Talk:The protonmotive force and respiratory control

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» Manuscript phases and versions

Manuscript phases and versions - an open-access apporach

COST Action MitoEAGLE
This manuscript on ‘Mitochondrial respiratory states and rates’ is a position statement in the frame of COST Action CA15203 MitoEAGLE. The list of coauthors evolved beyond phase 1 in the bottom-up spirit of COST.
The global MitoEAGLE network made it possible to collaborate with a large number of coauthors to reach consensus on the present manuscript. Nevertheless, we do not consider scientific progress to be supported by ‘declaration’ statements (other than on ethical or political issues). Our manuscript aims at providing arguments for further debate rather than pushing opinions. We hope to initiate a much broader process of discussion and want to raise the awareness on the importance of a consistent terminology for reporting of scientific data in the field of bioenergetics, mitochondrial physiology and pathology. Quality of research requires quality of communication. Some established researchers in the field may not want to re-consider the use of jargon which has become established despite deficiencies of accuracy and meaning. In the long run, superior standards will become accepted. We hope to contribute to this evolutionary process, with an emphasis on harmonization rather than standardization.
  • Phase 1: The protonmotive force and respiratory control
» The protonmotive force and respiratory control - Discussion
» MitoEAGLE preprint 2017-09-21 - Discussion
  • Phase 2: Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology Part 1
» MitoEAGLE Task Group States and rates - Discussion
  • Phase 4: Journal submission
  • Target: CELL METABOLISM, aiming at indexing by The Web of Science and PubMed.
Coauthors
  • 2017-09-21 Version 01: 105 coauthors
  • 2017-10-15 Version 10: 131 coauthors
  • 2018-01-18 Version 20: 168 coauthors
  • 2018-02-26 Version 30: 225 coauthors
  • 2018-08-20 Version 40: 350 coauthors - EBEC Poster
  • 2018-10-17 Version 44: 426 coauthors - MiPschool Tromso-Bergen 2018
  • 2018-12-12 Version 50: 517 coauthors - Submission to the preprint server bioRxiv not successful
  • 2019-02-12 Preprint version 1: 530 coauthors
  • 2019-03-15 Preprint version 2: 533 coauthors
  • 2019-04-24 Preprint version 3: 533 coauthors
  • 2019-05-20 Preprint version 4: 542 coauthors
  • 2019-07-24 Preprint version 5: 612 coauthors
  • 2019-08-30 Preprint version 6: 622 coauthors - Preprint publication doi:10.26124/mitofit:190001.v6


Phase 1: Work flow 44 versions until 2017-09-18

 » The protonmotive force and respiratory control
  • Few comments on the manuscript (attached).
  • I think it will be a great resource for mitochondrial research and help clarify terminology. We had a comment around "Sample concentration" (page 28), relating to the use of isolated mitochondria. Please see below.
  • Tissues can contain multiple cell populations which may have distinct mitochondrial subtypes. Mitochondria are also in a constant state of flux due to highly dynamic fission and fusion cycles, and can exist in multiple stages and sizes which may be altered by a range of factors. The isolation of mitochondria (often achieved through differential centrifugation) can therefore yield a subsample of the mitochondrial types present in a tissue, dependent on isolation protocols utilised (e.g. centrifugation speed). This possible artefact should be taken into account when planning experiments using isolated mitochondria. The tendency for mitochondria of specific sizes to be enriched at different centrifugation speeds also has the potential to allow the isolation of specific mitochondrial subpopulations and therefore the analysis of mitochondria from multiple cell lineages within a single tissue.
  • Wow you work fast! The document really is great and I can see that you've put a lot of work into it, so I hope that our very minor comments were of some use.
  • Herewith my comment to the mitoeagle consensus review. An impressive piece of work already and good for the community! I have a number of comments, and I hpope they are of use. Some of the comments are, :::: I think, quite fundamental and likely stem from the fact that I am not a mitochondrial bio-energeticist. I put them down, because I think that if I have comments, also others will and addressing them will help to improve the manuscript. So please consider them in a supportive manner.
Coupling states: “since this is the key of the paper, I think this has to be defined here. personally I am not sure that the term coupling state is actually the best, since this refers to coupling of OXPHOS only (and not energetic coupling in general). I would therefor prefer States of OXPHOS coupling, with coupling defined as linked in terms of energy transfer. 100 % coupling is full transfer of energy”
  • EG: You have a strong point here, which warrants consideration. ‘OXPHOS coupling’ might be too close a term to ‘OXPHOS state’. It is ‘mitochondrial coupling states’, which includes aspects of uncoupling (e.g. UCP1) which divert away from OXPHOS.
  • Keijer J: “we do not test it like this but by adding cytochrome c. I think this is the case for many labs. So maybe replace by: as shown by the lack of a response of respiration of isolated mitochndria to the addition of cytochrome c”
  • EG: This is a misunderstanding: A cytochrome c effect would show an injury of the outer mitochondrial membrane. “lack of a respiratory response of respiration of isolated mitochondria to the addition of such low concentrations of digitonin and saponin” shows that respiration is not affected by these detergents at the applied concentrations.
  • Keijer J: I am not sure regulation and control are only relevant for metabolic control analysis. Furthermore, I think metabolic control analysis is something different than what is is used for normally (copied form wikipedia): Metabolic control analysis (MCA) is a mathematical framework for describing metabolic, signaling, and genetic pathways. MCA quantifies how variables, such as fluxes and species concentrations, depend on network parameters. In particular it is able to describe how network dependent properties, called control coefficients, depend on local properties called elasticities. MCA was originally developed to describe the control in metabolic pathways but was subsequently extended to describe signaling and genetic networks. For control and regulation I would use the following (adapted form wikipedia): The regulation of a protein (or RNA) in a pathway or process is how its activity or abundance is increased and decreased in response to signals. The control exerted by this protein (or RNA) is the effect that these changes in its activity or abundance have on the overall rate of the pathway (the flux through the pathway) (Salter M, Knowles R, Pogson C (1994). "Metabolic control". Essays Biochem. 28: 1–12). For example, an enzyme may show large changes in activity (i.e. it is highly regulated) but if these changes have little effect on the flux of a metabolic pathway, then this enzyme is not involved in the control of the pathway.
  • EG: Consider the more general perspective provided by the citation from David Fell. We do not restrict these terms to enzymes, but consider substrates etc as well.
  • Keijer J: “in the intro coupling states is mentioned. Respiratory states is much better.”
  • EG: Respiratory states are coupling control states (topic of the present report) and pathway control states (future report). But I agree: the ‘coupling states’ was changed to respiratory states in the abstract.
  • Keijer J: coupling control state?
  • EG: Coupling state and coupling control state have the same implicit meaning with different explicit detail.
  • Keijer J: (‘uncontrolled state’)
  • EG: in inverted commas, following the classical literature. The reason for using these terms is historical, which is now explained.
  • Keijer J: ‘involve fewer than three coupling sites’
  • EG: Yes, again classical terminology, now explained.
  • Keijer J: Although there are very view search hits, I do not like the abbreviation ROX. It is just too similar to ROS. Can RCO (residual consumption of oxygen) be the alternative?
  • EG: although ROX is already in use in the literature?
  • Keijer J: Definition of coupling.
  • EG: What about this – ‘In this context, coupling means that two processes of energy transformation are linked such that the input power (exergonic input force times input flow) drives the power output (endergonic output force times output flow). In a fully or completely coupled process, output and input flows are related by a fixed stoichiometry. 100% coupling efficiency implies that no work (exergy) is lost and, therefore, does not only depend on complete coupling but also on a full balance of output and input forces.’
  • Keijer J: yes, in the part after 3.2.
  • Here are Eskil's and my comments on the article (attached)
  • I have read through the manuscript and find it extremely detailed and well written. Few of my comments in general are as follows:
1. Figure 1A: Please consider enlarging this figure. This is the main figure and from the perspective of students, this figure serves as an introduction to the field of mitochondrial bioenergetics.
2. Figure 1A: Please consider adding FADH in the figure as it is not evident on how electrons move from Succinate to Q-junction.
3. I work with mitochondrial mutants where we discuss a lot about reactive oxygen species. Other than in the ROX section, I did not find much information on ROS production. I understand that ROS could be a giant topic in itself. However, please consider adding a small section on the mechanism of ROS generation and antioxidants within the mitochondria.
  • Attached you will find a Pdf containing some suggestions for revisions.
Many of my previous comments in edition 38 have already been attended to and I therefore only have a few suggestions.
Overall I found the review to be very informative and easy understandable – as a medical doctor who have moved into this complex field I am sure that others during the same will find a lot of help in this review.
  • I just have some general remarks:
I do not know to what extend this is important before submitting the article to a journal, however, I think that it would improve readability if tables and figures were placed closer to their first reference in the text (preferentially on the same or on the following page).
I think that it would be beneficial to emphasize certain take-away messages (e.g. by underlining) that preferentially occur at the end of a chapter, for example sentences like We define respiratory capacities, comparable to channel capacity in information theory, as the upper bound of the rate of respiration measured in defined coupling and pathway control states of mitochondrial preparations (page 13, version 44).
Maybe it is possible to re-arrange the order of definitions in a way that minimizes the use of technical terms before they are actually defined (e.g. “coupling” is used many times before it is defined).
From a physicist’s point of view, it would be appreciated to incorporate in the manuscript the concept of energy conversion and mitochondria as the sites of energy conversion taking place. E.g. on page 31 (Version 44), where total power is mentioned, one could add that at a theoretical conversion efficiency of 1, lossless energy conversion takes place.
  • My comments attached as well. Overall reads well some parts stray a bit, likely caused by multiple versions and authors. The message is clear and the need to define terminology and reporting of data is critical as the field moves ever forward to human samples and disease.
  • In the meantime, I have read your ms, and globally it is fine for me, and I would be pleased to join as a co-author.
There are some points on which I could give feedback, e.g. concerning Cr-stimulated respiration, or comments to make the second part (conceptual/thermodynamic) part more “digestible” for non-specialists.
  • please find attached a few edits (as comments in Acrobat) on the most recent version of the MitoEAGLE manuscript. Overall it reads really well and clarifies a lot of the terminology. Please let me know, what steps to take and when you would like us to review another version.
  • I read the manuscript, its written very well I hardly found places for my inputs, still I tried my best. However, I highlighted certain places, where I either found things can be modified/changed, therefore, I added things at highlighted place in a text box. Please refer the next box in the PDF copy as a reference.
Just few comment, as I have mentioned in manuscript as well. In the introduction of manuscript we can write about the disease perspective of mitochondria and then the diagnosis. As, in conclusion we are mentioning about diagnosis. May be you can add if you think it will be justified.
Second, at many places references et. al is not in Italics, that can be modified.
Third, can we mention somewhere about the permiabilization specif effect: I mean that depending on the process of permiablization, flux can vary. Also, sometime it could be inherently different from one cell to other or one individual to other depending on the background (AGE, Origin etc).
  • I finished reading the paper "The protonmotive force..."
I am under the impression of this excellent review. Of course, my knowledge on this subject, especially at the moment of many corrected versions ...it is not enough to find any mistakes, etc. However, I read this review very carefully and I have to admit that I learnt a lot from this paper. In my opinion, there are some very, very important chapters which (I am sure) will be very helpful and valuable for those who deal with mitochondria. I mean: all definitions; very clearly explained the difference between states and rates as well as the excellent chapter dedicated to the normalization problems. Moreover, throughout the text there are a lot of very useful graphs and illustrations - they have a very good quality and are very informative. It is really a very good job! My congratulations to you and the other co-authors.
  • 2017-09-18 Catherine Trivigno
  • The standardization of terms and precise definitions provided by the paper are extremely helpful, and I wish I'd had this manuscript when I first began my postdoc.
One humble suggestion would be to briefly address the issue of optimization of density and arrangement (eg. confluent monolayer or clumps) of cells used for experiments carried out in wells. The following protocol papers may be useful references: Salabei et al -http://www.nature.com/nprot/journal/v9/n2/full/nprot.2014.018.html - and Zhang et al -(http://www.nature.com/nprot/journal/v9/n2/full/nprot.2014.018.html
The attached file contains a few editing suggestions
  • Ich habe "The protonmotive force and respiratory control: Building blocks of mitochondrial physiology Part 1." gelesen und bestätige hiermit, dass ich die Nomenklatur, wenn nicht sowieso schon in Anwendung, anwenden werde. Ich finde die Idee und das Konzept dieses Manuskripts überaus unterstützenswert, da auch tagtägliche Diskussionsthemen aus unserem Labor abgehandelt werden wie zum Bsp die Normalisierung. Dennoch habe ich noch ein paar Anmerkungen: Ich finde manchmal die Schreibweise etwas zu lapidar/ zu wenig wissenschaftlich: "Vague or ambiguous jargon can lead to confusion and may relegate valuable signals to wasteful noise." oder es fehlen mir Referenzen, hier nur Beispiele aufgelistet:
"The in-ner mitochondrial membrane contains the non-bilayer phospholipid cardiolipin, which is not present in any other cellular membranes and promotes the assembly of respiratory supercom-plexes."
Oder auch ihre These, dass "Gender" die Mitofunktion beeinflusst ist mir persönlich neu und würde ich deswegen gerne nachlesen, bezüglich Einfluss von "Sex" weiß ich schon Bescheid, aber "Gender"?
Außerdem wäre es vllt noch möglich ein paar Referenzen einzufügen nach Aussagen wie diesen:
"However, since mitochondrial quality changes under certain stimuli, particularly in mito-chondrial dysfunction, some markers can vary while other markers are unchanged."
Gerade für Beginner wäre es vllt gut zu wissen, was die Vorteile/Nachteile der möglichen Mitomarker wäre, wie z.B. dass die CS activität durch akute exercise reguliert wird.
Dennoch finde ich es eine gute Idee die Nomenklatur zu vereinheitlichen und unterstütze daher diese Publikation, auch wenn ich manche der Namengebungen nicht intuitive finde wie uncoupled, noncoupled and decoupled.
  • 2017-09-18 Gro Vatne Rosland
  • Thank you for the open invitation for providing input to your paper entitled : `The protonmotive force and respiratory control: Building blocks of mitochondrial physiology, Part 1`.
I had just a few comments to your well written and informative paper (please find a version with my comments attached). As our focus are on mitochondrial fuction in intact, living cells, I hope to contribute to a greater extent in the following parts of this article.
  • I am sending you my comments to version 44 of the manuscript. It became a long and complicated paper with many equations that requires a huge amount of time for reading and understanding.
The last part will come later.

