Schmidt 2021 J Biol Chem

From Bioblast
Publications in the MiPMap
Schmidt CA, Fisher-Wellman KH, Neufer PD (2021) From OCR and ECAR to energy: perspectives on the design and interpretation of bioenergetics studies. J Biol Chem 207:101140.

» PMID: 34461088 Open Access

Schmidt CA, Fisher-Wellman KH, Neufer P Darrell (2021) J Biol Chem

Abstract: Biological energy transduction underlies all physiological phenomena in cells. The metabolic systems that support energy transduction have been of great interest due to their association with numerous pathologies including diabetes, cancer, rare genetic diseases, and aberrant cell death. Commercially available bioenergetics technologies (e.g. extracellular flux analysis, high resolution respirometry, fluorescent dye kits, etc.) have made practical assessment of metabolic parameters widely accessible. This has facilitated an explosion in the number of studies exploring, in particular, the biological implications of oxygen consumption rate (OCR) and substrate level phosphorylation via glycolysis (i.e. via extracellular acidification rate (ECAR)). Though these technologies have demonstrated substantial utility and broad applicability to cell biology research, they are also susceptible to historical assumptions, experimental limitations, and other caveats that have led to premature and/or erroneous interpretations. This review enumerates various important considerations for designing and interpreting cellular and mitochondrial bioenergetics experiments, some common challenges and pitfalls in data interpretation, and some potential ‘next steps’ to be taken that can address these highlighted challenges.

Bioblast editor: Gnaiger E O2k-Network Lab: US NC Greenville Neufer PD

Selected quotes and comments

  • ".. 1) a potentiometric method that uses a platinum hydrogen (Clark) electrode,"
  • Comment: The Clark polarographic oxygen sensor consists of two electrodes, most frequently a silver-silver chloride anode and a gold (sometimes platinum) anode. Since current is measured as the primary signal, it is not a potentiometric method, but actually an amperometric method. See references:
  • Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96. - »Bioblast access«
  • 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 & Sons, Inc, Hoboken, NJ:327-52. - »Bioblast access«
  • "measurement of the electrochemical potential across the inner mitochondrial membrane (IMM) is proportional to the energetic state of the ETS and can be measured potentiometrically or with fluorescent organic cations (e.g. tetramethyl rhodamine methyl ester)".
  • Comment: Using ion-selective electrodes (TPP+ or TPMP+) for measurement of the mitochondrial membrane potential across the inner mt-membrane (mtIM) is a potentiometric method. The electrochemical potential difference across the mtIM consists of an electric and a chemical component. Only the electric component of the electrochemical potential difference is detected by the potentiometric method with TPP+ or TPMP+ used as reporter cations. The chemical component is related to the pH difference across the mtIM, and this is not usually measurable by a potentiometric method. Proportionality implies a linear relationship through the origin. Non-linear functions, however, prevail and have to be considered in the relation to the energetic state of the ETS. See reference:
  • Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2: 112 pp. doi:10.26124/bec:2020-0002
  • "normalization must be carefully considered early in the experimental design and several options should be tested to determine the best approach."
  • Comment: A more detailed discussion of normalization is provided in the MitoEAGLE Consortium Communication:
  • "respiratory control ratios described for isolated mitochondria studies (9, 48)"
  • Comment: a more detailed account of respiratory control ratios compared to references 9 and 48 is found in doi:10.26124/bec:2020-0002.
  • "To account for potential sources of variation, standardization practices should be incorporated into all bioenergetics designs (and reported when possible)."
  • Comment: The possibly most advanced instrumental background test should be considered. See references:
  • Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277-97. - »Bioblast access«
  • Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. doi: 10.1007/978-1-4939-7831-1_3 - »Bioblast access«
  • Comment: The handling of outliers is not addressed but represents a significant component of standardization practices:
  • Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8. doi:10.26124/mitofit:2021-0008 - »Bioblast access«
  • "the Gibbs free energy of the hydrolysis reaction (ΔGATP, kJ/mol)".
  • Comment: Why is thermodynamics scary? Confusion of Gibbs energy [kJ] and Gibbs force [kJ/mol] makes thermodynamics more scary than necessary. - See Chapter 8 in doi:10.26124/bec:2020-0002.

On terminology

» Mitochondrial states and rates - terminology beyond MitoEAGLE 2020
For harmonization of terminology on respiratory states and rates, see


Outdated terminology 

Labels: MiParea: Respiration, Instruments;methods 

Regulation: Aerobic glycolysis, Coupling efficiency;uncoupling, Flux control, mt-Membrane potential, Redox state 

HRR: Oxygraph-2k 

Comparison of respirometric methods, MitoFit2022QC 

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