Komlodi 2021 MitoFit AmR-O2

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Komlodi 2021 MitoFit AmR-O2

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Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. MitoFit Preprints 2021.10. doi:10.26124/mitofit:2021-0010

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Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed

Komlodi Timea, Sobotka Ondrej, Gnaiger Erich (2021-11-08) MitoFit Prep

Abstract:

Version 1 (v1) 2021-11-08 doi:10.26124/mitofit:2021-0010
Graphical abstract

The fluorometric Amplex UltraRed AmR assay is frequently used for quantitative assessment of hydrogen peroxide production. It is specific to H2O2, can be calibrated accurately, and allows continuous real-time measurement. Without correction for the background fluorescence slope, however, H2O2-independent formation of the fluorescent product UltroxRed (or resorufin) leads to artefacts.

We analysed (1) the medium specificity of the background fluorescence slope of the AmR assay, and (2) the oxygen dependence of H2O2 flux in baker´s yeast Saccharomyces cerevisiae. Apparent H2O2 flux, O2 concentration and O2 flux were measured simultaneously by high-resolution respirometry equipped with the fluorescence module. The apparent H2O2 flux of yeast showed a maximum under hypoxia when incubated in Dulbecco´s Phosphate Buffered Saline DPBS or KCl-medium. This hypoxic peak increased with the sequential number of normoxic-anoxic transitions. Even in the absence of yeast, the fluorescence slope increased at low O2 levels as a function of fluorescence intensity. The hypoxic peak was not observed in mitochondrial respiration medium MiR05.

Therefore, the hypoxic peak was a medium-specific background effect unrelated to cell physiology. In MiR05, H2O2 production of yeast decreased linearly from hyperoxia to hypoxia, with a steep decline towards anoxia. Respiration and oxygen dependence expressed as p50 of yeast were higher in MiR05 than DPBS. Respiration was a hyperbolic function of oxygen concentration in the low-oxygen range. The flux-dependence of oxygen affinity explained the higher p50 in MiR05.

Keywords: Amplex UltraRed, AmR, hydrogen peroxide production, H2O2 flux, respiration media, mitochondrial respiration medium, MiR05, oxygen dependence, yeast, reductive stress, anoxia, hypoxia, O2 kinetics, respiration, reoxygenation Bioblast editor: Komlodi T


Labels: MiParea: Respiration, Instruments;methods 

Stress:Oxidative stress;RONS, Hypoxia  Organism: Saccharomyces cerevisiae  Tissue;cell: Other cell lines  Preparation: Intact cells 

Regulation: Oxygen kinetics  Coupling state: ROUTINE 

HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

SUIT-013, SUIT-013 AmR ce D023, SUIT-009, SUIT-009 AmR mt D021, SUIT-009 AmR pce D019, SUIT-018, SUIT-018 AmR mt D031, SUIT-006, SUIT-006 AmR mt D048, SUIT-003 AmR ce D017, SUIT-003 AmR ce D058, AmR 