I send this  in parts because versions adapt rapidly.* 2017-09-18 Keijer J

  • Herewith I send you the pdf with a few comments of the mitoeagle recommendation manuscript.
More will follow later...
  • I have read the manuscript and I think this will be an important review which gain consensus of terms and a general understanding of mitochondrial respiratory physiology. For me, which is new in the field, this manuscript was very explanatory in understanding common terms related to the topic and the physiology and biochemistry that affect mitochondrial respiratory control. This paper will hopefully results in better consensus of terminology which increase the global response to experimental results carried out by different research groups. For students, this paper will be an useful knowledge building platform and a perfect starting point when they are entering the field.
I have not made any changes in the manuscript since I am quite new in this field and look at myself more like a “student” than a scientist in this specific area. I believe however that it is important to support this review to increase the joint consensus of terminology related to mitochondrial respiratory control and the protonmotive force.
  • I would like to submit to your attention my contribution to the joint MitoEAGLE review "The protonmotive force and respiratory control: Building blocks of mitochondrial physiology - Part 1".
I have annotated my comments and suggestions in the text of the .pdf Version_44 that was available online today, please find enclosed my file "MLG MitoEAGLE_1_Mitochondrial_respiratory_coupling_control.pdf".
Please also consider the attached .jpg file concerning some possible changes to Figure 1A.
  • Here my comment after reading carefully the final manuscript. It is a very interesting review which include much useful information.

In page number 6-7, in these sentences: "Mitochondria maintain their own genetic material known as mitochondrial DNA (mtDNA) that encodes subunits of the transmembrane respiratory Complexes CI, CIII, CIV and CV, 13 peptides and the mitochondrial 16S and 12S rRNA and 22 tRNAs, and is both regulated and supplemented by nuclear-encoded mitochondrially targeted proteins. Evidence has accumulated that additional gene content is encoded in mitochondrial genomes, e.g. microRNAs, smithRNAs, and even additional proteins ()".

I think that it will be useful if it is possible to add the following sentence: "Usually, in the mtDNA, mt-ND1 and mt-ND4 genes have been analysed. Both genes provide intructions for making a protein called NADH dehydrogenase 1 and 4, respectively (proteins of the CI)".
  • After reading the manuscript, I´m impressed on the great efforts done to define precisely bioenergetic terms. Theoretically, I feel that I´m not able to contribute with improvements to the present state of this work.
Nevertheless, I have attached some minor comments.
  • Below some more remarks:
One general impression I had was that at many occasions more references are necessary (examples in the pdf).
There was a mix-up of figures, 5 was before 3 but guess has been corrected already in this version.
In my view I am not so sure about the 2nd thermodynamic part. Is it necessary to advance our understanding and unify nomenclature? Seems very textbook-like and very hard to follow for a physiologist.
The highly important part of 4. Normalization: flows and fluxes could be moved forward and the thermodynamic part could be a supplement-appendix?
Although pan, this is still mainly a European initiative it seems. How about the US (and other countries) scientific communities? Especially relevant for publications and discussions with reviewers from these countries with regard to use of nomenclature?
  • 2017-09-16: Phase 1 completed - 44 versions in preparation of the MitoEAGLE preprint.
Download last update 2017-09-16 (Version 44): Bioblast pdf - » Versions
  • Please, find enclosed the next and updated summary of just single remarks of ours, mostly editorial, to the review paper on the recommendation part 1.
We refer to page numbers and the lining of the document attached;
  • Congratulations on this manuscript, and the idea of inviting feedback.
Attached are some comments from myself and JJ (Jujiao Kuang)
  • I have read it with great interest; it is a major piece of work, it's message is clear.
  • I have only made a few very minor comments (please see the attachment), which you might find useful. Among these, I'd like to ask you about the calculation of the total mitochondrial number in human body based on the total number of cells at the end of the manuscript. If I understand correctly, the calculation (300 mitochondria per cell x 37x10exp(12) cells = 11x10exp(15) mitochondria) was made without taking into account the number of erythrocytes in the body (approx. 25x10exp(12))? If I am not mistaken, the final figure is lower than the one provided in the current version of the manuscript if only cells that are not erythrocytes are used to calculate the total number of mitochondria. Of course, this does not affect the content or the message of the paper, but perhaps it would be worthwhile to take into account the very latest estimations of cell numbers in the human body (papers by Senders et al., Cell 2016 and PLOS Biol 2016).
  • The proposed mission of the MitoEAGLE network to develop harmonized protocols and serve as a central resource for studies of mitochondrial respiration is outstanding. I would have immensely enjoyed such a resource as a trainee, and I look forward to advancing the mission of the MitoEAGLE network moving forward. I confirm that I have read the manuscript and I look forward to implementing the recommended guidelines into my current and future research.
  • Section 2.1, Paragraph 2: Would we expect the beginner or new user to understand that the terms “respiratory state” and “coupling state” are synonymous with each other? - Reply by EG: These terms are not synonymous. I added: "Coupling control states and pathway control states are complementary, since mitochondrial preparations depend on an exogenous supply of fuel substrates and oxygen." Further explanations in Section 2.2.
  • Could it also help for clarity to state what the respiratory coupling states are at this particular stage of the manuscript? Although these respiratory states are clearly stated in Table 2, I would wonder if the beginner might ask (when getting to this section) what are examples of respiratory states (ETS, OXPHOS, LEAK), since they could be hearing these terms for the first time?
  • Sections 2.2-2.3: I enjoyed reading this section as it provided an overview of the classical terminology, and their extension toward a concept-driven terminology with the three coupling states. I suspect new users will find this information of value as well.
  • Mitochondrial markers and mitochondria-specific flux: Will the group be making any specific recommendations for selecting a mitochondrial marker (e.g. citrate synthase activity, mtDNA, OXPHOS protein expression etc.) in the present work? In other words, could it be worthwhile to state the different mitochondrial markers encountered in the literature, and under what specific scenarios a given marker would be ideal or appropriate (similar to selecting a given reference protein in western blotting). Or is this section intended to broach the topic broadly, and then provide more specific guidelines for selecting a mitochondrial marker in the future follow-up recommendations? - EG: Specific guidelines may be elaborated in a future report.
  • EG:
Thank you for your valuable input and feedback, integrated into the new version 42.
  • I found the manuscript excellently written, and more importantly, a very comprehensive yet clear and easily understandable guide to nomenclature and definitions.
Please find attached a .pdf with minor suggestions/comments incorporated.
  • The majority of the comments relate to the structure of the review. I find it sometimes very hard to follow the overall flow of the review. Would it make sense to make 3 distinct parts about 1) proton motive force, 2) respiratory states and 3) normalisation.
  • ".. cellular respiration is defined as the consumption of oxygen of which the majority is coupled to the physical and chemical processes of ATP production." - EG: Well, then LEAK respiration and respiration in the ETS state would not be cellular respiration? What about this definition: "cell respiration is defined as the consumption of oxygen coupled to electrochemical proton translocation across the inner mitochondrial membrane."
  • What is the difference between this uncontrolled state and uncoupled state, what most people will use when they talk about uncouplers. - EG: see Table 2. Distinction of terms related to coupling.
  • non- or uncoupled? If we talk about FCCP as uncouplers, it makes more sense to call this uncoupled respiration. - EG: Then you call LEAK respiration 'uncoupled' and respiration in the ETS state as 'uncoupled', propagating confusion? See Table 2.
  • Fig. 2: What is the difference between H+ slip (2x) and H+ leak? - EG: When moving the section on LEAK state up, these definitions come closer to Fig. 2.
  • Because of the relatively large intracellular diffusion distances in sk and cardiac muscle, this problem is less likely to occur in other preparations where cell dimensions are smaller, such as permeabilised cells and brain/kidney cells. so the Km is likely dependent, also, on cell size. - EG: There is no direct evidence supporting this statement.
  • Still find the difference between uncoupled and non coupled difficult, and is not explained so far. Here it seems that uncoupled refers to leak respiration, while non-coupled refers to exogenous addition of UNcouplers. It will be tough to convince others about talking about non-coupling when you use uncouplers. - EG: Which better alternative can you suggest to convince others?
  • Proton slip: can we make an estimate about the size of this? This obviously decreases P/O ratio. If it's <0.5%, the measurement errors are generally larger, and there's no practical way to correct for this in standard respirometry. - EG: We could refer to classical references by the group of L Azzone.
  • Cation cycling (as a component in the control of LEAK respiration): Also, an increase in mt Ca levels effectively decrease the proton motive force. We measured this recently (Wust et al PMID: 28028811), but I don't think anyone has clearly shown lower proton motive force when ca enters the mitos. Also: ca increases krebs' cycle enzyme activities and decrease the intrinsic resistance in the complexes. - EG: Context?
  • Section 2.3: I would move this section further up, so that we can make a better statement that these states are not compatible with the current knowledge. Before 2.2. - EG: This would be the historical approach. To be more helpful educationally, the constructive terminology is used first to explain the coupling states, which then makes the classical states and their rationale more clear.
  • State 5: If this paragraph is moved up, this also allows for a critical appraisal of these states that goes beyond this list. There is already something about state 3u above (which is confusing if the concepts of states is introduced here). Plus, the comment can be easily made that different substrates result to varying state 2 and 3 Jo2s. "Therefore, we propose the following states....." - EG: There is no fuel substrate added in the classical State 2. The terms state 3u and 4o have been removed from the upper section to Section 2.3.
  • cO2 - EG: See IUPAC.
  • Because the title is proton motive force and respiratory states, I would make 2 clear sections in the paper: one dealing with everything with PMF and the other with the respriatory states. Now they constantly interchange. - EG: The key to clarify the concept of the protonmotive force is the context of state and rate (Table 4; Eq. 3; etc.).
  • I always thought that changing from ph 6 to 7 is something completely different than from 2-3 or 9-10, because of the log scale in the H+. Does this make the change in V similar then? - EG: That follows from the thermodynamic definition of chemical potentials.
  • Comments
  • Please find attached a small contribution which I would like to stress on. After having gone through the work, I felt systems biology/simulation aspects is missing which I believe would serve as a good addendum.
  • I carefully read the manuscript which I found really clarifying in terms of concepts and nomenclature. I think my short contribution could fit well into the section 3.4 Normalization.
I focus the subject on the medium composition. Given the influence of cations on mitochondrial respiration and their variability in the solution composition, I think it could be a good point to harmonize among studies or at least to be taken into account. I think it would provide more conceptual strength to the manuscript and link to the concept of cation cycling mentioned in the 'classical terminology for isolated mitochondria' section.
  • Please find the attached my Review for MitoEAGLE_1_2017-09-10(39)_Mitochondrial respiratory coupling control-edited. I summarized the editing below:
1. On page 7, please consider changing “to interrelate results obtained in different studies” to “to corroborate results gathered across a spectrum of studies”
2. On page 9, please add at the end of mitochondrial preparation definition: “The term mitochondrial preparations do not include further fractionation of mitochondrial components, as well as submitochondrialparticles.”
3. On page 11, please include a short but detailed comparison of phosphorylation as described in OXPHOS and phosphorylation as a posttranslational modification.
4. Regarding normalization for mitochondrial content section, please improve on this part. The idea is vague and cannot be understood. Rephrase or expand more. Some improvements have also been made in this portion, as seen below:
a. Mitochondrial concentration, Cmte, and mitochondrial markers: It is important that mitochondrial content be quantified since its concentration serves an indicator for cellular oxidative capacity and normalization factor for functional analysis. Mitochondrial organelles comprise a cellular reticulum that is in a continual flux of fusion and fission. Hence the definition of an "amount" of mitochondria is often misconceived: mitochondria cannot be counted as a number of occurring elements. Therefore, quantification of the "amount" of mitochondria depends on measurement of a chosen mitochondrial markersof chosen mitochondrial markers. ‘Mitochondria are the structural and functional elemental units of cell respiration’ (Gnaiger 2014). The quantity of a mitochondrial marker can be considered as the measurement of the amount of elemental mitochondrial units or mitochondrial elements, mte. However, since mitochondrial quality changes under certain stimuli, particularly in mitochondrial dysfunction, some markers can vary while other markers are unchanged. (1) Structural markers areParameters such as mitochondrial volume or membrane area are considered to be structural markers, while. Mitochondrial mitochondrial protein mass is a marker frequently used as a marker for isolated mitochondria. (2) Mitochondrial marker enzymes (amounts or activities) and molecular markers can be selected as matrix markers, e.g., citrate synthase activity, mtDNA or inner mt-membrane markers, e.g., cytochrome c oxidase activity, aa3 content; TOM20. (3) Extending the measurement of mitochondrial marker enzyme activity to mitochondrial pathway capacity, measured as ETS or OXPHOS capacity, can be considered as an integrative functional mitochondrial marker.
in the attachment, please find my first comments for version 40 of the terminology manuscript.
Excellent comments, now included in the new Version.
“please explain me the driving force -470 kJ.mol-1 O2 for oxidation and 0.2 V in line 2 of the screenshot”
perhaps it is best if you explain me the question or difficulty, such that I can provide the explanation.
  • It is of very high quality and very well written. We are convinced that it will be a milestone for the scientific community and serve to foster future research in the field of mitochondrial respiration. Nonetheless, we have some suggestions which are mainly related to editing/wording, which you might consider in your revision to improve clarity. Please see attached file.
  • 2017-09-13 Orynbayeva Zulfiya
I have attached my comments to the manuscript. Please don’t hesitate to share your concerns and suggestions if you have any or if any additional supportive references would be needed.
Many thanks for your excellent comments and input, fully integrated into the new version.
  • Orynbayeva Zulfiya
I have attached the answer to your question.
  • 2017-09-12 Version 40
  • I have read version 38 and have the following two comments:
P 23 - The symbol ξ should be defined.
P 42 -“Mitochondrial markers must be evaluated critically, such as citrate synthase activity as an enzymatic matrix marker, that provides a link to the tissue of origin on the basis of calculating the mitochondrial yield, i.e., the fraction of mitochondrial marker obtained from a unit mass of tissue.”
I do not understand this sentence. I would have tried to rearrange it, but I am not sure what message is intended to be conveyed to the reader.
New: Mitochondrial markers, such as citrate synthase activity as an enzymatic matrix marker, provide a link to the tissue of origin on the basis of calculating the mitochondrial yield, i.e., the fraction of mitochondrial marker obtained from a unit mass of tissue.
The sentence is perfectly clear now!
  • I send you some comments for the “The protonmotive force and respiratory control”.

