Respirometry Analysis Essay

  • 1.

    Vacanti NM, Divakaruni AS, Green CR, Parker SJ, Henry RR, Ciaraldi TP, Murphy AN, Metallo CM (2014) Regulation of substrate utilization by the mitochondrial pyruvate carrier. Mol Cell 56:425–435CrossRefPubMedPubMedCentralGoogle Scholar

  • 2.

    Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435:297–312CrossRefPubMedPubMedCentralGoogle Scholar

  • 3.

    Huttemann M, Lee I, Pecinova A, Pecina P, Przyklenk K, Doan JW (2008) Regulation of oxidative phosphorylation, the mitochondrial membrane potential, and their role in human disease. J Bioenerg Biomembr 40:445–456CrossRefPubMedGoogle Scholar

  • 4.

    Mitchell P, Moyle J (1969) Estimation of membrane potential and pH difference across the cristae membrane of rat liver mitochondria. Eur J Biochem 7:471–484CrossRefPubMedGoogle Scholar

  • 5.

    Nicholls DG (1974) The influence of respiration and ATP hydrolysis on the proton-electrochemical gradient across the inner membrane of rat-liver mitochondria as determined by ion distribution. Eur J Biochem 50:305–315CrossRefPubMedGoogle Scholar

  • 6.

    Brand MD, Chien LF, Ainscow EK, Rolfe DFS, Porter RK (1994) The causes and functions of mitochondrial proton leak. BBA-Bioenergetics 1187:132–139CrossRefPubMedGoogle Scholar

  • 7.

    Rolfe DFS, Brand MD (1997) The physiological significance of mitochondrial proton leak in animal cells and tissues. Biosci Rep 17:9–16CrossRefPubMedGoogle Scholar

  • 8.

    Nelson DL, Cox MM (2013) Lehninger Principles of Biochemistry. W.H. Freeman and Company, New York, NYGoogle Scholar

  • 9.

    Jastroch M, Divakaruni AS, Mookerjee S, Treberg JR, Brand MD (2010) Mitochondrial proton and electron leaks. Essays Biochem 47:53–67CrossRefPubMedPubMedCentralGoogle Scholar

  • 10.

    Okun JG, Lummen P, Brandt U (1999) Three classes of inhibitors share a common binding domain in mitochondrial complex I (NADH: ubiquinone oxidoreductase). J Biol Chem 274:2625–2630CrossRefPubMedGoogle Scholar

  • 11.

    Sun F, Huo X, Zhai YJ, Wang AJ, Xu JX, Su D, Bartlam M, Rao ZH (2005) Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121:1043–1057CrossRefPubMedGoogle Scholar

  • 12.

    Lai B, Zhang L, Dong L-Y, Zhu Y-H, Sun F-Y, Zheng P (2005) Inhibition of Qi site of mitochondrial complex III with antimycin A decreases persistent and transient sodium currents via reactive oxygen species and protein kinase C in rat hippocampal CA1 cells. Exp Neurol 194:484–494CrossRefPubMedGoogle Scholar

  • 13.

    Thierbach G, Reichenbach H (1981) Myxothiazol, a new antibiotic interfering with respiration. Antimicrob Agents Chemother 19:504–507CrossRefPubMedPubMedCentralGoogle Scholar

  • 14.

    Bergmann F, Keller BU (2004) Impact of mitochondrial inhibition on excitability and cytosolic Ca(2+) levels in brainstem motoneurones from mouse. J Physiol 555:45–59CrossRefPubMedGoogle Scholar

  • 15.

    Penefsky HS (1985) Mechanism of inhibition of mitochondrial adenosine triphosphatase by dicyclohexylcarbodiimide and oligomycin: relationship to ATP synthesis. Proc Natl Acad Sci U S A 82:1589–1593CrossRefPubMedPubMedCentralGoogle Scholar

  • 16.

    Juthberg SKA, Brismar T (1997) Effect of metabolic inhibitors on membrane potential and ion conductance of rat astrocytes. Cell Mol Neurobiol 17:367–377CrossRefPubMedGoogle Scholar

  • 17.