References

LinkReferenceViewYear
Aon MA, Cortassa S, O'Rourke B (2010) Redox-optimized ROS balance: a unifying hypothesis. Biochim Biophys Acta 1797:865-77.PMID:20175987 Open Access2010
Bienert GP, Schjoerring JK, Jahn TP (2006) Membrane transport of hydrogen peroxide. Biochim Biophys Acta 1758:994-1003.PMID:16566894 Open Access2006
Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 134:707-16.PMID: 4749271 Open Access1973
Brand MD (2016) Mitochondrial generation of superoxide and hydrogen peroxide as the source of mitochondrial redox signaling. Free Radical Biol Med 100:14-31.PMID: 27085844 Open Access2016
Buettner GR, Wagner BA, Rodgers VG (2013) Quantitative redox biology: an approach to understand the role of reactive species in defining the cellular redox environment. Cell Biochem Biophys 67:477-83.Open access2013
Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998) Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci 95:11715-20.PMID:9751731 Open Access1998
Crowe JH, Carpenter JF, Crowe LM (1998) The role of vitrification in anhydrobiosis. Annu Rev Physiol 60:73-103.PMID:95584551998
Dawson TL, Gores GJ, Nieminen AL, Herman B, Lemasters JJ (1993) Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am J Physiol 264:C961-7.PMID:83864541993
Dikalov SI, Harrison DG (2014) Methods for detection of mitochondrial and cellular reactive oxygen species. Antioxid Redox Signal 20:372–82.PMID: 22978713 Open Access2014
Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. doi: 10.1007/978-1-4939-7831-1_3PMID: 29850993 »O2k-brief
O2k-Protocols
2018
Duong QV, Hoffman A, Zhong K, Dessinger MJ, Zhang Y, Bazil JN (2020) Calcium overload decreases net free radical emission in cardiac mitochondria. Mitochondrion 51:126-39.PMID: 319826142020
Dutton DR, Reed GA, Parkinson A (1989) Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase. Arch Biochem Biophys 268:605-16.PMID:24643381989
Dębski D, Smulik R, Zielonka J, Michałowski B, Jakubowska M, Dębowska K, Adamus J, Marcinek A, Kalyanaraman B, Sikora A (2016) Mechanism of oxidative conversion of Amplex® Red to resorufin: pulse radiolysis and enzymatic studies. Free Radic Biol Med 95:323-32.PMID:27021961 Open Access2016
Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97.PMID: 11718759
Bioblast pdf
2001
Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52.Bioblast pdf
O2k-Protocols contents
2008
Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Berlin, Heidelberg:431-42. https://doi.org/10.1007/978-3-662-04162-8_45Bioblast pdf - Springer link
O2k-Protocols contents
2000
Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96.PMID: 8746845
Bioblast Access
1995
Grivennikova VG, Kareyeva AV, Vinogradov AD (2018) Oxygen-dependence of mitochondrial ROS production as detected by Amplex Red assay. Redox Biol 17:192-9.PMID:29702406 Open Access2018
Guzy RD, Mack MM, Schumacker PT (2007) Mitochondrial complex III is required for hypoxia-induced ROS production and gene transcription in yeast. Antioxid Redox Signal 9:1317-28.PMID:176274642007
Hernansanz-Agustin P, Izquierdo-Álvarez A, Sánchez-Gómez FJ, Ramos E, Villa-Piña T, Lamas S, Bogdanova A, Martínez-Ruiz A (2014) Acute hypoxia produces a superoxide burst in cells. Free Radic Biol Med 71:146-56.PMID: 246372632014
Hoffman DL, Salter JD, Brookes PS (2007) Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling. Am J Physiol Heart Circ Physiol 292:H101-8.PMID:16963616 Open Access2007
Kalyanaraman B, Darley-Usmar V, Davies KJA, Dennery PA, Forman HJ, Grisham MB, Mann GE, Moore K, Roberts LJ 2nd, Ischiropoulos H (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radical Biol Med 52:1-6.PMID:22027063 Open Access2012
Koga S, Echigo A, Nunomura K (1966) Physical properties of cell water in partially dried Saccharomyces cerevisiae.. Biophys J 6:665-74.PMID:5970569 Open Access1966
Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55.PMID:298509982018
Korge P, Calmettes G, Weiss JN (2015) Increased reactive oxygen species production during reductive stress: the roles of mitochondrial glutathione and thioredoxin reductases. Biochim Biophys Acta 1847:514–25.PMID:25701705 Open Access2015
Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61.PMID: 256310192015
Li Puma LC, Hedges M, Heckman JM, Mathias AB, Engstrom MR, Brown AB, Chicco AJ (2020) Experimental oxygen concentration influences rates of mitochondrial hydrogen peroxide release from cardiac and skeletal muscle preparations. Am J Physiol Regul Integr Comp Physiol 318:972-80.PMID: 32233925 »O2k-brief2020