I worked in previous versions.

In general, I like more this version that the “first” ones, it is good to go deep in the topic, and to learn. However, I think a few sections can be complicate to understand (states and rates), and this can be a challenge if we want to reach a lot of people following the recommendations of the manuscript.
Anyway, it is a very great work, which is really needed for the scientific community.
  • I had been reading the 39th version of the ms ‘Mitochondrial respiratory control’ and thought the initiative really interesting and necessary. I already use most of the terminology that are refereed in the text, but I will be happy to follow this consensus-oriented recommendations. I think that all researchers in the field can gain with the initiative... in my opinion it is already a high quality text.
  • I have read through the terminology article again and I think, this is the best and most clearly written and understandable version so far.
Here you can find my minor comments to the topic:
In Fig.7. the flow is given in [pmol/s], although flux is in nmol/s*L. Is pmol written on purpose?
You make a good point. In such a general context, the units should be just given in their base form, which is now on the page
  • Timea Komlodi: I would support the changes what you have done in Fig.8. and also the table below for the description- it was a great idea, it makes it more understandable.
  • I think this has the potential to become and influential paper in the field. Please see attachment with my comments and suggestions. Please let me know if you would like me to elaborate on certain parts.


  • What a great idea to have a large group of mitochondrial investigators working collaboratively to create a consensus-orientated manuscript.
  • I have added my comments to the pdf using the comment tools in adobe.
I found it accessible and informative. I think that it will be especially useful for beginners in the field, such as new PhD students or postdocs embarking in the area.I Wish you every success with the manuscript.
Attached are my comments on the manuscript. While the technical arguments are a bit beyond my level of expertise, most of what's there reads quite well. My comments therefore are mostly all stylistic.
  • Thank you very much for writing it up, good read! Here are the changes that I would like to propose for inclusion:
1. End of mitochondrial preparations definition: I suggested to specify that control study corresponds to the case in the chamber. We can study also in vivo system that wouldn't need mitochondrial reparation as such. Proposed wording and comment at page 9 of PDF
2. High Km in permeabilized fiber. I would like to stress that the diffusion is restricted in the cells (not aggregates in chamber or unstirred water layers), that conductivity of mitochondrial outer membrane is reduced in some preparations and that tubulin interaction is a possibility. I hope you'll accept the changes with the included references. Proposed wording and comment at page 19 of PDF
3. You use steady-state for what I was taught as stationary state (thermodynamics). However, it looks to me that most in the field use steady-state these days. So, feel free to ignore it, please.
4. In one unit conversion, I would expect that division by number of cells stays (typo, I expect). Shown by comment at page 41 of PDF.
  • I have attached the edited version of the manuscript v29. Two issues of style, one no italics and the for example and that is are , e.g., and ,i.e., no parenthesis either.
  • I think it is very important and well written article to summarize previous concepts and clarify terminology. The only main question I have (maybe you have strong concept beside that) is about the paragraphs in part 3. The 3.1 is bioenergetic states , then 3.2 and 3.3 is biophysics and then 3.4 and 3.5 are normalization and conversation in general. In my personal opinion - maybe it is better to sum up 3.2 and 3.3 ? And do we need Table 9 SI prefixes (IUPAC)? - EG: Sections restructured, normalization as new Section 4.
  • First of all I would like to thank you for this really nice initiative. See attached my annotated comments, and I hope it will further strenghten the manuscript.
  • I would like to offer a few comments to the manuscript, which I found to be very good. I always appreciate your thoughtfulness and approach to science.
  • Just glancing at it, it seems to be in a great shape and have improved significantly since the last time I read it.
2017-09-04 Version 35
  • As already cited, I am very interested to possible/potential influences on so-called ROX. Specifically, the level of ROX may and may be influenced by various O2 consuming enzyme activities which in turn may be modified by the concentrations of oxygen in medium. For these considerations I began to highlight this possible reciprocal influences. Thereby and briefly ( the text may be also further expanded), I would like to suggest to modify the paragraph on ROX on your manuscript as follows:
  • P 20. ROX:… oxygen-consuming reactions in mitochondria not related to ETS, such as oxygen consumption in reactions catalyzed by monoamine oxidases.., (type A and B), monooygenases ( cytochrome P450 monooxygenases), dioxygenase (sulfur dioxygenase and trimethyllysine dioxygenase), several hydoxylases and so on. It is important to stress that the interplay between mitochondrial respiratory complexes, oxygen concentrations and the important enzymes activities has a significant reciprocal regulatory role, which should be clarified in all its physiological and pathophysiological implications, thereby also influencing oxygen consumption measurements.
  • Because of my brief career in the world of mitochondrial breathing, the article has served me to my training much more than my modest contributions can serve to you. .. I would like to thank you again for the opportunity that you have offered me in my personal formation in the exciting world of mitochondrial breathing that is fascinating me. At the moment we have done some modest works but I want to implement this line of investigation in the laboratory that I’m leading. I hope you can read some of our work in near future. For now, our work have been accepted for communication at an international congress (ECTRIMS 2017) and our plan is implement this work in order to perform an original article.
  • To make a clearer differentiation between the figure feet and the body text. Personally, sometimes it has cost me to differentiate it and consequently has made my reading less fluid. As a possible solution I suggest framing the figure feet or use a different font size than the rest of the text.
  • Personally the section that talks about the UCP I think is not very informative. I think that remaking it could be more informative, my proposal is to change that section by: There are proton channels in the inner mitochondrial membrane that allow the passage of protons from the intermembrane space to the mitochondrial matrix. As consequence there is a loss of membrane potential resulting in heat production instead of ATP.
  • EG: Your comment is included in the following form: “Uncoupling mediated by uncoupling protein 1 (UCP1) is physiologically controlled, e.g. in brown adipose tissue. UCP1 is a proton channel of the inner mitochondrial membrane facilitating the conductance of protons across the inner mitochondrial membrane. As consequence of this effective short-circiut, the protonmotive force collapses, resulting in stimulation of electron transfer to oxygen and heat dissipation without phosphorylation of ADP.”
Moreover I agree to implement the recommendations into future manuscripts, presentations and teaching materials.
  • I know, that it is non physiological situation (in swollen mitochondria) but it is possible to measure in plant mitochondria (e.g. potato tuber) a cytochrome c dependent, antimycin-A resistant respiration thanks to the NADH-cytochrome c reductase. This phenomenon was described between 1970 and 1980. Later there is no paper directly connecting an outer mitochondrial membrane enzyme with the complex IV. What do you think? Can we mention about this? Such phenomenon was not described for liver or hart mitochondria. - EG: It would be helpful to widen the perspective to plant mitochondria.
  • EG: It would be helpful to widen the perspective to plant mitochondria.
  • Wieckowski Mariusz: I made a deep search in this topic. Unfortunately, the situation I described above is very artificial. If we have intact outer mitochondrial membrane there is no (for 100%) flow of electrons from the NADH-cytochrome c reductase to the complex IV. This occurs only if we swell mitochondria (break OMM) and add external cytochrome c. Normally cytochrome c is bound to the cardiolipin… so no way in this case that such reaction will exist under physiological condition. So sorry for that.
  • I think that it is a mistake and hexokinase should be deleted. Reading this sentence I have a feeling that hexokinase among other mentioned here enzymes is DIRECTLY involved in the phosphorylation of ADP. It is well known that e.g based on the paper of Nelson and Kabir (BBA; 1985) that in mitochondria from hepatoma adenylate kinase provides about 50% of ATP that IS USED by Hexokinase. I do not know the metabolic situation in which hexokinase phosphorylates ADP. I propose to delete hexokinase.
  • EG: Hexokinase: Perhaps we need a figure for explanation. In my understanding the mitochondrial hexokinase reaction serves a function in metabolic channeling analogous to mitochondrial creatine kinase. What do you think? This could be an extension to Fig. 1B.
  • Wieckowski Mariusz: I agree with you, but reading this sentence „Page 10- ….and phosphorylation of ADP catalysed by phosphoenylpyruvate carboxykinase, adenylate kinase, creatine kinase, hexokinase and nucleoside diphosphate kinase (NDPK)." I understood that hexokinase phosphorylates ADP. Of course it is written something about kinase cycles, but still for me the next sentence has no direct connection with the info about hexokinase. Can you ask someone else if he or she has the same feeling as me?
  • 2017-09-02 EG
  • Changes in Table 4. Protonmotive force matrix
  1. ΔΨH+ to ∆Ψ
  2. Fel,H+/e to Fel/e
  3. Fel,H+/n to Fel/n
  • Whereas ∆μH+ = Fd,H+/n is the partial protonmotive force specific for proton displacement (d,H+), ∆Ψ = Fel/e is the partial protonmotive force (el) acting on charged motive molecules (ions that are displaceable across the inner mitochondrial membrane) in general.
  • Rows: isomorphic formats, e and n
  • Columns: partial isomorphic forces, Fel and Fd,H+
I really enjoyed reading the manuscript.It is great.
Please find attached PDF file with my first comments comments and proposals of additions.
  • Erich: There should be a change in writing the unit of volume (on the axes, in the text of various windows): from ml to mL.
We are not yet sure if we should change from pmol.s-1.mL-1 to nmol.s-1.L-1 – let’s be conservative (user-friendly) and stick with pmol.s-1.mL-1 for the time being.
  • Andras: Erich, one (philosophical) question: I am currently making figures, where both JV,O2 and Icell,O2 is expressed for comparison. Should I stick then to pmol.s-1.10-6 cells and pmol.s-1.mL-1 (to have pmol as the common point), or to amol.s-1.cells-1 and nmol.s-1.L-1 (with the idea to be platform-independent)? The volume-specific flux unit amol.s-1.L-1 does not seem to be a good idea, since it would mean a JV,O2 of 10.000.000.000 amol.s-1.L-1 instead of 10 pmol.s-1.mL-1.
  • Erich: Sorry for my mistake, corrected above - of course, it should be nmol.s-1.L-1. I have started to like the idea of amol.s-1.cell-1 (not amol.s-1.cells-1), since it is much simpler than pmol.s-1.10-6 cells, which more correctly might be written as pmol.s-1.(10^6 cells)-1 (??). If we do that, then we can as well go for nmol.s-1.L-1 and nmol.s-1.g-1. What do you think?
  • Andras: You are right in both above points. I am sorry, "amol.s-1.cells-1" was a typo, and pmol.s-1.(10^6 cells)-1 is the correct form.
  • I was thinking about the "per cell" approach and I also like it, since (at least for me) it gives a more direct feeling that we are expressing oxygen consumption for single cells (it is the property of the cell) and not for a bunch of cells. Expressing the flow per million cells is much more instrument related, as you pointed out previously.
  • As for nmol.s-1.L-1 and nmol.s-1.g-1: the SI-derived unit for volume is m3 (IUPAC “Green Book” 2012, p13) and liter (1 dm3) is a “non-SI unit accepted for use with the SI” (p107). In this sense, I am not sure if L is more appropriate than cm3 (=mL), as both of these units are only related to m3 (centi is a “SI prefix”: what about nmol.s-1.cm-3 ?).
  • Along these lines, the SI base unit for mass is kg (p14), and g is a CGS unit (p136), so to use nmol.s-1.g-1 instead of nmol.s-1.mg-1 is not necessarily more coherent with the IUPAC recommendations. What do you think?
2017-09-01 Version 30
This sounds like a very exciting Project! Congratulations for that terrific milestone!
  • 2017-08-30:
I have to say that I found it very interesting, and useful. Enclosed you will find some minor comments (that you will find as sticky notes in the pdf file).
The manuscript flows well although I need to tell you that in some sections (3.2, and 3.3) I have no critical input, and more biophysical knowledge than the one I have would be required to do so.
  • In my very personal taste, I would restyle the draft focusing on very few, clear and essential take home messages coupled if possible with few experiment images, more than adding further things.
  • I always bear in mind that too many informations are not digested by readers, especially from general audience but even from specialists! Basically, in my very personal view, too many sophistications risk to increase confusion to the existing confusion.
  • Just going through the new version of the MitoEAGLE manuscript. I like the figure 8 added, but I was not about the meaning of the abbreviation and the legend did not clarify it for me. Then I realized that you need Table 6 to fully understand. I was wondering if some of the definition in Table 6 that are fully needed to understand Fig. 8 should not be simply added in Fig. 8 (a little table included in a box with some symbols and expression used in the figure). I think it would make it way easier for the reader to understand the figure quickly (and to not have to look on a separate page to understand).
  • As discussed in a previous workshop together, I believe this is a great initiative since so much confusion is in the general science community, especially since the recent resurrection of the interest for mitochondria from different specialists not previously involved in mitochondrial research. My very personal point of view is that the manuscript should be a bit restyled in order to focus on LESS but VERY clear concepts that can be easily digested by non specialists, in order to have a larger audience and impact, otherwise the paper will mainly talk to specialist, many of which are at least partly familiar with these informations. Please have a look how effectively these papers addressed methodological issues on cell death and autophagy. They are very easy to read and had huge impact. I would personally try to simplify the key concepts, cut energically overclassifications and formulae, limit historical citations, and possibly add a couple of experiments to illustrate the different fluxes. ::::Limit abbreviations to a minimum. And more clearly explain what are OXPHOS and ETS, what it relies on, and what it means if 1 or both are decreased. Possibly with an illustrative trace. For the rest, with this first read, I have some other, more circumscribed comments:
  • Page 6: I don’t understand where the content of page 6 goes. The IM separates the IMS from matrix. In some place of the manuscript I would more clearly states what the Respiratory chain includes (CI-CIV) compared to OXPHOS system (Respiratory chain + CV). Also not enough highlighted that OXPHOS rely also on Krebs. - EG: see Fig. 1.
  • In page 6, other important mitochondrial components in addition to OXPHOS are mentioned, but many others have not: calcium metabolism, lipid, Coq10 and steroid biosynthesis, vitamin metabolism, etc…. Maybe just simpler to mention that the mitochondrial proteome comprises more than 1200 proteins (Mitocharta), mostly encoded by nDNA, with a fantastic variety of functions, many of which still under investigation… - EG: added.
  • I personally do not like ETS, and prefer ETC=electron transfer capacity.
  • EG: ETS - system: this is the structural and functional unit; Capacity: this relates to flux. A distinction is required.
