in this population under the certain conditions, which should be considered by responsible race managers and medical advisors.

As the knowledge base and importance of mitochondrial physiology to human health expands, the necessity for harmonizing the terminologyconcerning mitochondrial respiratory states and rates has become increasingly apparent. Thechemiosmotic theoryestablishes the mechanism of energy transformationandcoupling in oxidative phosphorylation. Theunifying concept of the protonmotive force providestheframeworkfordeveloping a consistent theoretical foundation ofmitochondrial physiology and bioenergetics.We followguidelines of the International Union of Pure and Applied Chemistry(IUPAC)onterminology inphysical chemistry, extended by considerationsofopen systems and thermodynamicsof irreversible processes.Theconcept-driven constructive terminology incorporates the meaning of each quantity and alignsconcepts and symbols withthe nomenclature of classicalbioenergetics. We endeavour to provide a balanced view ofmitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes.Uniform standards for evaluation of respiratory states and rates will ultimatelycontribute to reproducibility between laboratories and thussupport the development of databases of mitochondrial respiratory function in species, tissues, and cells.Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery.

[PDF] [Full Text] [Abstract] , March 1, 2010; 95 (3): 411-421. Exp Physiol Robert F. Chapman, Jonathon L. Stickford and Benjamin D. Levine Altitude training considerations for the winter sport athlete [PDF] [Full Text] [Abstract] , October , 2010; 109 (4): 1140-1147. J Appl Physiol Santiago Lorenzo, John R. Halliwill, Michael N. Sawka and Christopher T. Minson Heat acclimation improves exercise performance [PDF] [Full Text] , January , 2011; 110 (1): 278-293. J Appl Physiol Taylor, Bruce D. Johnson, Jeeser Alves de Almeida, Herbert Gustavo Simões and Thomas Seifert Stephen M. Roth, Jos J. de Koning, Hein A. Daanen, Joanna Majerczak, Bruno Grassi, Bryan J. Boussuges, Pascal Rossi, Hidetaka Okada, Yasunori Morioka, Zhichao Zhou, Nathan T. Jenkins, Davinia Vicente-Campos, Kenneth W. Rundell, Ryan M. Broxterman, Thomas J. Barstow, Alain Jonas R. Mureika, Denis E. O'Donnell, Abigail Laymon, Carsten Lundby, Benjamin D. Levine, Andrew G. Edwards, Guido Ferretti, Andres E. Carrillo, Lawrence I. Sinoway, Michael J. White, Benjamin T. Corona, Fabiola C. Nunes, Shane P. Esau, R. John Holash, Brian R. MacIntosh, Chin Leong Lim, Asker E. Jeukendrup, Olivier Girard, Andrew M. Jones, Scott J. Montain, Carl Foster, Jerzy A. Zoladz, Thomas P. Olson, Daniel Alexandre Boullosa, Henning Bay Nielsen, Delliaux, Alun G. Williams, Wissam H. Joumaa, Yutaka Kano, Daphne Merkus, Erik D. Hanson, Frank E. Marino, Jose L. Chicharro, Charles L. Dumke, James M. Smoliga, Carl J. Ade, Stephane Andreas D. Flouris, Conrad P. Earnest, Rachel C. Drew, Jordan A. Guenette, Robert F. Chapman, Braga, Jared R. Fletcher, Sandra K. Hunter, Martin Buchheit, Anthony J. Donato, Carlo Capelli, Philip F. Skiba, Claudio Marabotti, Matthew R. Ely, Beth A. Parker, Chad D. Markert, Valdir A. Trent Stellingwerff, Stephane Perrey, Roy J. Shephard, Matthew M. Schubert, Gregoire P. Millet, Commentaries on Viewpoint: The two-hour marathon: Who and when? [PDF] [Full Text] [Abstract] , October , 2011; 301 (4): R1078-R1087. Am J Physiol Regul Integr Comp Physiol Kobel, Walther Parson, Martin Burtscher, Michael Schocke and Erich Gnaiger Dominik Pesta, Florian Hoppel, Christian Macek, Hubert Messner, Martin Faulhaber, Conrad and endurance training in normoxia and hypoxia in sedentary humans Similar qualitative and quantitative changes of mitochondrial respiration following strength

Aim. The use of resistance training by adolescents has been an area of controversy. The aim of the present work was therefore to evaluate the degree of strength trainability in adolescents compared to adults.Methods. Thirteen healthy male adolescents (AL) and eight adults (AD) volunteered to participate in a 10-week training program. Subjects performed supervised exercises for the legs, calf raise, leg curl and leg extension three times a week. Maximal strength, explosive power and anaerobic power were assessed prior and after the 10-week training program.Results. Significant interaction effects (time * age group) were found only for explosive strength as improvements of squat jump and counter movement jump performance (P<0.05) in favor of the AL group. No between-group changes were found for maximal strength and anaerobic power. However, significant time effects were observed for these parameters within both groups.Conclusion. Taken together, adolescents show distinct muscular adaptations by a higher gain in explosive power in response to resistance training when compared to adults. This might be related to peak height velocity (PHV) which is a "sensitive" period of trainability and accelerated adaptation to resistance training in adolescents.

