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== Affiliations ==
1-Pharmacobiochem Lab, Fac Med, Comenius Univ, Bratislava, Slovakia; 2-Oroboros Instruments; 3-Dept Visceral, Transplant Thoracic Surgery, D. Swarovski Research Lab, Medical Univ Innsbruck, Austria. – firstname.lastname@example.org
==Abstract continued ==
In rat brain mitochondria, M inhibited CII-linked ET-pathway respiration slightly more than CII-linked OXPHOS respiration (-22 and -15% respectively in 2 mM M) (Fig. 1A). In the comprehensive SUIT protocol, the inhibitory effect of M on SDH became only evident at CII-linked ET-pathway respiration (Fig 1B). The relative share of CI-linked respiration in total respiration with all substrate combinations was highest with 5 mM M (Fig 2A). The combination of P (5 mM) and M (2 or 5 mM) best reflected the maximum capacity of CI-linked respiration with the least involvement of CII-linked respiration (less than 8%). Addition of G (10 mM) to this combination increased total respiration (Fig 2A), but this apparent increase reflected just higher involvement of CII-linked respiration (Fig 2B).
For the proper evaluation of CII-linked ET-pathway respiration, the inhibitory effect of 2 mM M could be overcome by additional titration of succinate (to a final concentration of 50 mM) after rotenone. The inhibition caused by 5 mM M could not be overcome by addition of S.
Based on these data, we suggest using 2 mM M in the SUIT protocols. It appears that 50 mM S could be generally applicable as a single titration in the SUIT protocols right after CI-linked substrates as it does not negatively affect the CI&II-linked OXPHOS and ET-pathway respiratory rates. Nevertheless, we suggest testing the effect of 50 mM S on these rates in each type of preparation before using it routinely in the SUIT protocol.
== Figures ==
[[Image:MitoFit Training Camp 2016 Sumbalova Figure1.jpg|left|600px]] '''Figure 1. A:'' The effect of malate on the respiration with 10 mM succinate + 0.5 µM rotenone (CII protocol) in OXPHOS and ET-pathway state in rat brain mitochondria.
''B:'' Respiratory rates determined with the comprehensive protocol with 3 concentrations of malate (M), 0.5, 2 and 5 mM, in mitochondrial respiration medium MiR05 , at 37°C. In the protocol, 5 mM pyruvate (P), 10 mM glutamate (G), M, 2 mM ADP, 10 mM succinate (S) and FCCP 0.5-1 µM were titrated to determine ET capacity with CI&II substrates. Subsequently, 0.5 µM rotenone was added for evaluation of CII-linked ET-pathway, followed by antimycin A for evaluation of residual oxygen consumption (ROX). For comparison, the value of CII-linked ET-pathway determined in the CII protocol is shown in the graph. All data are corrected for ROX shown in the graph and are means ± SEM of experiments conducted with 3-4 preparations.
[[Image:MitoFit Training Camp 2016 Sumbalova Figure2.jpg|left|600px]] ''Figure 2. A:'' The respiration in OXPHOS state determined in rat brain mitochondria with CI-linked substrates, CI&II-linked substrate combinations, and CII-linked substrate, and the respective malonate-insensitive part obtained after addition of 5 mM malonate. B: The malonate-sensitive part of respiration determined in A expressed as fraction of total OXPHOS respiration. Data are ROX-corrected and are means ± SEM of experiments conducted on 11 preparations with minimum number of experiments for each substrate combination being 3.
== References and Support ==
#Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopsies of human muscle. Methods Mol Biol 810:25-58.
#Ackrell BA, Kearney EB (1974) Mayr M. Role of oxalacetate in the regulation of mammalian succinate dehydrogenase. J Biol Chem 249:2021-7.
#Moser MD, Matsuzaki S, Humphries KM (2009) Inhibition of succinate-linked respiration and complex II activity by hydrogen peroxide. Arch Biochem Biophys 48:69-75.
#Sumbalova Z, Vancova O, Krumschnabel G, Gnaiger E (2014) Optimization of malate concentration for high-resolution respirometry: mitochondria from rat liver and brain. Mitochondr Physiol Network 19.13:p37.
Supported by Erasmus and Action Austria-Slovakia
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