  • Page 8: I don’t like to define mitochondrial preparations tissue homogenates, permeabilized fibers/tissues, but rather would define as they are : tissue homogenates, permeabilized fibers/tissues. Mitochondrial preparations are in my opinion mitochondrial enriched fraction or purified mitochondria (either by Percoll, sucrose or analogues). I don’t agree on the fact that these preparation largely maintain the mitochondrial structure, and very often not even the function. On the contrary most of these treatments introduce modifications in both, that can often be even quite drastic. This is one of the main limitations of the mitochondrial in vitro studies we have to accept until in vivo methods will be available, if ever. In fact one of the major plagues in mitochondrial papers, is that many groups basically measure O2 consumption on screwed out mitochondria, because of poorly prepared samples!!
  • Page 9: found not clear. Personally don’t like »P«
  • ROX: not necessarily non mitochondrial respiration in absolute terms, but in the practice the ROX should be almost undectable if the instrument has been properly calibrated. - EG: There are non-ETS enzymes catalyzing O2-consuming reactions.
  • It may be useful to mention how tricky it is to express fluxes by mass in weight g. The tissue composition in fact can change drastically between different diseases or individuals so that 1 g of dystrophic muscle will have half of its muscle mass replaced by fibrous tissue. Same is true for many other conditions, not to mention how drastically can differences in hydratation or even in the scale measurements (if very small pieces are used) of the tissue lead to errors.
  • Not really clear how to normalize for mitochondrial mass: (a) stress the importance to measure the normalizer on the SAME prep introduced into the chamber, which can be sometime difficult or impossible. (b) I don’t think TOM20 can be used to normalize respirometric data (with which technique?) (c) mtDNA quantification has been shown to be a poor marker of mitochondrial mass, and is frequently altered in diseases indedndently from mass. Better use CS or caldiolipin. (d) I personally don’t agree on recommending using FCR as internal marker, it clearly changes between individuals and even in the preparation phase
Of course I do not expect that everybody should agree, and open to discussion.
  • 1. Would it be useful to evoke/include the beta-oxidation cycle in the general framework of Fig1A ? - EG: We should do this in a follow-up part focussing on ETS pathways.
  • 2. In Fig1, H+out/O2 and H+in/»P ratios are presented as fixed ratios. My understanding is that these are the ratios expected while one NADH (and not one succinate) is oxydised in theory, e.g. not taking into account proton slipping. If I am correct, this should be precised in the fig legend ?
  • EG edited the caption: H+out/O2 is the ratio of outward proton flux from the matrix space to catabolic O2 flux in the NADH-linked pathway. ::::H+in/»P is the ratio of inward proton flux from the inter-membrane space to the endergonic flux of phosphorylation of ADP to ATP. Due to proton leak and slip these are not fixed stoichiometries.
  • 3. At the bottom of page 10, « In isolated mammalian mitochondria ATP production catalysed by adenylate kinase…. » => what is the point of this sentence? particularly regarding mitochondrial exploration ?
  • 4. Page 19, paragraph 3.1 : « The protonmotive force is high in the OXPHOS state …, elevated in the LEAK state… » I would change elevated into very high or maximum to make it clearer. - DONE.
  • Thanks again for letting me review this great manuscript.
  • As a whole the manuscript reads easily. I have just few comments/suggestions. Please note that I worked on the version 24’ / 2017-08-25.
  • After all the suggestions that have been added, I just checked in the MitoEAGLE the last version, at the moment I have no additional comments. I think it is a great contribution to the field, and it would be great if the community will start to following the recommendations.
  • I think it is time that the scientific community finally address this topic and tries to unify nomenclature and protocols so reproducible data can generated and compared across labs worldwide.
  • This is extremely well crated manuscript. Considering the expansion of the field of mitochondrial biology, standardization of nomenclature, terminology, and units will help the understanding of students and entering scientist to the field. I have attached a file with my suggestions hoping to contribute a little bit to your manuscript. My suggestions appear on pages 7 and 31.
  • With an emphasis on quality of research, data gathered can be useful far beyond the specific question of an experiment.
  • Considering mte is referring to the quantity of mitochondria, why don't we use MtQ (with subscript Q). Q being the first letter of quantity, this might deliver the message to the point more easily.
  • EG: It is not easy to introduce a symbol if it does not exist in the literature. Q is a difficult decision, being used with so many different meanings. Perhaps some other suggestions will improve on the present “mte”.
  • I would like to make two very brief comments:
  • 1. On page 28, section “Sample concentration”, the sentence “Erich will add isolated mitochondria …” is to be found. This probably waits to be accordingly adjusted.
  • 2. Based on my own microcalorimetric experience, I was wondering whether the “crowding effect”, i.e., the decrease in mass-specific metabolic rate with increasing sample size, should not have (at least briefly) been mentioned. To guarantee that the mass-specific O2 flux is independent of the size of a muscle specimen (as well as of the volume density of a cell suspension) in the measuring chamber (page26), the tissue slice must be small (thin) enough (or the cell density low enough, or the stirring rate high enough) to allow unrestricted O2 diffusion. Otherwise, the specific metabolic rate could – apparently – decrease with increasing sample size, probably due to an increasing proportion of anaerobic glycolysis in the depth of the tissue sample (or at the bottom of the cell suspension, respectively).
The problem has been extensively discussed by Richard Kemp in The Handbook of Thermal Analysis and Calorimetry [1], referring to a model calculation that I had published some years before [2].
  1. Kemp RB, Guan YH: Microcalorimetric studies of animal tissues and their isolated cells. In: Handbook of Thermal Analysis and Calorimetry, ed by Gallagher PK, vol 4, From Macromolecules to Man, ed by Kemp RB; Elsevier, Amsterdam 1999, chapt 11, pp 557-656
  2. Singer D, Schunck O, Bach F, Kuhn HJ: Size effects on metabolic rate in cell, tissue, and body calorimetry. Thermochim Acta 1995; 251: 227-240
  • I agree with the almost final version of the text. I will follow the final position of all the partners but better BBA and Cell Metabolism if accepted. I find the text very pedagogic indeed.
  • I find the text clearly exposed even if the publicity for Mitoeagle is to much pronounced (but that is a choice).
Fig. 8A. Mitochondrial yield, Ymt, in preparation of isolated mitochondria.
Fig. 8B. Respirometry with tissue homogenate.
  • 2017-08-28 EG
  • Title: from 'Mitochondrial respiratory control: MitoEAGLE recommendations' to 'The protonmotive force and respiratory control: Building blocks of mitochondrial physiology'
  • Figure 8 and Table 6: mte vs. mt
This looks great. Thanks for the edits.
I think the project is an excellent idea and very timely.
  • 2017-08-27
  • Table 4 update by EG
  • Protonmotive force, symbol ∆pH+ changed from previous symbol ∆pmt, for consistency of force and flow, FH+/e and IH+/e
pH+ = ∆Ψmt + ∆µH+ / F ; (Eq. 1)
  • 2017-08-27 Morten Scheibye-Knudsen
  • I think the mitochondrial marker section is a bit simplistic and I would suggest including something about appropriate markers (for example use markers of beta-oxidation if you are investigating beta oxidation etc.).
  • EG: Perhaps TOM20 is the better marker, if fatty acid oxidation (FAO) capacity is selectively stimulated relative to NADH- and succinate-linked pathway capacity, then enzymes involved in FAO are a good marker for FAO but a bad marker for mitochondrial content.
  • I think these are excellent points and I think the sentence you have added is adequate. Regarding a symbol for mitochondrial content based on markers, I do not think such a symbol exists and I think you can therefore use the "mte" abbreviation as you do now.
  • Regarding the journal, if you get a large consensus which includes a large contingency of authors I guess this could be of interest for a higher level journal because it could act as a reference point. Perhaps even something like Cell Metabolism. - I have attached a version with a some suggestions as you can see. It is obviously a great reference manuscript for mitochondrial investigations and I have only a few corrections/comments.
2017-08-26 Version 25: see Table 6
  • Is there a general symbol available for amount of mitochondria, as estimated by a mitochondrial marker?
  • Amount of mt-elements, mte (= quantity of mt-marker)
  • Mitochondrial concentration in the instrumental chamber with volume V, Cmt=mte∙V-1
  • Specific mitochondrial density in tissue X with tissue mass mX, Dmt=mte∙mX-1
  • Mitochondrial content per cell, mtecell=mte∙Ncell-1 (where Ncell is the number of cells)
  • 2017-08-25 Hernansanz Pablo:
I have received the open invitation for the manuscript 'Mitochondrial respiratory control' and I think it is worth to mention the influence of monovalent cations on respiration.
  • 2017-08-24
  • 2017-08-24 David Fell
  • I've read through your draft and I think there is an issue that needs to be clarified, which is the distinction between 'responds to' and 'controlled by'. In MCA terms, this is equivalent to the difference between (flux) control coefficients and response coefficients. I could prepare a short document for you and your co-authors to explain my thinking on this issue. If you accept my points, the second step would be to work out how to alter the text without increasing its length. A question I have for you as well is: when you say 'respiratory control' are you exclusively concerned with control of oxygen consumption flux, or do you mean more generally it and other variables of the system, such as phosphorylation flux, or even PMF? If the latter, then there needs to be some clarification about that, in my view.
  • EG: You raised two good and important points. I would welcome clarification between ‘responds to’ and ‘controlled by’, and editing critically the present version of the ms. will be a big improvement. Yes, ‘respiratory control’ should be clarified in a wider sense not restricted to oxygen flux, since we explicitely talk about states and rates. Again, clarification will help.
  • The terminology here is a bit loose, and come close to invoking the idea of the "high energy" phosphate bond. See discussion in Nicholls's book on this term. Furthermore, all chemical processes in the cell occur in the "eneretic downhill direction". I am not sure if these two symbols are needed. But if needed, then I suggest the definition is that >>P refers to ATP synthesis from ADP and Pi, and >>P refers to ATP hydrolysis.
  • EG: Is this better?: phosphorylation in the context of OXPHOS is clearly defined as phosphorylation of ADP to ATP. On the other hand, the term phosphorylation is used in the general literature in many different contexts, e.g. protein phosphorylation. This justifies consideration of a symbol more discriminative than P as used in the P/O ratio (phosphate to atomic oxygen ratio), where P indicates phosphorylation of ADP to ATP or GDP to GTP. We propose the symbol »P for the endergonic direction of phosphorylation coupled to catabolic reactions, and likewise the symbol «P for the corresponding exergonic hydrolysis (upwards and downwards arrows, respectively, in Fig. 2).
  • Which ATPase activity are we talking about here? I think it is a contaminating ATP hydrolysis activity, not the ATP synthase activity. Need to clarify. - YES-CLARFIED.
  • In this situation, ATP synthesis is greater than 0 because ATP hydrolysis is greater than zero. Thus, the inequality here is correct, but could lead to confusion as an incomplete statement.
  • EG: Oxygen consumption in State 4 is an overestimation of LEAK respiration if the contaminating ATP hydrolysis activity recycles some ATP to ADP, J«P, which stimulates respiration coupled to phosphorylation, J»P>0.
  • By this definition, would calcium current be interpreted as a proton leak? Ca current is balanced by Na/Ca exchange, which is balanced by Na/H exchange or K/H exchange.
  • EG: No, this is cation cycling, not a property of the membrane. Calcium current is balanced by Na/Ca exchange, which is balanced by Na/H exchange or K/H exchange. This is another effective uncoupling mechanism different from proton leak and slip. Should this reference be added?
Vinnakota KC, Singhal A, Van den Bergh F, Bagher-Oskouei M, Wiseman RW, Beard DA (2016) Open-loop control of oxidative phosphorylation in skeletal and cardiac muscle mitochondria by Ca2+. Biophys J 110:954-61.
  • I think that this would be a better reference for the contribution of calcium cycling to leak current: Stucki and Ineichen. Eur. J. Biochem 48:365-375 (1974). I wonder if we should still call calcium cycling a “leak”. It is a “leak current” in the language of electrophysiology. I wonder if it would be worth make this distinction clear: Proton leak is a leak current of protons. There can be other cation contributors to leak current including calcium and probably magnesium (but I don’t know the magnitude.) Under physiological conditions the proton leak is the dominant contributor to the overall leak current.
  • Figure 1A: It might be simpler to show just one single arrow rather than multiple arrows between CI/CII and Q. I like the introduction of the term ">>P”, but when I first looked at Figure 1 I didn't understand what the ">>" represented because it hadn’t yet been defined, so consider defining it in the figure legend.
  • EG: Yes, the definitions are added to the figure legend, where the multiple convergent electron transfer pathways are also explained.
  • Figure 1B: The use of "-“ to reflect stoichiometry could be confusing to some readers, and it might be worth explaining it in the legend.
  • Page 9: I find the term "evolutionary background" confusing. In this context, "evolutionary or acquired differences in the genetic basis of mitochondrial function (or dysfunction) between subjects" would be clearer, at least from my perspective.
  • Figure 3: Should ATPase activity and the J<<P arrow be shown here? Could it be removed or somehow shown as lower than the rate of phosphorylation? Showing it as it is currently is relevant to intact cells, but less relevant to isolated mitochondria with low ATPase activity.
  • EG: – But highly relevant for permeabilized cells and fibres.
  • Page 16: I think a statement, either here or earlier, that explicitly distinguishes the terms uncoupled, noncoupled, and dyscoupled would be valuable. As written, the distinction is only made implicitly, but I think it would be valuable to new researchers to have the meanings spelled out clearly.
  • Page 18, last two sentences: I think the point of these two sentences could be made more clearly.
  • Page 35, “300 mitochondria per cell”: I think it would be better to express this idea by instead using the volume density of mitochondria per cell.
First, I want to thank you for sharing the manuscript. Since I am more a "mitochondrial geneticist" than a mitochondrial physiologist, I found the manuscript on mitochondrial respiratory control very clear and well written, and it will be very helpful for my students (and also for myself actually)!! I have very few edits (attached is the manuscript).
  • 2017-08-24 Buettner Garry
I fully appreciate pushing the community to a common language and lab approaches so that data gathered can be useful far beyond the specific question of an experiment.
I will review the manuscript and pass along my comments.
Please find the attached word document with some minor suggestions for your consideration.
  • 2017-08-24 Gan Zhenji:
The review looks great.
I will read the present manuscript (that looks great) and give my comments if necessary.
  • 2017-08-24 Orynbayeva Zulfiya
I am glad to make my contribution to this important work.
Congratulations for this great piece of work...This work will be very helpful to me because I am invited at the next European College of Equine Internal Medicine congress with an one hour presentation dealing with mitochondrial function in equine neuromuscular disorders.