Longo VD, Gralla EB, Valentine JS (1996) Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae. Mitochondrial production of toxic oxygen species in vivo. J Biol Chem 271:12275-80.PMID: 8647826 Open Access1996
Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38.PMID: 26131977 Open Access
O2k-Protocols contents
2015
Mishin V, Gray JP, Heck DE, Laskin DL, Laskin JD (2010) Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes. Free Radic Biol Med 48:1485-91.PMID:20188819 Open Access2010
Miwa S, Treumann A, Bell A, Vistoli G, Nelson G, Hay S, von Zglinicki T (2015) Carboxylesterase converts Amplex red to resorufin: Implications for mitochondrial H2O2 release assays. Free Radic Biol Med 90:173-83.PMID: 26577176 Open Access2015
Mohanty JG, Jaffe JS, Schulman ES, Raible DG (1997) A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Meth 202:133-41.PMID: 91073021997
Paniker NV, Srivastava SK, Beutler E (1970) Glutathione metabolism of the red cells. Effect of glutathione reductase deficiency on the stimulation of hexose monophosphate shunt under oxidative stress. Biochim Biophys Acta 215:456-60.PMID:55073671970
Piwonski HM, Goomanovsky M, Bensimon D, Horovitz A, Haran G (2012) Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles. Proc Natl Acad Sci U S A 109:E1437-43.PMID:22562794 Open Access2012
Robb EL, Hall AR, Prime TA, Eaton S, Szibor M, Viscomi C, James AM, Murphy MP (2018) Control of mitochondrial superoxide production by reverse electron transport at complex I. J Biol Chem 293:9869-79.PMID: 29743240 Open Access2018
Sies H (1997) Oxidative stress: oxidants and antioxidants. Exp Physiol 82:291-5.PMID:9129943 Open Access1997
Sies H, Jones DP (2020) Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21:363-83.PMID:322312632020
Skulachev VP (1996) Role of uncoupled and non-coupled oxidations in maintenance of safely low levels of oxygen and its one-electron reductants. Q Rev Biophys 29:169-202.PMID:88700731996
Smith KA, Waypa GB, Schumacker PT (2017) Redox signaling during hypoxia in mammalian cells. Redox Biol 13:228-34.PMID:28595160 Open Access2017
Stepanova A, Galkin A (2020) Measurement of mitochondrial H2O2 production under varying O2 tensions. Methods Cell Biol 155:273-93.PMID: 321839622020
Stepanova A, Kahl A, Konrad C, Ten V, Starkov AS, Galkin A (2017) Reverse electron transfer results in a loss of flavin from mitochondrial complex I: Potential mechanism for brain ischemia-reperfusion injury. J Cereb Blood Flow Metab 37:3649-58.PMID: 289141322017
Stepanova A, Konrad C, Guerrero-Castillo S, Manfredi G, Vannucci S, Arnold S, Galkin A (2018) Deactivation of mitochondrial complex I after hypoxia-ischemia in the immature brain. J Cereb Blood Flow Metab 39:1790-802.PMID: 296296022018
Stepanova A, Konrad C, Manfredi G, Springett R, Ten V, Galkin A (2018) The dependence of brain mitochondria reactive oxygen species production on oxygen level is linear, except when inhibited by antimycin A. J Neurochem 148:731-45.PMID: 305827482018
Szibor M, Schreckenberg R, Gizatullina Z, Dufour E, Wiesnet M, Dhandapani PK, Debska-Vielhaber G, Heidler J, Wittig I, Nyman TA, Gaertner U, Hall AR, Pell V, Viscomi C, Krieg T, Murphy MP, Braun T, Gellerich FN, Schlueter KD, Jacobs HT(2020) Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia. J Cell Mol Med 24:3534-48.PMID: 32040259 Open Access  »O2k-brief2020
Tretter L, Ambrus A (2014) Measurement of ROS homeostasis in isolated mitochondria. Methods Enzymol 547:199-223.PMID:254163602014
Verkhovsky MI, Morgan JE, Puustein A, Wikström M (1996) Kinetic trapping of oxygen in cell respiration. Nature 380:268-70.PMID:86375791996
Votyakova TV, Reynolds IJ (2004) Detection of hydrogen peroxide with Amplex Red: interference by NADH and reduced glutathione auto-oxidation. Arch Biochem Biophys 431:138-44.PMID:154647362004
Waypa GB, Chandel NS, Schumacker PT (2001) Model for Hypoxic Pulmonary Vasoconstriction Involving Mitochondrial Oxygen Sensing. Circ Res 88:1259–1266.PMID:11420302 Open Access2001
Xiao W, Loscalzo J (2020) Metabolic responses to reductive stress. Antioxid Redox Signal 32:1330-47.PMID:31218894 Open Access2020
Zhao B, Ranguelova K, Jiang J, Mason RP (2011) Studies on the photosensitized reduction of resorufin and implications for the detection of oxidative stress with Amplex Red. Free Radic Biol Med 51:153-9PMID: 22576106 Open Access2011
Zhao B, Summers FA, Mason RP (2012) Photooxidation of Amplex Red to resorufin: implications of exposing the Amplex Red assay to light. Free Radical Biol Med 51: 153–9.PMID:22765927 Open Access2012
Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP (1997) A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem 253:162-8.PMID:9367498 Open access1997