Talk:Rogers 2011 PLoS One

Comments by Erich Gnaiger

Rogers et al 2001 describe the application of a strongly advertised multi-well system (XF, 'Seahorse Bioscience') for respirometry with isolated mitochondria. Whereas multi-well systems do provide high throughput, it must be critically assessed if this is equivalent to high output of useful data and information.

Based on long-term expertise in high-resolution respirometry (HRR) and mitochondrial physiology, the paper by Rogers et al (2011) is discussed. Most arguments raised in this discussion are not restricted to applications with isolated mitochondria, but address general deficiencies of the multi-well system.

Conflicts of interest:

• Rogers GW and Ferrick DA are employees of Seahorse Bioscience (at the time of publication).
• Gnaiger E is founder and director of OROBOROS INSTRUMENTS.

Instrumental specifications of the XF multi-well system are missing

1. No specifications are provided on the limit of detection of respiratory flux, and no information is given on the validation of the large corrections applied for oxygen backdiffusion. The substantial problems involved can be deduced from Figure 2. Fig. 2B shows oxygen pressures dropping to the (impossible) level of about -10 mmHg, while the corresponding respiratory oxygen flux ('OCR') is high (in the range of 800 pmol/min; Fig. 2A).

• Comparison with HRR: In the Oxygraph-2k (O2k for HRR), plastic with high oxygen solubility is strictly avoided as a material in contact with the sample and incubation medium. Thus oxygen backdiffusion is minimized. Routine instrumental background tests are described for HRR as a SOP, to validate instrumental performance and use the resulting parameters for on-line correction of respiratory flux (Gnaiger et al 1995; Gnaiger 2001; Gnaiger 2008).

2. The effective volume of the closed chamber is not given accurately, but is stated to be "~7 µl" (p. 3) or "approximately 7 µl" (p. 10). Any uncertainty of the actual volume has a direct effect on the inaccuracy of the measured respiratory flux.

Lack of validation with high-resolution respirometry

Low-resolution instruments (compare HRR) with Clark-type polarographic oxygen sensors were used for validation of data obtained with the XF.

Lack of quality control of isolated mitochondria

1. Although the RCR is an accepted parameter for the quality control of isolated mitochondria, injuries of the outer mitochondrial membrane are only poorly reflected by the RCR (except in highly damaged mitochondria). Instead, a cytochrom c respiratory test is required (Kuznetsov et al 2004, which has not been applied by Rogers et al (2001). Additional wells would be required for such a test with the XF, furthrer reducing the effectived throughput and increasing the cost of application of dischargeable plates.

Conclusion

It must be concluded that the multi-well format yields high throughput,

1. without providing experimental flexibility: serial injections are restricted to 2 or 4 per well, in the 96 or 24 well system;

2. without facilitating the quantity of information: a large number of parallel replica is required to compensate for the poor reproducibility (contradictory results in Fig. 5C with P/E=1, but P/E>1.0 in Fig. 7B for mouse liver mitochondria respiring on succinate)

3. without minimizing the experimental time: an immense number of quality control tests is required before a high-throughput experimental series can even be started, and more quality control tests have to be performed for each experimental variation of pathophysiological state: narrow range of mitochondrial or cell density for reliable respiratory data, saturating substrate and inhibitor concentrations, optimum uncoupler concentrations, instrumental background correction, correction for autooxidation of TMPD and ascorbate.