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Talk:High-resolution respirometry

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Physiological temperature

->A new entry in MitoPedia including a table with recommended temperatures for different organisms will be created (name: "MitoPedia: Temperatures"?). Provide links to the pages O2k-Publications: Temperature: hypothermia; hyperthermia and Block 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.


Oxygen dependence of respiration

->will be updated and moved to Oxygen kinetics

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.

->perhaps include link to page Bezuidenhout 2016a Abstract MitoFit Science Camp 2016?

ADP dependence of respiration

->will be updated and moved to ADP

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 (MitoPedia: 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.


Titration of chemicals

->will be updated and moved to MiPNet09.12 O2k-Titrations; the page MiPNet03.02 Chemicals-Media should be integrated or linked to this page

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

Various

>> MiPNet19.03 O2k-cleaning and ISS
>> Cleaning the TPP+ electrodes
>> O2k-technical support and open innovation


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