The review will help me since I will try (as your review) to be educational, basic and consensus-oriented.

This is great news! I have gone through the paper very broadly and it seems that a lot of the text is already there!
This is a great idea.
I am sending part of my feedback regarding the publication on the MitoEAGLE recommendations.
2017-08-23 Version 22' (see Table 4)
  • Is there a general symbol available for amount of mitochondria, as estimated by a mitochondrial marker?
  • Amount of mt-elements, mte (= quantity of mt-marker)
  • Mitochondrial concentration in the instrumental chamber with volume V, Cmte=mte∙V-1
  • Specific mitochondrial density in tissue X with tissue mass mX, Dmte=mte∙mX-1
  • Mitochondrial content per cell, mtecell=mte∙Ncell-1 (where Ncell is the number of cells)
  • I think this is a strong and highly useful treaty (more than a paper) that I would be glad to participate in. I agree with the removal of the part of the text that mentions a "second definition" of state 2.
  • I also have some comments and corrections (see my comments and suggestions in attached file):
  1. Figure 1 - since fatty acids are mentioned previously, I would include also fatty acid oxidation pathway in Figure
  2. p. 8 - the "protein phosphorylation" is more common term, that also includes phosphorylation of enzymes. Suggestion: change "phosphorylation of enzymes" to "protein phosphorylation"
  3. p 12 State 4 definition. What about state 4 (or state 4o) as state after inhibition of phosphorylation system by Omy or Catr? suggestion is to mention this conditions also at state 4 definition.
  4. p 30. Add sentence of example - "For example, endurance exercise training increases citrate synthase activity."
  5. p 34 description of substrate-level phosphorylation - I believe there is a mistake. There should be "glucose" instead of "glycogen"
  6. p 35 include also respiratory reference state as mitochondrial marker
  7. The thing I missing is scheme of connection of classical Chance terminology to the recommended one. If you don't mind I'll think a little bit how to make this visible, and provide version of scheme or modification of Figure 6 later.
  • I really enjoyed the manuscript - clear definitions that are about meaning of processes not just numbers.
  • About Table - super idea!
  • 2017-08-23 Buettner Garry
Your editing to include our thoughts is wonderful. I offer a few suggestions and minor edits.
very good initiative, I fully support it.
I think this is a very important project, and it will be fantastic to have this resource (and your future MitoEAGLE work) available to mitochondrial physiologists. I’m happy to add my name as a co-author if it is helpful. I don’t have much to add to the manuscript, its already very clear and I agree whole-heartedly with nearly all of what is written. I have a few editorial comments for you to consider below.
I am excited to be a part of such an important endeavor.
  • I gave it a quick reading and it looks really great!
  • I think that this is a great service to the field. I have made some brief comments in the attached.
  • I agree with you and the purpose and philosophy that the paper pursues.
  • The manuscript is attached with some suggestions included.
  • This is a timely and important manuscript... I will send you my comments on the manuscript in a few days.
  • 2017-08-22 Orynbayeva Zulfiya
  • I found this initiative very important and timely from both scientific and educational perspectives. In our days in many schools the bioenergetics does not get an appropriate attention on its fundamental role in normal physiology and pathology. Unfortunately, many Master and PhD programs do not include mitochondria-centered research of metabolic processes, while the static molecular biology and genomic aspects are intensively studied. The conventional mitochondriology methods are considered the old style and not trivial. I strongly believe that this manuscript is of absolute importance.
  • In collaboration with my supervisor, Professor Pedro F. Oliveira, we have read the latest version of the manuscript and made some suggestions mentioned as annotations in the PDF file (annex). We want to congratulate you and all the contributing co-authors for the excellent work.
  • EG: Many thanks for your helpful suggestions. I have incorporated almost all of your changes in the upcoming version 22, contributing together with previous updates to an improvement of our ms.
  • I attached the PDF file with my comments. The paper is excellent material for all oxygraphists and bioenergetics. It is also in perfect agreement with the scientific viewpoints of our laboratory.
  • Particularly important is the part of thermodynamics, because of many researchers tend to forget this component of bioenergetics.
  • EG: I would like to share Tuuli’s point of view, representative of several comments received during the past few days. Nevertheless, I hope that we can move more ‘heavy’ text into tables and notes to tables, to make the final version more easily readable. I invite our co-author’s comments on the suggestions for ‘new’ symbols (see Tab. 4), which we have to introduce if IUPAC guidelines are insufficient (I am not aware of consistent symbols in the literature). I am particularly fond of Table 3, which gives so much more ‘straight’ insight into the protonmotive force, when ‘seen’ together with flux in the two ‘isomorphic’ formats.
  • Overall, I think it is a valuable position statement, which neatly conveys the need for a universal nomenclature on respiration states (i.e. not restricted to a particular protocol) whilst giving necessary context to the historical states. Thank you for bringing this all together.
  1. I can see that the section on normalisation has greatly expanded. I think this is a really valuable section and gives a very thoughtful consideration to the different options for normalisation. I particularly appreciated the section on the use of mitochondrial markers, and the warnings about the potential pitfalls of using such markers. It is not absolutely necessary but under the problem of accuracy of measurement, I would be tempted to highlight the potential issues of measuring a marker in the contents of the oxygraph chamber at the end of a respirometry protocol, when recovery of e.g. enzyme activity may be less than 100%. Overall, I think this section makes a strong case for the use of FCR without being forceful.
  2. It would be my own personal preference, but I would switch the order of the two sections "Mitochondrial concentration, Cmt, and mitochondrial markers" and "Mass-specific flux, JmX,O2" (Page 27). This way, the discussion of the use of mitochondrial markers follows on from the introduction of possible markers.
  3. I agree with the focus of the manuscript being on mitochondrial preparations (with reference to intact cells where appropriate), but what might be missing for the non-expert reader might be the purpose of using such preparations, as opposed to intact cells. I would suggest a sentence or two following the definition of mt-preparations (pages 5 and 6), which can lead into the next section. Feel free to edit, but perhaps something along the lines of: "The (non-permeabilized) plasma membrane prevents the free passage of many water-soluble mitochondrial substrates and inhibitors, limiting the scope of investigations into mitochondrial respiratory function, whilst the cycling of ATP/ADP in intact cells prohibits an analysis of the control exerted by ADP on respiration."
  4. A minor, pedantic point on page 14. Proton slip can also happen at the ATP-synthase, in which case the proton will not slip back to the original compartment (as stated in the definition), but slip downhill to the matrix without contributing to ATP synthesis. Would you class this as proton leak or proton slip? I have seen both terms used in the literature.
  5. Page 20, last line... change spelling of "Mitchel's" to "Mitchell's".
  • I think it looks really good! It does get very complex at times, but so is the underlying information that is to be communicated. I have suggested a small addition to the normalization chapter with basically just a short paragraph trying to tie the chapter together and also add a few examples to clarify. In addition to this I have also suggested a few minor changes that I think increases the readability of a few sentences. As I said, these are complex questions and will probably benefit from being clear and easy to read.
  • One thing that I realized as I read the manuscript is that brackets [] are used quite freely and somewhat irresponsibly. In several cases the brackets should be exchanged for parenthesis () as to not be confused with concentrations. It is important to remember the target audience, and even if a lot of the information goes closer to physics, most of the readers are likely to be biologists, chemists and such. Therefore I think we should try and look over where the brackets are actually necessary and exchange them for parentheses where they aren't.
  • A few “minor” comments. The manuscript is looking pretty clear and comprehensive.
  • EG: Thanks – it’s in version 21‘ on the page. ‘Conservation of energy’ – of course, it should be ‘exergy’ (not in the sense of First-Law energy conservation), but this will be later in the ms. To avoid unnecessary confusion, I just deleted the ‘exergy conservation’ part of the sentence, it is not necessary to explain ‘uncontrolled’
  • I have carefully red version 20 and in my opinion is a fantastic peace of work. Up to this moment I did not have any recommendation for changes or improvements. I will follow the new versions. It is a honor for me to participate in this project.
  • I wish you could add the following article as a reference: "Cellular allometry of mitochondrial functionality established the optimal cell size" by Miettinen TP and Bjorklund M, Developmental Cell, 39:370-382, 2016.
  • Here is my revision and suggestions to the manuscript. Thank you for putting all that work together in a very helpful review for mitochondrial scientists. I enjoy reading the review and found it incredibly helpful. Some section, however, are a little harder to follow and not easy to read, mostly because the amount of formulas and units. I felt like the main message is lost in the details. I added my comments directly in the document.
  • Again, thank you very much for the invitation in this excellent and seminal work. I am sending the PDF file with some comments and additions.
The term "ATP production" itself is wrong because one is not measuring real ATP production (ex: nmol ATP produced/min/mg protein). This should also be stressed!
  • EG: Thanks a lot for your very positive feedback, being incorporated into Version 21.
– YES - can you make a helpful suggestion?
How about: "When refering to oxygen fluxes, the term "ATP production" is itself misleading. Measuring oxygen fluxes during active ADP phosphorylation cannot be used as a direct measurement of the amount of ATP synthesized by mitochondria (i.e. nmol ATP produced/min by mg protein or by any mitochondrial marker)."
Would this be clear?
  • Been through the previous version a time or two and mostly understand and agree. The primary part that is giving me pause is the discussion on correcting flux values for external mitochondrial markers. Working on formulating my thoughts coherently.
  • EG: Here an important distinction:
•Correction: for instrumental or chemical background, to eliminate methodological artefacts.
•Normalization: for external or internal markers – this is different from ‘correction’, it is putting the data into specified contexts.
  • My biggest concern is the approach to normalization. As articulated in the comments within the manuscript, I do not believe that we currently have an external marker that meets the requirements of representing mitochondrial abundance, either from a qualitative or quantitative point of view. No external marker can be assured to have a fixed relationship with mitochondrial quality or quantity in order to serve as a surrogate or indicator for those properties, so to suggest that normalization in this manner is necessary and valid is not correct (in my opinion). This does not mean that expressing flux as a function of these proposed markers is incorrect – depending on the question being asked, it may be quite valid and enlightening to express flux as a function of enzyme X, Y, or Z. But I don’t believe that such approaches should be used at this time to extrapolate to mitochondria as a complex functional organelle.
  • 2017-08-21 Garry Buettner
  • On manuscript pages 11 and 12, you use the term “saturating levels of oxygen”. My first reaction was that the atmosphere was 100% O2. I thought you should rather have air-saturated.
  • But I soon realized this was meant to refer to Km. Perhaps use an appositive such as , >>Km, in the text and figure caption. The term saturating with gases can be confusing.
  • In a redox biology work, I take great care on the use of “reduce” -- lower values? or gain electron(s)? - EG: I agree. Since ‚saturating‘ may be insufficiently discriminated from ‘saturated’ levels of oxygen, I added now throughout the text ‘kinetically saturating’ .. Chuck Hoppel (whom I regard as one of our top experts) suggested to replace ETS state by oxidative state (OX). I guess this would not be welcome by redox biologists?
  • Ich finde die Tabellen generell sehr hilfreich und Informativ. Ich muss aber sagen, das das Teil „Normalisation: flows and fluxes“ ein bisschen vielleicht zu viel ist. Ist natürlich korrekt und ausführlich, aber für genau so ein Teil wäre meiner Meinung nach auch eine sehr klare, sehr direkte Zusammenfassung für LeserInnen nötig, wer schnell Referenz und klare Richtlinien suchen.
  1. I think, the noncoupling, uncoupling and discoupling should be defined. We discussed it with the Andras, Luiz and Zuzana on Thursday that a brief description would be helpful for the reader to clarify everything. It might be written in the 2.1 Section (definitions). The term controlled and uncontrolled might be confusing. We might distinguish what is the difference between coupled and controlled versus noncoupled and non-controlled.