    Tretter L, Chinopoulos C, AdamVizi V (1997) Enhanced depolarization-evoked calcium signal and reduced ATP/ADP ratio are unrelated events induced by oxidative stress in synaptosomes. J Neurochem 69:2529–2537CrossRefPubMedGoogle Scholar

  • 18.

    Loomis WF, Lipmann F (1948) Reversible inhibition of the coupling between phosphorylation and oxidation. J Biol Chem 173:807–808PubMedGoogle Scholar

  • 19.

    Quaranta M, Borisov S, Klimant I (2012) Indicators for optical oxygen sensors. Bioanal Rev 4:115–157CrossRefPubMedPubMedCentralGoogle Scholar

  • 20.

    Clark LC, Wolf R, Granger D, Taylor Z (1953) Continuous recording of blood oxygen tensions by polarography. J Appl Physiol 6:189–193PubMedGoogle Scholar

  • 21.

    Zhang J, Nuebel E, Wisidagama DRR, Setoguchi K, Hong JS, Van Horn CM, Imam SS, Vergnes L, Malone CS, Koehler CM, Teitell MA (2012) Measuring energy metabolism in cultured cells, including human pluripotent stem cells and differentiated cells. Nat Protoc 7(6):1068–1085. doi:10.1038/nprot.2012.1048CrossRefPubMedGoogle Scholar

  • 22.

    Ceroni P, Lebedev AY, Marchi E, Yuan M, Esipova TV, Bergamini G, Wilson DF, Busch TM, Vinogradov SA (2011) Evaluation of phototoxicity of dendritic porphyrin-based phosphorescent oxygen probes: an in vitro study. Photochem Photobiol Sci 10:1056–1065CrossRefPubMedPubMedCentralGoogle Scholar

  • 23.

    Owicki JC, Wallace Parce J (1992) Biosensors based on the energy metabolism of living cells: the physical chemistry and cell biology of extracellular acidification. Biosens Bioelectron 7:255–272CrossRefPubMedGoogle Scholar

  • 24.

  • 25.

    Xun Z, Rivera-Sanchez S, Ayala-Penã S, Lim J, Budworth H, Skoda EM, Robbins PD, Niedernhofer LJ, Wipf P, McMurray CT (2012) Targeting of XJB-5-131 to mitochondria suppresses oxidative DNA damage and motor decline in a mouse model of Huntington’s disease. Cell Rep 2:1137–1142CrossRefPubMedPubMedCentralGoogle Scholar

  • 26.

    Walls KC, Coskun P, Gallegos-Perez JL, Zadourian N, Freude K, Rasool S, Blurton-Jones M, Green KN, LaFerla FM (2012) Swedish Alzheimer mutation induces mitochondrial dysfunction mediated by HSP60 mislocalization of amyloid precursor protein (APP) and beta-amyloid. J Biol Chem 287:30317–30327CrossRefPubMedPubMedCentralGoogle Scholar

  • 27.

    Siuda J, Jasinska-Myga B, Boczarska-Jedynak M, Opala G, Fiesel FC, Moussaud-Lamodière EL, Scarffe LA, Dawson VL, Ross OA, Springer W, Dawson TM, Wszolek ZK (2014) Early-onset Parkinson’s disease due to PINK1 p.Q456X mutation – clinical and functional study. Parkinsonism Relat Disord 20:1274–1278CrossRefPubMedPubMedCentralGoogle Scholar

  • 28.

    Lange ML, Zeng Y, Knight A, Windebank A, Trushina E (2012) Comprehensive method for culturing embryonic dorsal root ganglion neurons for Seahorse Extracellular Flux XF24 Analysis. Front Neurol 3:175–182CrossRefPubMedPubMedCentralGoogle Scholar

  • 29.

    Horak Z, Kolarik J, Sipek M, Hynek V, Vecerka F (1998) Gas permeability and mechanical properties of polystyrene-polypropylene blends. J Appl Polym Sci 69:2615–2623CrossRefGoogle Scholar

  • 30.

