Makrecka-Kuka 2015 Biomolecules

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

Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) Biomolecules

Abstract: Whereas mitochondria are well established as the source of ATP in the process of oxidative phosphorylation (OXPHOS), it is debated if they are also the major 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 Ultrared 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 succinate-linked LEAK state, reduced with NS-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in uncoupled mitochondria respiring at electron transfer-pathway capacity. Simultaneous measurement of mitochondrial respiration and H₂O₂ flux requires careful optimization of assay conditions and reveals information in functional OXPHOS analysis beyond these assays carried out separately.

Terminology and abbreviations edited according to MitoEAGLE recommendations (Gnaiger E). • Keywords: High-resolution respirometry, H₂O₂ flux, Amplex Red, HEK 293T, Mouse brain homogenate, Mouse cardiac mitochondria

• O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, LV Riga Makrecka-Kuka M

Labels: MiParea: Respiration, Instruments;methods 

Stress:Oxidative stress;RONS  Organism: Human, Mouse  Tissue;cell: Heart, Nervous system, HEK  Preparation: Intact cells, Permeabilized cells, Homogenate, Isolated mitochondria 

Regulation: Inhibitor  Coupling state: LEAK, ROUTINE, OXPHOS, ET  Pathway: N, S, NS, ROX  HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

O2k-Demo, O2k-MultiSensor, MitoFit news, Amplex UltraRed, SUIT-009, SUIT-009 AmR mt D021, SUIT-009 AmR pce D019, SUIT-013, SUIT-013 AmR ce D023 

MitoFit news 2015#10

• 2015-07-01: Light in the powerhouse of the cells - SUIT protocols for measurement of respiration and H₂O₂ production. » MitoFit news
• Breitenbach 2016 Biomolecules