  2. The definition of coupling, efficiency and power should be moved before the coupling versus bound processes. Firstly, the definition should be explained and after that a difference between coupling and bounding. Moreover, I found this sentence in the coupling versus bounded processes redundant (“Coupling occurs in an energy transformation between processes, if a coupling mechanism allows work to be performed on the endergonic or uphill output process (work per unit time is power; dW/dt [J/s] = Pout [W]; with a positive partial Gibbs energy change) driven by the exergonic or downhill input process (with a negative partial Gibbs energy change.)”) with the following in the next section: “In energetics (ergodynamics) coupling is defined as an exergy transformation fuelled by an exergonic (downhill) input process driving the advancement of an endergonic (uphill) output process.”
  3. From my point of view, Table 3 is excellent and helpful according to our Thursday session, which I found interesting and now it is much more understandable.
  4. I think that the paragraph Molar quantities should be moved to the 3.4 section, because you are speaking in this paragraph about intensive and extensive quantities which is defined only later in section 3.4. It might be moved after the Extensive quantity paragraph.
  5. At page 28: “This problem is avoided when O2 fluxes measured in substrate-uncoupler-inhibitor titration protocols are normalized for flux in a defined respiratory reference state, which is used as an internal marker and yields flux control ratios, FCR (Fig. 7).”
  6. Does it refer only to the SUIT protocols? I think, we should write there for example in SUIT protocols.
  7. From my point of view, these new tables are quite helpful, the understanding is much more easier and the text is more traceable.
Table Protonmotive force matrix.jpg
2017-08-21 New Table 3 in Version 21
  • Bernard Tandler and I went through the manuscript and edited for English style. Additionally, suggest that this is two manuscripts. One the terminology and the second a biophysical discussion of the subject with representative equations as required for explanations. I suggest to separate so we make a clear and declarative statement about terminology. Then the important biophysical component stands on its own.
  • Also in Figure 2 I find the use of ETS under transport &..., Membrane-ETS, and to describe the overall process confusing. From substrates moving through the outer mitochondrial membrane, transported through the inner mitochondrial membrane, and metabolism by dehydrogenases producing NADH or FADH to enter the electron transport system, this seems to me to be the oxidative system.
  • I have read the MS, thank's for putting things together. I have added my comments in the pdf attached. I like the newly added part on mitochondria in generell. The part on protonmotive force is ambitious.
  • I added very few changes in the manuscript. Also, I think that could be useful to include a definition for control of metabolism and regulation. In one of my commentaries, I wrote a couple of sentences from David Fell's book that could be interesting. I know that is adding more text but is just a few words.
  • 2017-08-19 Garry Buettner
  • Ad MITOEAGLE: Have all CAPS is a bit gaudy. It makes text look strange and uninviting. Because „ito“ are not „first“ letters, I suggest lower case.
  • EG: I agree. MITOEAGLE was originally used in our successful COST Action grant application, accoring to the COST rules to use capital letters for the project acronym. Since then I have seen several COST projects using small and capital letters for their acronym. MitoEAGLE looks better. Probably, we will need a Management Committee E-vote. Let's try MitoEAGLE for the manuscript, and decide on the general strategy. .. You hit exactly the target of MitoEAGLE. With an emphasis on quality of research (and life), “data gathered can be useful far beyond the specific question of an experiment.” .. Added in the intro: The mission of the MitoEAGLE network aims at developing harmonized experimental protocols and implementing a quality control and data management system to interrelate results obtained in different studies and to generate a rigorously monitored database on mitochondrial respiratory function under varying experimental conditions. With an emphasis on quality of research, data gathered can be useful far beyond the specific question of an experiment.
    Fig. 7. Different meanings of rate may lead to confusion, if the normalization is not sufficiently specified.
  • I would use L and g rather than mL and mg. Although l for liter is still in use, L is better and is the future. For example, the American Chem Soc Journals go with L as do many others. Elsevier is lagging, but becoming flexible.
  • EG: This makes sense, and we should get used to it. I added a conversion table, to facilitate harmonization.
  • Attached are my specific comments. I have added them to Garry's version, in the hope that this may make it easier for you.
  1. Concerning normalization, i.e., the "mg protein" versus "cell" issue Garry raised, "cell" is limited to cellular experiments, whereas "mitochondrial preparations" (introduced earlier) refer to tissue and cells. As there are many applications using (permeabilized) tissue, fibers etc (we have even used zebrafish hearts), I suggest to either use a single normalisation that serves all purposes or then give specific recommendations for different applications. Also, there are arguably additional parameters that one may wish to use to standardise respiration, e.g., mitochondrial mass. The choice of normalisation may well depend on the biological question the user wishes to address, and I suggest to make this point clear.
  2. Figure 2 refers to "ROS", though this aspect is not discussed further in the article. If you wish to retain reference to electron leak to oxygen in the Figure, I recommend that you refer to the univalent reduction of oxygen to superoxide anion radical instead, as this is the known product, whereas "ROS" is not well defined, and we probably don't want to recommend its use.
  3. It may be worth considering using the term dioxygen instead of oxygen; in any case, I suggest to use one term only if possible, and the abbreviation (O2) consistently after its introduction.
  • 2017-08-18 Garry Buettner
  • I think in the section on whole cells a somewhat different approach to normalization would be better. I offer my suggestions and some edits with that, but the edits are not complete as your reaction to the suggestion is needed.
  1. In our work with whole cells, be it bioenergetics or other goals, we normalize everything to cell number to produce mol cell-1 or mol cell-1 s-1 . With oxygen consumption by cells attomoles is most convenient, so we use amol cell-1 s-1 . This allows scaling to be simple. We use cell density asl cell L-1 . Results mesh directly with M. Scaling and manipulations are easy with many fewer mistakes by beginners. See Wagner BA, Venkataraman S, Buettner GR. (2011) The rate of oxygen utilization by cells. Free Radic Biol Med. 51:700-712. PMID: 21664270 http://dx.doi.org/10.1016/j.freeradbiomed.2011.05.024 PMCID: PMC3147247
  2. In cell culture, mol cell-1 should be the coin of the realm See Doskey CM, van ‘t Erve TJ, Wagner BA, Buettner GR. (2015) Moles of a substance per cell is a highly informative dosing metric in cell culture. PLoS ONE 10(7): e0132572. PMID: 26172833 http://dx.doi.org/doi:10.1371/journal.pone.0132572 Open Access PMCID: PMC4501792
  3. An application of this is in: Doskey CM, Buranasudja V, Wagner BA, Wilkes JG, Du J, Cullen JJ, Buettner GR. (2016) Tumor cells have decreased ability to metabolize H2O2: Implications for pharmacological ascorbate in cancer therapy. Redox Biology. 10:274-284. PMID: 27833040 http://dx.doi.org/10.1016/j.redox.2016.10.010 Open Access PMCID: PMC5106370
  4. On manuscript pages 11 and 12, you use the term “saturating levels of oxygen”. My first reaction was that the atmosphere was 100% O2. I thought you should rather have air-saturated. But I soon realized this was meant to refer to Km. Perhaps use an appositive such as , >>Km, in the text and figure caption. The term saturating with gases can be confusing.
  5. In a redox biology work, I take great care on the use of “reduce” -- lower values? or gain electron(s)?
  • I have completed the review and English editing with my colleague Bernard Tandler, Ph.D.. I am working to add these in edit mode. I worked on version 9 and when the version came from Javier I switched to using that version. I realize version 10 has now come. I will complete the editing on version 9 and send. Then I will compare to version 10.
  • The manuscript appears to be two different stories. One on physiological terminology and the other more biophysical. I will recommend making these two different manuscripts.
  • We have read the most updated version of the file (version 18), and we do not want to add further comments. Therefore, the article has our approval.
  • I applaud you and the co-authors on an excellent first step toward (re)defining terminology and concepts on mitochondrial respiratory control. While the definitions and explanations are very carefully stated, I agree with previous comments that the overall length and level of detail might limit how well the key concepts and terms are grasped and adopted by what we hope will be a broad readership of physiologists and biochemists.
  • To help with this, I suggest including a "Table 2" that summarizes essential aspects of the 4 primary coupling states defined in section 2, which would compliment the already included classic Chance and Williams states in Table 1. I've attached working version that would be useful from my perspective that includes a succinct definition of LEAK, OXPHOS, ETS and ROX, with information on corresponding MMP, resp. rate, limiting and induction factors).
  • It also seems appropriate, given the topic of this review, to include a careful definition of the general coupling control factor (1-L/P) here a surrogate for RCR still widely used in the literature as an index of "coupling control" ... I have also included a couple of minor comments/questions on the attached pdf for your consideration.
  • 2017-08-18 Erich edited some equations in Version 18:
  • Protonmotive force, ∆pmt: The protonmotive force, ∆pmt,
pmt = ∆Ψmt + ∆µH+ / F ; (Eq. 1)
is composed of an electric part, ∆Ψmt, which is the difference of charge (electrical potential difference) across the inner mitochondrial membrane, and a chemical part, ∆µH+/F, which stems from the difference of pH (chemical potential difference) across the inner mitochondrial membrane and incorporates the Faraday constant, F. In other words, the protonmotive force is expressed as the sum of two terms, with somewhat complicated symbols in Eq. 1, which can be more easily explained as isomorphic partial protonmotive forces.
  • Electrical, el: Fe,el = ∆Ψmt is the electrical pat of the protonmotive force expressed in units joules per coulomb, i.e., volt [V=J/C], and defined as partial Gibbs energy change per motive elementary charge of protons, e [C]. Fn,el = ∆ΨmtF is the electrical force expressed in units joules per mole [V=J/mol], and defined as partial Gibbs energy change per motive amount of charge, n [mol].
  • Chemical, diffusion or dislocation, d: Fn,d = ∆µH+ is the corresponding chemical part of the protonmotive force expressed in units joules per mole [J/mol], and defined as partial Gibbs energy change per motive amount of protons, n [mol]. Fe,d = ∆µH+/F is the chemical force expressed in units joules per coulomb, i.e., volt [V=J/C], and defined as partial Gibbs energy change per motive amount of protons expressed in units of electric charge, e [C].
  • Faraday constant, F: The Faraday constant is the product of the elementary charge and the Avogadro (or Loschmidt) constant, F = e∙NA [C/mol], and yields the conversion between protonmotive force, Fe = ∆pmt [J/C], expressed per motive charge, e [C], and protonmotive force or chemiosmotic potential difference, Fn = ∆pmtF [J/mol], expressed per motive amount of substance, n [mol],
Fn = FeF ; (Eq. 2.1)
Fe = Fe,el + Fe,d = ∆Ψmt + ∆µH+/F ; e-isomorph [J/C=V] (Eq. 2.2)
Fn = Fn,el + Fn,d = ∆ΨmtF + ∆µH+ ; n-isomorph [J/mol] (Eq. 2.3)
  • Protonmotive means that protons are moved across the mitochondrial membrane at constant force, and the direction of the translocation is defined in Fig. 2 as H+in → H+out,
Fn,d = ∆µH+ = -ln(10)∙RT∙∆pHmt ; (Eq. 3)
where RT is the gas constant times absolute temperature. ln(10)∙RT = 5.708 and 5.938 kJ∙mol­-1 at 25 and 37 °C, respectively. ln(10)∙RT/F = 59.16 and 61.54 mV at 25 and 37 °C, respectively. For a ∆pH of 1 unit, the chemical force (Eq. 3) changes by 6 kJ∙mol­-1 and the protonmotive force (Eq. 2.2) changes by 0.06 V.
  • Since F equals 96.5 (kJ∙mol­-1)/V, a membrane potential difference of -0.2 V (Eq. 2.2) equals a chemiosmotic potential difference, Fn, of 19 kJ∙mol-1 H+outSubscript text (Eq. 2.3). Considering a driving force of -470 kJ∙mol-1 O2 for oxidation, the thermodynamic limit of the H+out/O2 ratio is reached at a value of 470/19 = 24, compared to a mechanistic stoichiometry of 20 (H+out/O=10).
  • In general this is very well written text!
  • Attached my comments to the manuscript (version 17 from the MitoEAGLE homepage).
  • Many thanks for your kind offer, putting together a review on Mitochondrial respiratory control is such a great idea and would be of great value to the community. I’d be delighted to be a part and support this review.
  • "Alternatively, coupling of electron flow and phosphorylation is disengaged by uncouplers which induces a burst of oxygen consumption without performance of biochemical work (Fig. 1)."
  • What I intended to say is that "performance of biochemical work" could be an ambiguous term “Energy production“? – I do not like it (1st law of thermodynamics?). i agree with you if we follow first law of thermodynamics we probably should link uncoupling to HEAT and coupling to WORK or BIOCHEMICAL WORK.
  • Great job!
  • The amendment to the abstract is excellent. I also agree with the force/energy argument - I was being a bit "physicist" about it.
  • Leak:“when oxygen flux is maintained mainly to facilitate to compensate for the proton leak at a high chemiosmotic potential, when ATP synthase is not active.” - EG: NOT facilitate the proton leak, but to drive protons back, i.e. compensate for the proton leak. - Brown RA answer: Trying to be helpful; I see facilitate is not a good word but is there not a better way of describing what is in yellow – the addition of to drive protons back made it clearer to me than the word compensate – many thanks.
  • “when oxygen flux is maintained mainly to try to compensate for loss of PMF due to proton leak at a high chemiosmotic potential, when ATP synthase is not active.” (comment - if compensation does not work mito fails; no? This phrasing allows for return to steady state, failure and inbetween)
