Pecina 2014 Biochim Biophys Acta Clinical

Pecina P, Houstkova H, Mracek T, Pecinova A, Nuskova H.Tesarova M, Hansikova H, Janota J, Zemanc J, Houstek J (2014) Noninvasive diagnostics of mitochondrial disorders in isolated lymphocytes with high resolution respirometry. Biochim Biophys Acta Clinical 2:62–71.

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Pecina P, Houstkova H, Mracek T, Pecinova A, Nuskova H.Tesarova M, Hansikova H, Janota J, Zemanc J, Houstek J (2014) BBA Clinical

Abstract: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts.

To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitoninpermeabilized cells from48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue Native electrophoreses.

Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory Complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed Complex I defects; changes in p50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of Complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations.

Based on our results, we propose the best biochemical parameters predictive for defects of respiratory Complexes I, IV andVmanifesting in peripheral blood lymphocytes.

The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients. • Keywords: Lymphocytes, Respirometry, Oxidative phosphorylation, Mitochondrial diseases, Diagnostics

• O2k-Network Lab: CZ Prague Houstek J

Coupling control and the Q-junction

Mitochondrial coupling control states are measured without simultaneous change of a selected pathway control state, i.e. coupling control is separated from pathway control. Biochemical coupling efficiencies (E-L coupling efficiencies) and P-L coupling efficiencies are, therefore, studied at a defined pathway control state that must not change between measurement of LEAK respiration L, OXPHOS capacity P, and electron transfer capacity E.

A physiologically relevant pathway control state for partial reconstitution of TCA cycle function is obtained by supply of NADH-linked substrates (e.g. pyruvate&malate PM; N-pathway) in combination with succinate (S; S-pathway), supporting convergent electron transfer through Complexes I and II into the Q-junction (NS-pathway). OXPHOS- and ET-capacities are higher in the combined NS-pathway than in the separate N- or S-pathway (Gnaiger 2020). Is the NS-pathway control state appropriate for the analysis of coupling control?

Partial additivity in OXPHOS capacity NS_P or ET capacity NS_E implies that there is competition between the N- and S-pathway, when the NS-pathway capacity is less than the arithmetic sum of the constituent pathway capacities. In mitochondria with lower OXPHOS than ET capacity (P<E; when the phosphorylation system is limiting), the competition in NS_E is increasingly pronounced in NS_P, and when respiration is further reduced by complete inhibition of the phosphorylation system (e.g. by oligomycin), competition between the N- and S-pathways is maximal in LEAK respiration. Different levels of competition imply that the ratio of the effective N- and S-pathway in the NS-pathway state may shift to the extent that the dominant pathway may fully outcompete the other in the LEAK state. Convergent electron input into the Q-junction in NS_E, therefore, may shift to single electron input through either the dominant N- or S-pathway in NS_L, which then would effectively correspond to either N_L or S_L. This has deep implications on LEAK respiration, since the N-pathway has three coupling sites (H⁺ pumps: CI, CIII, CIV) with a correspondingly higher H⁺/O₂ ratio compared to the S-pathway with two coupling sites (H⁺ pumps: CIII, CIV). A higher rate of the proton leak is implied when measuring the same rate of LEAK respiration in N_L than when observing an identical oxygen consumption rate in S_L.

When inhibiting O₂ consumption by oligomycin in the NS-pathway state, the relative contribution of the N- and S-pathways to LEAK respiration is not known. By subsequent uncoupler titrations, the relative contribution of these pathways is likely to change, thus obtaining an undefined combination of pathway control and coupling control. In conclusion, the NS-pathway state is not appropriate for studying coupling control. Coupling control is best studied in the separate N- or S-pathway (Gnaiger et al 2000; 2015).

Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002
Gnaiger E, Boushel R, Søndergaard H, Munch-Andersen T, Damsgaard R, Hagen C, Díez-Sánchez C, Ara I, Wright-Paradis C, Schrauwen P, Hesselink M, Calbet JAL, Christiansen M, Helge JW, Saltin B (2015) Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and caucasians in the arctic winter. https://doi.org/10.1111/sms.12612
Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci U S A 97:11080-5. https://doi.org/10.1073/pnas.97.20.11080

Labels: MiParea: Respiration, Instruments;methods, Patients

Organism: Human Tissue;cell: Blood cells, Lymphocyte Preparation: Permeabilized cells

Regulation: Inhibitor Coupling state: LEAK, OXPHOS Pathway: N, Gp, CIV, Other combinations, ROX HRR: Oxygraph-2k

Blood cell preparation