    Gerencser AA, Neilson A, Choi SW, Edman U, Yadava N, Oh RJ, Ferrick DA, Nicholls DG, Brand MD (2009) Quantitative microplate-based respirometry with correction for oxygen diffusion. Anal Chem 81:6868–6878CrossRefPubMedPubMedCentralGoogle Scholar

  • 31.

    Brewer GJ, Torricelli JR (2007) Isolation and culture of adult neurons and neurospheres. Nat Protoc 2:1490–1498CrossRefPubMedGoogle Scholar

  • 32.

    Banker G, Goslin K (1998) Culturing nerve cells. London, CambridgeGoogle Scholar

  • 33.

    Trushina E, Nemutlu E, Zhang S, Christensen T, Camp J, Mesa J, Siddiqui A, Tamura Y, Sesaki H, Wengenack TM, Dzeja PP, Poduslo JF (2012) Defects in mitochondrial dynamics and metabolomic signatures of evolving energetic stress in mouse models of familial Alzheimer’s disease. PLoS One 7:e32737CrossRefPubMedPubMedCentralGoogle Scholar

  • 34.

    Viollet B, Guigas B, Sanz Garcia N, Leclerc J, Foretz M, Andreelli F (2012) Cellular and molecular mechanisms of metformin: an overview. Clin Sci 122:253–270CrossRefPubMedPubMedCentralGoogle Scholar

  • 35.

    Desler C, Hansen TL, Frederiksen JB, Marcker ML, Singh KK, Juel Rasmussen L (2012) Is there a link between mitochondrial reserve respiratory capacity and aging? J Aging Res 2012:192503–192503CrossRefPubMedPubMedCentralGoogle Scholar

  • 36.

    Nobes CD, Brown GC, Olive PN, Brand MD (1990) Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes. J Biol Chem 265:12903–12909PubMedGoogle Scholar

  • 37.

    Brand MD, Harper ME, Taylor HC (1993) Control of the effective P/O ratio of oxidative phosphorylation in liver mitochondria and hepatocytes. Biochem J 291:739–748CrossRefPubMedPubMedCentralGoogle Scholar

  • 38.

    Jekabsons MB, Nicholls DG (2004) In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate. J Biol Chem 279:32989–33000CrossRefPubMedGoogle Scholar

  • 39.

    Amo T, Yadava N, Oh R, Nicholls DG, Brand MD (2008) Experimental assessment of bioenergetic differences caused by the common European mitochondrial DNA haplogroups H and T. Gene 411:69–76CrossRefPubMedPubMedCentralGoogle Scholar

  • Abstract

    Whereas mitochondria are well established as the source of ATP in oxidative phosphorylation (OXPHOS), it is debated if they are also the major cellular sources of reactive oxygen species (ROS). Here we describe the novel approach of combining high-resolution respirometry and fluorometric measurement of hydrogen peroxide (H2O2) production, applied to mitochondrial preparations (permeabilized cells, tissue homogenate, isolated mitochondria). The widely used H2O2 probe Amplex Red inhibited respiration in intact and permeabilized cells and should not be applied at concentrations above 10 µM. H2O2 fluxes were generally less than 1% of oxygen fluxes in physiological substrate and coupling states, specifically in permeabilized cells. H2O2 flux was consistently highest in the Complex II-linked LEAK state, reduced with CI&II-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in uncoupled mitochondria respiring at electron transfer system capacity. Simultaneous measurement of mitochondrial respiration and H2O2 flux requires careful optimization of assay conditions and reveals information on mitochondrial function beyond separate analysis of ROS production. View Full-Text

    Keywords: high-resolution respirometry; H2O2 flux; Amplex Red; HEK 293T; mouse brain homogenate; mouse cardiac mitochondriahigh-resolution respirometry; H2O2 flux; Amplex Red; HEK 293T; mouse brain homogenate; mouse cardiac mitochondria

    ►▼ Figures

    0 thoughts on “Respirometry Analysis Essay

    Leave a Reply

    Your email address will not be published. Required fields are marked *