Fasching 2011 Abstract Berlin

Fasching M, Harrison DK, Tretter L, Gnaiger E (2011) Combination of high-resolution respirometry and fluorimetry for continuous monitoring of hydrogen peroxide production by mitochondria with resolution in the nanomolar range. Abstract Berlin.

Link: MiP2011 Bordeaux, FR, Targeting Mitochondria Berlin 2011

Fasching M, Harrison DK, Tretter L, Gnaiger E (2011)

Event:

Combining fluorimetric methods with high-resolution respirometry enabled a wide range of analytical parameters of major interest in mitochochondrial physiology. The advantages of measuring two parameters simultaneously are discussed and we describe the simultaneous high-resolution measurement of oxygen consumption and fluorimetric determination of hydrogen peroxide production in the O2k, using Amplex Red®.

• O2k-Network Lab: AT Innsbruck Gnaiger E, HU Budapest Tretter L

Labels:

Organism: Mouse, Other Mammal"Other Mammal" is not in the list (Human, Pig, Mouse, Rat, Guinea pig, Bovines, Horse, Dog, Rabbit, Cat, ...) of allowed values for the "Mammal and model" property.  Tissue;cell: Neurons; Brain"Neurons; Brain" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property.  Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

HRR: Oxygraph-2k, Spectrofluorimetry"Spectrofluorimetry" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

Full abstract

High-resolution respiromety [1] is extended by MultiSensor techniques allowing the simultaneous measurement of oxygen consumption and one additional parameter (mitochondrial membrane potential, pH, Ca2+, or NO concentration [2,3]). Combining optical measurements with high-resolution respirometry further increased the range of analytical opportunities. In particular, fluorimetric methods are available for a wide range of analytical parameters of major interest in mitochochondrial physiology: H2O2 production, mitochondrial membrane potential, intracellular pH, Ca2+, Mg2+, and NADH levels.

The simultaneous measurement of additional parameters in a single respirometric chamber under strictly identical conditions offers important advantages: (i) respirometric performance provides a quality control of cells or mitochondrial preparations; (ii) overtitration of uncouplers, incomplete action of inhibitors, or non-saturating substrate concentrations are evaluated by the simultaneous response of multiple parameters, providing objective exclusion criteria; (iii) side effects of TPP+ or fluorophores (inhibition, dyscoupling) are detected by respiration and artifacts are, therefore, excluded; (iv) the direct relationship between the different parameters eliminates artifacts of normalization for a mitochondrial marker; (v) additional information is acquired for a limited amount of sample. In addition, there are practical and economical advantages, saving handling time and money.

The glass chamber of the OROBOROS Oxygraph-2k (O2k) allows transmitting optical signals through the chamber wall. This extends MultiSensor applications, obtaining the optical signal in addition to the signals of the oxygen sensor and of other electrodes inserted through the stopper (e.g. respiration, mitochondrial membrane potential and H2O2 production).

We describe the simultaneous high-resolution measurement of oxygen consumption and fluoroimetric determination of hydrogen peroxide production in the O2k, using Amplex Red®. Sensitivity to detect H2O2 production in relation to signal drift and noise is optimized. Low nanomolar concentrations of hydrogen peroxide were detected by integrating the components of a fluorimeter into the O2k. Potential problems are discussed for a three-parameter measurement, e.g. interaction of fluorophores with the TPP+ electrode.

Supported by MitoCom Tyrol.

[1] 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: 327-352.

[2] Aguirre E, Rodríguez-Juárez F, Bellelli A, Gnaiger E, Cadenas S (2010) Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells. Biochim. Biophys. Acta 1797: 557-565.

[3] http://www.oroboros.at/index.php?mipnetanalyzer

[4] Original presentation at MiP2011

Product information

• Fluorescence O2k-MultiSensor Module