Dorit: Attached is the conclusion part in which I suggest to introduce 2 sentences marked in yellow.
  • 2017-08-17 EG
  • Perspective added in Version 17: To provide an overall perspective of mitochondrial physiology we may link cellular bioenergetics to systemic human respiratory activity, without yet addressing cell- and tissue-specific mitochondrial function. A routine O2 flow of 234 µmol∙s-1 per individual or flux of 3.3 nmol∙s-1∙g-1 body mass corresponds to -110 W catabolic energy flow at a body mass of 70 kg and -470 kJ/mol O2. Considering a cell count of 514∙106 cells per g tissue mass and an estimate of 300 mitochondria per cell (Ahluwalia 2017), the average oxygen flow per million cells at Jm,O2peak of 45 nmol·s-1·g-1 (60 ml O2·min-1·kg-1) is 88 pmol∙s­-1∙10­-6 cells, which compares well with OXPHOS capacity of human fibroblasts (not ETS but the lower OXPHOS capacity is used as a reference; Gnaiger 2014). We can describe our body as the sum of 36∙1012 cells (36 trillion cells). ::::Mitochondrial fitness of our 11∙1015 mitochondria (11 quadrillion mt) is indicated if O2 flow of 0.02 pmol∙s­-1∙10-6 mt­ at rest can be activated to 0.3 pmol∙s-1∙10-6 mt at high ergometric performance.
Fig. 6. Four-compartmental model of oxidative phosphorylation with respiratory states (ETS, OXPHOS, LEAK) and corresponding rates (E, P, L). Modified from Gnaiger (2014).
  • 2018-08-16 EG - added Fig. 6 and text in Version 16
  • Fig. 6 summarizes the three coupling states, ETS, LEAK and OXPHOS, and puts them into a schematic context with the corresponding respiratory rates, abbreviated as E, L and P, respectively. This clarifies that E may exceed or be equal to P, but E cannot theoretically be lower than P. E<P must be discounted as an artefact, which may be caused experimentally by (i) using high and inhibitory uncoupler concentrations (Gnaiger 2008), (ii) non-saturating [ADP] or [Pi] (State 3), (iii) high oligomycin concentrations applied for measurement of L before titrations of uncoupler, when oligomycin exerts an inhibitory effect on E, or (iv) loss of oxidative capacity during the time course of the respirometric assay with E measured subsequently to P (Gnaiger 2014). On the other hand, E>P is observed in many types of mitochondria and depends on (i) the excess ETS capacity pushing the phosphorylation system (Fig. 1B) to the limit of its capacity of utilizing ∆pmt, (ii) the pathway control state with single or multiple electron input into the Q-junction and involvement of three or less coupling sites determining the H+out/O2 coupling stoichiometry (Fig. 2A), and (iii) the biochemical coupling efficiency expressed as (E-L)/E, since any increase of L causes an increase of P upwards to the limit of E. The excess E-P capacity, ExP=E-P, therefore, provides a sensitive diagnostic indicator of specific injuries of the phosphorylation system, when E remains constant but P declines relative to controls (Fig. 6). ::::Substrate cocktails supporting simultaneous convergent electron transfer to the Q-junction for reconstitution of TCA cycle function stimulate ETS capacity, and consequently increase the sensitivity of the ExP assay.
  • ... In general, it is inappropriate to use the term ATP production for the difference of oxygen consumption measured in states P and L. The difference P-L is the upper limit of the part of OXPHOS capacity which is free (corrected for LEAK respiration) and is fully coupled to phosphorylation with a maximum mechanistic stoichoimetry, ≈P = P-L (Fig. 6).
  • I think that it is integrating well the 'State 1-5 - nomenclature' with the 'new terminology'. In 2.2 or in the final paragraph: A schematic view similar with the one from the previous versions of the manuscript (April 2017, on which I have sent feedback at the time) (see above) may still be useful for readers that are new to the field of mitochondria physiology, but are starting to employ such measurements in their work.
  • It clarifies and summarises the terminology and what it means in an experimental setting.
  • In 3.3 page 20 we may need to add more detail, may be an example of additional normalization that could be employed.
  • EG: I agree. I just asked one of our co-authors to consider an example of normalization which elicited a good discussion during the past MiPschool.
  • I have attached the scheme from previous version for your reference. The Ideea is to have something visual and simple that integrates the new 'terms'.
  • I went through your interesting review which for me also enlightened new aspects on the mitochondrial chemophysical properties. I completely agree with the need to establish a common language and standard criteria in the field of mitochondria, so that different studies can be compared and abnormalities associated with diseases will be defined and maybe used in the future as biomarkers. I introduced several changes and asked for several clarifications by comments and notes in the text. As I am not an expert in fluxes and flows, forces and rates my input to these parts is limited.
  • I do suggest to include a short paragraph on the crosstalk between mitochondria and the cells via Ca, and other signaling pathways which may affect mitochondrial activity in the conclusion part. Although it is beyond the scope of the review it puts it in a wider physiological context.
  • Here are my minor comments on the manuscript. .., but I hope that you find them useful. As I comment in the abstract, I am not sure to what extent prior knowledge of the existing terminology can be assumed. However, it is clearly aimed at a readership already active in the field.
  • EG: Would this be correct: „Do students expect researchers of bioenergetics explain Mitchell's theory of chemiosmotic energy transformation?”
  • David Harrison: I thought that the original ... was ok... or "Do students expect researchers of bioenergetics to explain Mitchell's theory of chemiosmotic energy transformation?" would be fine - it depends on the type of expectation
  • As for our concern, this version can get our approval. However, we think the text can be improved in the aspect of linguistic coherence.
  • There are sentences that seem to not connect well, maybe because they were written by different authors. For instance, the last paragraph of the conclusion, seems to be more suitable for an introduction, rather than for a conclusion. The content of the text, in our opinion, is very good, very clear and useful for an harmonization of terms. The major terminology issues about mitochondrial states are addressed, and the text sets the paradigm for the upcoming parts. As mentioned in the ms, there will be the need of a "part 2", totally dedicated to mitochondrial enzyme nomenclature. We hope we are able to contribute with more critic comments on that subject, as it is more related to our expertise thus far, and a more active role on the endeavor.
  • 2018-08-16 Leo Sazanov
  • Yes, will be happy to contribute - I think some changes to Fig. 1 are needed.
  • 2018-08-16 Yau-Huei Wei
  • I have completed reading and revising this manuscript (attached). I appreciate your enthusiasm and great effort in preparing this manuscript. I agree with you that it is a very important concept in mitochondrial physiology. However, there are misunderstandings and misuse of this term.
EG: Due to feedback by other co-authors asking for a short and more explanatory section and figure on coupling states, I added new Fig. 6 and text, specifically shown on the website, such that it is not necessary to screen the entire pdf for news.
  • Thank you for your reply to my suggested revision of the manuscript that you drafted nicely to review important parameters that have been widely used in characterizing the quality and function of isolated mitochondria. I am sure that this is a valuable paper and reference for life scientists, researchers in pharmaceutical companies and clinicians.
  • 2017-08-16 Nicholls David G
  • Thank you for the invitation to contribute to your review. However, there is too great a distance between your in-depth approach and the minimalistic ‘need-to-know’ proton circuitry that I have promulgated for the past 40 years or so, and so I must decline. Good luck with the review.
  • 2017-08-15 EG: New Fig. 1 in Version 15
  • Happy to be a co-author and provide critical feedback on this important paper. Just glancing at it now – it is in pretty good shape.
  • I added in a few comments on the attached PDF. I think it looks great and I’m glad to have been a part of the process.
  • Find attached my comments after reading the manuscript. There is a lot of work behind the content of this manuscript. Some parts will be more difficult to follow for scientists new to the field of mitochondrial physiology but the information supply is correct, careful and hopefully an starting point to start unifying terminology.
  • I have read joint review, it is very nice manuscript, and very useful for teaching. I really like the chapter Normalization: flows and fluxes, its very good expalined.
  • I appreciate the opportunity to contribute to this joint review. Please give me a few days to examine the most recent version – if I feel that I understand the material well enough to comment and/or approve, I will be happy to do so and join the co-author list.
  • One comment that I do have based on the preliminary reading that I did over my morning coffee is that for someone like me who views all of this as more of a means to an end (the end being understanding physiology and pathophysiology on a more macroscopic level) instead of an end unto itself, I find that when I am struggling to understand something it is because it lacks a context for me – either from a methodological standpoint (if you add X and Y to the reaction mixture, the results represent Z) or a physiological standpoint (adding X and Y and getting Z represents these in vivo conditions). Context is not necessary for someone like yourself and many of the co-authors who live in the world of mitochondrial physiology, but in order to gain a broader acceptance of the revised terminology and concepts, that additional context may be necessary.
In oxidative phosphorylation the electron transfer system (A) is coupled to the phosphorylation system (B). See Eqs. 4 and 5 for further explanation. Modified from (A) Lemieux et al (2017) and (B) Gnaiger (2014).
  • EG: Abstract modified according to your suggestion. Pi* added to Fig. on OXPHOS. “Energy production“? – I do not like it (1st law of thermodynamics?). I like the ‘exogenous’ uncoupler, versus endogenous uncoupling.
  • What happens when the ETS is branched at the oxidase level - need to define what "uncoupled" is!
  • EG added text: "Such uncoupling is different from switching to mitochondrial pathways which involve less than three coupling sites with electron entry into the Q-junction bypassing Complex I (Fig. 1; including a bypass of CIV by alternative oxidases, not shown). This may be considered as a switch of gears (stoichiometry) rather than uncoupling (loosening the stoichiometry)."
  • The symbol »P: Ambiguous and likely to cause confusion! In my view you need to also define that you have changed this to »P/O2 and not O indicating you are referring to the full 4 electron reduction of oxygen!
  • How are you going to define the ROX state in tissues that have had additional oxidase added i.e. AOX following Gene therapy.
  • EG: AOX has to be inhibited, too, to measure ROX, since AOX is part of the (genetically modified) electron transfer system. See original text: "ROX is measured either in the absence of fuel substrates or after blocking the electron supply to cytochrome c oxidase and alternative oxidases."
  • FO2 versus F
  • Added after Eq. 1: ".. and a chemical part incorporating the Faraday constant"
  • 2017-08-14 EG added to Version 14 many suggestions of GC Brown, and
  • Coupled versus bound processes: Since the chemiosmotic theory explains the mechanism of coupling in OXPHOS, it may be interesting to ask if the electrical and chemical parts of proton translocation are coupled processes. This is not the case according to the definition of coupling given above (in the manuscript). It is not possible to physically uncouple the electrical and chemical processes, which are only theoretically partitioned as electrical and chemical components (Eq. 1) and can be measured separately. If partial processes (fluxes, forces) are non-separable, i.e. cannot be uncoupled, then these are not coupled but are defined as bound processes. The electrical and chemical part of Eq. 1 are tightly bound partial forces of the protonmotive force.
  • I have very fond memories of the MiP summer schools.I can’t help much with this, but I enclose some thoughts. In general, the purpose is a bit unclear, and it rambles over a variety of areas, without clear definitions, recommendations or focus. If I were doing it, I would half the length and sharpen the focus, and make the definitions and recommendations crystal clear. But I realise that doing this within a large consortium is not easy!
  • EG: As always, I fully appreciate your sharp and to-the-point level of discussion. Like me, many of us consider you as a teacher. Therefore, I respond with full appreciation that you ‘help us with this’. And yes: the large consortium has by now a history of dedicated meetings with good discussions, resulting in an evolutionary approach that needs to be appreciated without the claim of full-power selective optimization. I thank you so much for your detailed feedback. Many suggestions that you made are implemented in the new version. In addition my comments are summarized here:
  1. Abstact: This was written before the group-dynamics changed the focus of the ms. With more and more questions on ‘clarification’, ‘definition of terms’, the attempt to summarize some simple recommendations shifted to ‘educational’. So far, more ‘explanatory’ was the result, without reassertion if this was also more ‘educational’. We need the help of experts in science writing who are firm with the basic concepts.
  2. I agree (without necessarily speaking of other contributors) that we should reduce ‘crazy’ recommendations on abbreviations. It is a cancerous phenomenon of scientific literature, and we better stay away of too much of the same. imt etc are not even used in later sections, thus we can get rid of it.
  3. I suggest an exception of the above agreement toavoid abbreviations: mt. Would you suggest that we recommend to use ‘mitochondrial DNA’ and skip mtDNA?
