Difference between revisions of "High-resolution respirometry"

Revision as of 14:32, 10 May 2012

high-resolution terminology - matching measurements at high-resolution

High-resolution respirometry

Description

High-resolution respirometry (HRR) is based on the OROBOROS Oxygraph-2k, combining chamber design, application of oxygen-tight materials, electrochemical sensors and electronics, Peltier-temperature control and software features (DatLab) to obtain a unique level of quantitative resolution of oxygen concentration and oxygen flux, with a closed-chamber or open-chamber mode of operation (TIP2k). Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the experimental basis for high accuracy of quantitative results and quality control in HRR.

Abbreviation: HRR

Reference: MiPNet06.01; Gnaiger_2008_POS; Gnaiger_2001_Respir Physiol

MitoPedia methods: Respirometry, Fluorometry, Spectrophotometry

Links

Oxygraphy: Linear versus non-linear slope

Oxygen dependence of respiration

In isolated mitochondria and many types of small cells (such as endothelial cells, fibroblasts etc), zero-order kinetics with respect to oxygen pressure (i.e. independence of flux with declining oxygen concentration) applies over a wide range down to very low oxygen levels, where then a hyperbolic oxygen dependence is observed. With a p50 (c50, apparent Km) in the order of <1 µM and a hyperbolic oxygen dependence of flux, 99% saturation of flux and hence zero-order kinetics is observed at an oxygen concentration >19 µM (i.e. >2 kPa, >2% oxygen saturation or >10% air saturation). For reviews see Gnaiger et al 1995, Scandurra and Gnaiger 2010.

Compared to the difficulties with classical chart recorder tracings, our 'modern' approaches over the past 20 years allowed us to drop the linearity assumption and statistically test for it, frequently rejecting this assumption to describe a non-linear oxygen dependence beyond the low-oxygen range governed by cytochrome c oxidase. For review see Gnaiger 2003.

In permeabilized muscle fibres (30 to 37 °C), oxygen kinetics is shifted 100-fold due to artifially high oxygen gradients, forcing us to apply elevated oxygen levels for obtaining near-zero-order kinetics. For details, see Pesta and Gnaiger 2012, Gnaiger 2003, and Discussion.

ADP dependence of respiration

The assumption of linearity (linear regression of oxygen concentration over time) is frequently not valid for various reasons other than oxygen kinetics. In classical ‘State 3’, ADP levels are ‘high’ (Chance and Williams 1955), but not necessarily saturating (Glossary: Respiratory states). Then, an ADP-dependent decline of respiration is observed immediately after titration of a sub-saturating concentration of ADP, which is obscured by any linear regression. This has caused in the past a tremendous underestimation of the apparent Km for ADP, perpetuated even today with the uncritical application of non-adequate software implementing the simple linearity approach only. For a critical approach to ADP kinetics, see Gnaiger et al 2000 and Gnaiger 2001.

@@ Line 17: / Line 17: @@
 * [http://www.oroboros.at/index.php?respirometry Respirometry @OROBOROS]
 * [http://en.wikipedia.org/wiki/Respirometry Respirometry @Wikipedia]
+== Oxygraphy: Linear versus non-linear slope ==
+=== Oxygen dependence of respiration ===
+In isolated mitochondria and many types of small cells (such as endothelial cells, fibroblasts etc), zero-order kinetics with respect to oxygen pressure (i.e. independence of flux with declining oxygen concentration) applies over a wide range down to very low oxygen levels, where then a hyperbolic oxygen dependence is observed. With a ''p''50 (''c''50, apparent ''K''m) in the order of <1 µM and a hyperbolic oxygen dependence of flux, 99% saturation of flux and hence zero-order kinetics is observed at an oxygen concentration >19 µM (i.e. >2 kPa, >2% oxygen saturation or >10% air saturation). For reviews see [[Gnaiger_1995_J_Bioenerg_Biomembr|Gnaiger et al 1995]], [[Scandurra_2010_Adv_Exp_Med_Biol|Scandurra and Gnaiger 2010]].
+Compared to the difficulties with classical chart recorder tracings, our 'modern' approaches over the past 20 years allowed us to drop the linearity assumption and statistically test for it, frequently rejecting this assumption to describe a non-linear oxygen dependence beyond the low-oxygen range governed by cytochrome c oxidase. For review see [[Gnaiger_2003_Adv_Exp_Med_Biol|Gnaiger 2003]].
+In permeabilized muscle fibres (30 to 37 °C), oxygen kinetics is shifted 100-fold due to artifially high oxygen gradients, forcing us to apply elevated oxygen levels for obtaining near-zero-order kinetics. For details, see [[Pesta_2012_Methods_Mol_Biol|Pesta and Gnaiger 2012]], [[Gnaiger_2003_Adv_Exp_Med_Biol|Gnaiger 2003]], and [[Permeabilized_muscle_fibres#Oxygen_kinetics_of_permeabilized_fibres|Discussion]].
+=== ADP dependence of respiration ===
+The assumption of linearity (linear regression of oxygen concentration over time) is frequently not valid for various reasons other than oxygen kinetics. In classical ‘State 3’, ADP levels are ‘high’ (Chance and Williams 1955), but not necessarily saturating ([[Glossary: Respiratory states]]). Then, an ADP-dependent decline of respiration is observed immediately after titration of a sub-saturating concentration of ADP, which is obscured by any linear regression. This has caused in the past a tremendous underestimation of the apparent Km for ADP, perpetuated even today with the uncritical application of non-adequate software implementing the simple linearity approach only. For a critical approach to ADP kinetics, see [[Gnaiger_2000_Proc_Natl_Acad_Sci_USA|Gnaiger et al 2000]] and [[Gnaiger_2001_Respir_Physiol|Gnaiger 2001]].