High-resolution respirometry

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

• Respirometry
• Glossary: Respirometry
• @OROBOROS: Respirometry
• @Wikipedia: Respirometry

Physiological temperature

We recommend determining mitochondrial respiratory activity at the physiologic temperature of a biologic system, no matter whether you work with biopsies, cultivated cells or isolated mitochondria. When measuring in an appropriate medium (protecting and stabilizing mitochondrial function) stability is preserved even in isolated mitochondria.

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.

Contamination by hydrophobic inhibitors

Problem

Lasting effect of inhibitors (i.e. rotenone, antimycin A), as observed by persistent low fluxes, when working with permeabilized fibers.

Cleaning the chamber between experiments

Clean the chamber after an experiment involving lipid-soluble inhibitors (such as oligomycin, rotenone, or antimycin A):

• The chamber must be cleaned rigorously with water (washing water-soluble inhibitors such as azide), and ethanol (100%), since lipid-soluble inhibitor(s) are difficult to be washed out from the chamber and may inhibit mitochondrial respiration in subsequent experiments.

1. Siphon off the cell/mitochondrial suspension at the end of the experiment and rinse the chamber with distilled water 5 times, by filling the chamber up to the rim. During all washing steps of the chamber, stirring has to be switched on.
2. Rinse the surface and capillary of the stopper with distilled water several times properly.
3. Fill the water-cleaned chamber with 70 % ethanol and replace the stopper making sure that the ethanol fills up the receptacle, and leave for 5 min.
4. Remove the stopper and siphon off the ethanol to empty the chamber. Repeat these cleaning steps with 70 % ethanol three times.
5. Then fill the chamber with absolute ethanol (99.6 %) and insert the stopper making sure that the ethanol fills up the receptacle. Place the perspex cover on top of the stopper and leave for 15-20 min.

• If you are finished with experiments for the day, replace absolute ethanol with 70% ethanol for storage.

• If you will start a new experiment, rinse chamber with distilled water 5 times, and rinse the surface and capillary of the stopper with distilled water several times properly.

Rinse the stopper by holding it at the receptacle, not at the shaft that fits into the chamber to avoid contamination.

• After measurements on permeabilized fibers it is advisable to remove the stirrer from the chamber and to clean it mechanically, as fibers may be very sticky and may not be removed by the standard celaning procedure.

Cleaning with "Dead Cells":

• Every once in a while it might be necessary to wash the chambers with "dead cells" obtained from a e.g. former cell-experiment (frozen at -20°C). Inhibitors will be taken up by the 'dead cells' when filling the chamber up to the rim with a 'dead cell' suspension. The suspension should be left in the chambers with inserted stoppers for at least 30 min.

Unintentional introduction of inhibitors to the O2 chamber

Inhibitors may also be introduced unintentionally, one example being 70% ethanol used in hospital settings containing antiseptics. Such inhibitors may accumulate e.g. in plastic parts and inhibit subsequent experiments.

>> Cleaning the TPP+ electrodes

>> Troubleshooting

Titration of chemicals

Do overshoots or short interruptions of the flux have a meaning after injection of chemicals?

Neither the overshoot nor the short signal disturbance is representing mitochondrial respiration. The disturbance of the signal is due to the injection of the chemical. With the addition of chemicals solved in ethanol a little bit of oxygen is always introduced, since ethanol has higher oxygen solubility than water.

>> MiPNet09.12 O2k-Titrations