  4. tr: consider spelling this out in all following symbols.
  5. Section 2.2. “You need a section here, outlining why the classical nomenclature is not sufficient. This is important!“ - Thanks, there should be more detail in the first introductory sentences.
  6. LEAK state - thanks, would you agree on this: “A state of mitochondrial respiration when oxygen flux is maintained at saturating levels of oxygen and respiratory substrates, and zero ATP-turnover without addition of any experimental uncoupler, as an estimate of the maximal proton leak rate.”
  7. Your excellent comments on detail have all been incorporated in Version 14.
  8. "∆pmt - not clear why you are using mt here." Protonmotive force could be used for any membrane, but in the context of this manuscript, confusion is very unlikely." – But think of all the literature not taking into account the plasma membrane potential. - Later versions: "∆pmt changed to "∆pH+
  • “The protonmotive force is maximum in the LEAK state” - EG: I agree with your comment – is “elevated” better?
  • "Forces and flows in physics and irreversible thermodynamics: Why?" – EG: Can we ignore IUPAC recommendations?
  • I have added several corrections and comments to the manuscript, mainly according to the small handout that I received at the MiPschool.
  • I was interested in the ROUTINE state, but the section 2.3 for Intact cells versus mitochondrial preparations has been deleted.
  • After coming back from Obergurgl, I was interested in the patch-clamp analysis of mitochondria. I noticed that Bertholet et al applied a patch-clamp method to analyze UCP1-positive and UCP1-negative beige adipocytes. They detected "Creatine Cycling” in mitochondria from UCP1-negative beige adipocytes (Fig. 7 D/E).
  1. On Fig. 1A: Perhaps we should simplify it for the present purpose, since the chapter on ‘pathway control’ has been shifted away form the present Part 1 to another manuscript (Part 2), where these issues should be discussed and controversies resolved in full detail.
  2. Intact cell respiration was also shifted to another future manuscript.
  3. I added ‘intermembrane space’ to Fig. 1B.
  4. V[dot]O2max: I fully agree, I just do not know how to add the dot in Microsoft Word. In the old style, it was simple to just put such a dot on paper.
  5. Definition of V: It is unfortunate that V[dot]O2max has been introduced, but we cannot expect to change the sport science symbols (they should change from volume to amount of substance for metabolic oxygen consumption). And we cannot change V. Therefore V[dot] needs to be distinguished from the other definitions of V. I added: “.. whereas maximum mass-specific oxygen flux, V[dot]O2max or V[dot]O2peak, is constant across a large range of individual body mass (Weibel and Hoppeler 2005). V[dot]O2peak of human endurance athletes is 60 up to 80 ml O2·min-1·kg-1 body mass, converted to Jm,O2peak of 45 to 60 nmol·s-1·g-1 (Gnaiger 2014).”
  • The review is coming together nicely. Much more advanced than a couple weeks ago. Great job. I added a few comments.
  • 2017-08-12 EG added to Version 12
  • Proton leak: Proton leak is the process in which protons are translocated across the inner mt-membrane in the direction of the downhill protonmotive force without coupling to phosphorylation. The proton leak flux depends on ∆pmt and is a property of the inner mt-membrane. Proton slip: Proton slip is the process in which protons are only partially translocated by a proton pump and slip back to the original compartment. The proton slip is a property of the proton pump and depends on the turnover rate of the proton pump.
  • I managed to set aside some time to work on the manuscript. My suggested revisions and comments are embedded in the document using the “track changes” mode and margin comments.
  • On »P (»P/O ratio) and «P: Can a stronger statement be made here? I like the suggested symbols.
  • On ADP concentration: It may be useful to discuss the potential confusion between high ADP and saturating ADP. The arbitrariness of some commonly used protocols is a problem in the field, particularly when using plate-based systems for measuring respiration.
  • I’ve just finished with the last version (9) of the paper. I really like how it looks like now and I have let some of our PhD students to read (as a test for a beginner) and she enjoyed a lot the paper. I added very few suggestions which I attached in the word document.
  • Thank you for your generous invitation to become an author of this historical paper. This is a great honor to me. I have some comments to make;
  • I wish you would elaborate more on the respiratory 'control', I always wondered how and why this term is adopted, instead of other terms, such as mitochondrial functional characteristics or functional anatomy.
  • Addition to the MS by EG: Control and regulation: The terms metabolic control and regulation are frequently used synonymously, but are distinguished in metabolic control analysis (Fell 1997). Respiratory control may be exerted by (1) ATP demand (Fig. 2), (2) fuel substrate, pathway competition and oxygen availability (starvation and hypoxia), (3) the protonmotive force, redox states, flux-force relationships, coupling and efficiency, (4) mitochondrial enzyme activities and allosteric regulation by adenylates, phosphorylation of regulatory enzymes, Ca2+ and other ions including pH, (5) inhibitors (e.g. NO or intermediary metabolites, such as oxaloacetate), (6) enzyme content, concentrations of cofactors and conserved moieties) such as adenylates, NADH/NAD+, coenzyme Q, cytochrome c); (7) metabolic channeling by supercomplexes, (8) mitochondrial density and morphology (fission and fusion), (9) hormone levels, gender, life style (influencing all control mechanisms listed before), and (10) genetic or acquired diseases causing mitochondrial dysfunction (for reviews see Brown 1992; Gnaiger 1993a, 2009; 2014; Morrow et al 2017).
  • I was really happy to find a section "Size-specific quantities", where you wrote "The well-established scaling law in respiratory physiology reveals a strong interaction of oxygen consumption and body mass by the fact that mass-specific basal metabolic rate (oxygen flux) does not increase proportionally and linearly with body mass, whereas maximum mass-specific oxygen flux, VO2max, is constant across a large range of body mass (Weibel and Hoppeler 2005)." However, I understand the mass-specific (basal) metabolic rate (oxygen flux) decrease proportionally if not linearly with body mass.
  • I found the discussion on the protonmotive force very challenging. I wonder if you could make it simpler for more wide audiences.
2017-08-10 Version 10
  • Integration of suggestions and corrections by T Komlodi and A Meszaros.
  • Phosphorylation, »P: Although phosphorylation in the context of OXPHOS is clearly defined as phosphorylation of ADP to ATP, potentially involving substrate-level phosphorylation as part of the tricarboxylic acid cycle (succinyl-CoA ligase), in the matrix (phosphoenylpyruvate carboxykinase) and in the cytosol (pyruvate kinase, phosphoglycerate kinase). ADP is formed in the adenylate kinase reaction, 2 ADP <--> ATP + AMP. In isolated mitochondria high adenylate kinase related ATP production can be detected in the presence of ADP and without respiratory substrates (Komlódi and Tretter 2017). On the other hand, the term phosphorylation is used in the general literature in many different contexts (phosphorylation of enzymes, etc.). This justifies consideration of a symbol more discriminative than P as used in the P/O ratio (phosphate to atomic oxygen ratio), where P indicates phosphorylation of ADP to ATP or GDP to GTP. We propose the symbol »P for the energetic uphill direction of phosphorylation coupled to catabolic reactions, and likewise the symbol «P for the corresponding downhill reaction (Fig. 2).
  • Coupling and efficiency: In an energy transformation, tr, coupling occurs between processes, if a coupling mechanism allows work to be performed on the endergonic or uphill output process (work per unit time is power; dW/dt [J/s] = Pout [W]; with a positive partial Gibbs energy change) driven by the exergonic or downhill input process (with a negative partial Gibbs energy change). At the limit of maximum efficiency of a completely coupled system, the (negative) input power equals the (positive) output power, such that the total power equals zero at an efficiency of 1. If the coupling mechanism is disengaged, the output process becomes independent of the input, and both proceed in their downhill direction (Fig. 2). - (added by EG)
  • Extensive quantities: An extensive quantity increases proportional with system size. The magnitude of an extensive quantity is completely additive for non-interacting subsystems, such as mass or flow expressed per defined system. The magnitude of these quantities depends on the extent or size of the system (Cohen et al 2008).
  • Size-specific quantities: ‘The adjective specific before the name of an extensive quantity is often used to mean divided by mass’ (Cohen et al 2008). A mass-specific quantity (e.g. mass-specific flux is flow divided by mass of the system) is independent of the extent of non-interacting homogenous subsystems. Tissue specific quantities are of fundamental interest in comparative mitochondrial physiology, where specific refers to the type rather than mass of the tissue. The term specific, therefore, must be clarified further, such that tissue mass-specific (e.g. muscle mass-specific) quantities are defined. - (added by EG)
  • here is my feedback on the manuscript. It is very detailed, indeed. Thank you for the monumental work! My suggestions relate to the manuscript organization at the beginning and you'll find them in the annotated PDF document as well as in the Word file. Happy to look at later versions of it (or the final draft, likely after the November meeting) as well.
  • 2017-08-08 to 09
  • Updated versions by [Gnaiger E|EG]], with Sections 3.2. Normalization: flows and fluxes and 3.3. Conversion: oxygen, protons, ATP
  • Forces and flows in physics and irreversible thermodynamics: According to definition in physics, a potential difference and as such the protonmotive force, ∆pmt, is not a force (Cohen et al 2008). The fundamental forces of physics are distinguished from motive forces (e.g. ∆pmt) of statistical and irreversible thermodynamics. Complementary to the attempt towards unification of fundamental forces defined in physics, the concepts of Nobel laureates Lars Onsager, Erwin Schrödinger, Ilya Prigogine and Peter Mitchell (even if expressed in apparently unrelated terms) unite the diversity of ‘isomorphic’ flow-force relationships, the product of which links to the dissipation function and Second Law of thermodynamics (Prigogine 1967; Schrödinger 1944). A motive force is the change of potentially available or ‘free’ energy (exergy) per isomorphic motive unit (force=exergy/motive unit; in integral form, this definition takes care of isothermal and non-isothermal processes). A potential difference is, in the framework of flow-force relationships, an isomorphic force, Ftr, involved in an exergy transformation, defined as the partial derivative of Gibbs energy, ∂trG, per advancement, dtrξ, of the transformation, tr (the isomorphic motive unit in the transformation): Ftr = ∂trG/dtrξ (Gnaiger 1993a,b). This formal generalization represents an appreciation of the conceptual beauty of Peter Mitchel’s innovation of the protonmotive force against the background of the established paradigm of the electromotive force (emf) defined at the limit of zero current (Cohen et al 2008).
  • Coupling, efficiency and power: In energetics (ergodynamics) coupling is defined as an exergy transformation fuelled by an exergonic (downhill) input process driving the advancement of an endergonic (uphill) output process. The (negative) output/input power ratio is the efficiency of a coupled energy transformation. Power, Ptr = ∂trG/dt [W=J∙s­-1], is closely linked to the dissipation function (Prigogine 1967) and is the product of flow, Itr=dtrξ∙dt-1 [xtr∙s-1] times generalized force, Ftr = ∂trG/∂trξ [J∙xtr-1] (Gnaiger 1993b).
  • 2017-08-08: Updated paragraph by [Gnaiger E|EG]] on ‚P/O‘
Many thanks for your very valuable comments. The nomenclature on ETS complexes (including not only CI to CIV) versus mt-enzymes versus the gene-linked terminology should indeed be discussed thoroughly. I will add your comments under your name to the website for discussion. The pathway control states have been removed from our first part and will be the focal topic of Part 2. Perhaps Fig. 2A should be simplified for Part 1, to avoid the terms CGpDH and CETF.
  • 2017-08-02 Updated ms summarizing WG1 input by J Iglesias-Gonzalez.
  • 2017-07-29 Version 3 on website: Mitochondrial_respiratory_control:_MitoEAGLE_recommendations_1
  • 2017-07-28 to 29: Obergurgl MitoEAGLE Workshop: WG1 – group discussion and edits based on printed Version 2.
  • State 1: depending on the fact that mitochondrial extract of isolated mitochondria is crude or purified (for exemple on Percoll gradients or sucrose gradients), the isolated mitochondria have still somes endogenous substrats or not that makes a pulse at the start during their early dissipation. Could this be taken into consideration or being indiscted for the users.
  • State 4: I do not find this very well writen since after the addition of ADP (state 3) the mitochondrial membrane potential drop and reacquire its high value (at the state 4) when all the ADP has been transformed in ATP has been used? am I wrong. So this shoul be writen clearly.
  • Why escaping to state about CR (respiratory control) and ADP/O measurements and definitions?
  • Again, I perfectly understand the reference to the bioblast link by since we do not live outside teh real world the reference are usually referred to pubmed also... so we should refer to both...
  • For te publication, the suggestion is nice but by tradition our journals are more....
  1. BBA general subjects or bioenergetic: 6554 occurrences
  2. FEBS J: 2911 FEBS letters 2563 (so 1+2 = 5474 Occurrences)
  3. Journal of Biological Chemistry: 7730 occurrences
  • My personnal preference will go To BBA general subjects... But should be decided in commun
2017-04-18 Version 1 circulated to MitoEAGLE by E Gnaiger.
  • 2017 03 21 to 23: Barcelona MitoEAGLE Workshop: WG1 – presentations and group discussions.


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