Until recently, assessment of muscle metabolism was only possible by invasive sampling. 31P magnetic resonance spectroscopy (31P MRS) offers a way to study muscle metabolism non-invasively. The aim of the present study was to use spatially-resolved 31P MRS to assess the metabolism of the quadriceps muscle in sprint-trained, endurance-trained and untrained individuals during exercise and recovery. 5 sprint-trained (STA), 5 endurance-trained (ETA) and 7 untrained individuals (UTI) completed one unlocalized 31P MRS session to measure phosphocreatine (PCr) recovery, and a second session in which spatially-resolved 31P MR spectra were obtained. PCr recovery time constant (τ) was significantly longer in STA (50±17 s) and UTI (41±9 s) than in ETA (30±4 s), (P<0.05). PCr changes during exercise differed between the groups, but were uniform across the different components of the quadriceps within each group. pH during recovery was higher for the ETA than for the UTI (P<0.05) and also higher than for the STA (P<0.01). Muscle volume was greater in STA than in UTI (P<0.05) but not different from ETA. Dynamic 31P MRS revealed considerable differences among endurance and sprint athletes and untrained people. This non-invasive method offers a way to quantify differences between individual muscles and muscle components in athletes compared to untrained individuals.

Skeletal muscle is a highly adaptable tissue that can adjust to different stimuli. In the present study we investigated the impact of endurance training on muscle oxidative capacity with high resolution respirometry and 31P magnetic resonance spectroscopy (31P MRS). 40 healthy untrained subjects (UG) who performed an endurance training program 3 times a week lasting for 10 weeks were included in the study. 17 highly trained athletes (AG) were studied for comparison. Spatially-resolved dynamic 31P MRS measurements were obtained from the upper leg and biopsy samples were taken from the vastus lateralis to assess mitochondrial capacity with high-resolution respirometry. Subsequently, endurance and strength capacities of the subjects were determined via motor performance tests. After 10 weeks, the initial tests and muscle biopsies were repeated. We observed a significant increase in mass specific OXPHOS flux with training in the UG from 78.97 ± 16.05 to 101.39 ± 19.19 pmol.s-1.mg-1 (p<0.01). Flux between the UG and AG, both before and after training, respectively, was significantly different (AG: 120 ± 32.79 pmol.s-1.mg-1, UG see above, p<0.01). The capacity of the mitochondria to oxidize MCFA was significantly increased with training, observed as an increase in absolute flux (from 12.79 ± 4.67 pre-training to 29.58 ± 7.25 pmol.s-1.mg-1 post-training, p<0.01) and in the flux control ratio (FCR=fraction of a given flux relative to the maximal flux) of octanoyl-carnitine (0.14 ± 0.05 pre-trainig to 0.28 ± 0.04 post-training, p<0.01). However, no difference in the FCR of MCFA oxidation was found between UG after training and the AG (0.28 ± 0.04 post-training vs 0.26 ± 0.06). The FCR of Oxphos was increased after training (0.95 ± 0.09, p<0.01) but was not different between UG before training and AG (0.86 ± 0.09 pre-training vs. 0.86 ± 0.11). To date, analysis of the 31P MRS was still in progress. In conclusion, mitochondria seem to adapt to endurance training in a quantitative and qualitative way. The qualitative adaptations can most prominently be observed in the capacity of MCFA oxidation, which is increased due to training. The limitation of the OXPHOS system seems to be decreased temporarily in untrained subjects exposed to exercise training. Yet, this reversed decreased limitation in athletic subjects is unknown.

Endurance and strength training are established as distinct exercise modalities, increasing either mitochondrial density or myofibrillar units. Recent research, however, suggests that mitochondrial biogenesis is stimulated by both training modalities. To test the training "specificity" hypothesis, mitochondrial respiration was studied in permeabilized muscle fibers from 25 sedentary adults after endurance (ET) or strength training (ST) in normoxia or hypoxia [fraction of inspired oxygen (Fi(O(2))) = 21% or 13.5%]. Biopsies were taken from the musculus vastus lateralis, and cycle-ergometric incremental maximum oxygen uptake (VO(2max)) exercise tests were performed under normoxia, before and after the 10-wk training program. The main finding was a significant increase (P < 0.05) of fatty acid oxidation capacity per muscle mass, after endurance and strength training under normoxia [2.6- and 2.4-fold for endurance training normoxia group (ET(N)) and strength training normoxia group (ST(N)); n = 8 and 3] and hypoxia [2.0-fold for the endurance training hypoxia group (ET(H)) and strength training hypoxia group (ST(H)); n = 7 and 7], and higher coupling control of oxidative phosphorylation. The enhanced lipid oxidative phosphorylation (OXPHOS) capacity was mainly (87%) due to qualitative mitochondrial changes increasing the relative capacity for fatty acid oxidation (P < 0.01). Mitochondrial tissue-density contributed to a smaller extent (13%), reflected by the gain in muscle mass-specific respiratory capacity with a physiological substrate cocktail (glutamate, malate, succinate, and octanoylcarnitine). No significant increase was observed in mitochondrial DNA (mtDNA) content. Physiological OXPHOS capacity increased significantly in ET(N) (P < 0.01), with the same trend in ET(H) and ST(H) (P < 0.1). The limitation of flux by the phosphorylation system was diminished after training. Importantly, key mitochondrial adaptations were similar after endurance and strength training, regardless of normoxic or hypoxic exercise. The transition from a sedentary to an active lifestyle induced muscular changes of mitochondrial quality representative of mitochondrial health.