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Mitochondrial and Energy Metabolism Dysfunction in ME/CFS — Myhill, Booth and McLaren-Howard Papers

paolo

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The best thing you could probably do in this situation is to contact her directly and ask precisely targeted questions
that would reveal the truth of the matter, such as 'why have you not done any follow up studies to this one'.
I think that Myhill and colleagues hope for an indipendent study. In the 2009 paper they wrote: "It would be good to confirm the biochemical test results in a second (perhaps government-supported) laboratory".
 

realturbo

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Thanks. Actually, it has taken me all week to read through and write up the 3 Myhill et al papers!
Hip, amazing piece of work pulling together all the research. I might be able to read and understand a small fraction of it in a week!

Are the ATP tests valid/reliable etc, were the results skewed in any manner due to bias. So many questions which I think are very relevant related to this research.
It's useful to be made aware of possible conflicts of interest that may have had the potential of impacting the research. Seems a perfectly reasonable position to take ...
 
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Hip

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Great blog, @paolo. Your blog is very readable using Google translate to English, in case anyone else here wants to have a look.



I am particularly interested in group B (Hi Blk) which seems to have a dysfunction in ADP/ATP translcase (both functions of this enzyme are impaired) and also a bigger percentage of energy produced in an anaerobic way. I belong to this group.
Did you get yourself tested by the "ATP Profiles" test from Acumen Labs? Is that how you know that you are a Group B ME/CFS patient?
 

Hip

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Just for everyone's reference, the mitochondria dysfunction ME/CFS patient subtypes that Myhill et al discovered are these:

ME/CFS Patient Mitochondria Dysfunction Subtype Groups:

Group A Patients — oxidative phosphorylation running normally. Group A then divides into two subgroups:
  • Group A1 Patients ('no HIs') — These are Group A patients who have normal or sub-normal TL IN values (and 87% of these patients also have sub-normal TL OUT values).
  • Group A2 Patients ('HI TL IN') — Group A patients who have super-normal TL IN value.
Group B Patients ('HI Blk') — oxidative phosphorylation partially blocked and running at low efficiency.
  • All of the Group B patients were found have sub-normal TL IN values (ie, they have reduced translocator protein transfer of ATP from mitochondria to the cytosol of the cell). And 78% of the Group B patients have sub-normal values of TL OUT (decreased translocator protein ferrying of ADP from the cell to the mitochondria).

Group A patients try to compensate for the mitochondrial ATP shortage by increasing glycolysis to make ATP.

Group B patients try to compensate for the shortfall in mitochondrial ATP most likely by using the adenylate kinase reaction to make ATP.

If what we see in neutrophil mitochondria also applies to muscle cells we would predict the following: Group A patients will have large PCr depletion, excess lactate production and high acidosis (depressed pH). Group B patients will have low PCr depletion (the shuttle is not needed for the ADK reaction), no excess lactate production and less acidosis for the same work load.
Source: Myhill 2012.

Group A2 patients appear to be short in Krebs cycle substrates. Ref: Myhill 2013.

The Krebs cycle substrates are: citrate, isocitrate, alpha-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate. The way the Krebs cycle works, replenishing any one of these Krebs cycle substrates will serve to replenish all of them (because the substrates are converted from one to the other in the Krebs cycle).

Group B patients in particular may benefit from D-ribose supplementation.




Note that Myhill et al found that the group a patient falls into is not permanently fixed: a patient can sometimes change group, especially as a result of treatment which leads to an improvement in their ME/CFS symptoms.

As patients get better, Dr Myhill found that they tend to change groups in the following sequence:

Group B ➤ Group A2 ➤ Group A1

In Myhill 2013 they say:
clinical improvement follows the pattern of switching from Group B to Group A2 and then to Group A1. That is to say, these patients move from being blocked to unblocked, and then from substrate deficiency to no substrate deficiency.
 
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paolo

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Great blog, @paolo. Your blog is very readable using Google translate to English, in case anyone else here wants to have a look.



Did you get yourself tested by the "ATP Profiles" test from Acumen Labs? Is that how you know that you are a Group B ME/CFS patient?
Yes, I did. In 2013. I am 'pazien. 1' (which means patient 1) in figure 5 of my blog post. I have both a low TL IN and a low TL OUT. And, as you can see, my ATPini is high (23.2%), which means that I rely more than what would be expected on anaerobic energy production. Low TL IN and high ATPini (or low ATPoss, as you wish) qualify me as Group B (HI Blk).
 

Mark

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On the face of it, it seems to me that the new paper from Stanford contradicts the McLaren-Howard findings. The Stanford paper found no mitochondrial abnormalities (with one relatively minor caveat) and normal ATP production in mitochondria, but excess ATP production from an unknown source. Whereas McLaren-Howard seem to have reported multiple mitochondrial abnormalities. But those are just the abstract headlines, as they seem to me; there is a lot more detail needed to understand whether the two perspectives are reporting on the same questions, and whether they are contradictory or compatible. I'd like to hear on this thread from someone who understands the detail of this science who can clarify how the findings from the two research groups compare.

I think the ideal thing would be if someone could contact the Stanford researchers and ask for a comment which can be posted here, explaining whether their findings are compatible with McLaren-Howard's findings.
 

paolo

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On the face of it, it seems to me that the new paper from Stanford contradicts the McLaren-Howard findings. The Stanford paper found no mitochondrial abnormalities (with one relatively minor caveat) and normal ATP production in mitochondria, but excess ATP production from an unknown source. Whereas McLaren-Howard seem to have reported multiple mitochondrial abnormalities. But those are just the abstract headlines, as they seem to me; there is a lot more detail needed to understand whether the two perspectives are reporting on the same questions, and whether they are contradictory or compatible. I'd like to hear on this thread from someone who understands the detail of this science who can clarify how the findings from the two research groups compare.

I think the ideal thing would be if someone could contact the Stanford researchers and ask for a comment which can be posted here, explaining whether their findings are compatible with McLaren-Howard's findings.
The finding of an abnormally high production of anaerobic ATP described in the paper by Stanford is what Myhill and colleagues found in Group B.

In the paper by Stanford University they describe a complessive high production of ATP in perpheral blood mononuclear cells (PBMC) which are lymphocytes and macrophages, whereas Myhill and colleagues found low complessive ATP production in neutrophils. This, to me, means that there is an activation of PBMC, and not of neutrophils.

In conclusion, both papers found an increase in anaerobic ATP production, and the recent paper by Stanford University describe also an activation of PBMC.

As the electron transport chain seems normal according to the study by Standford, the defect could be in Kreb's cycle, as described in a recent metabolomic Japanese paper.
 
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bel canto

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Is the only way to determine what group a patient fits into through the Acumen test? Do amino acid levels in urine give clues? Do high versus low pyruvate levels in urine reflect one group vs. another?

I'm thinking that the Acumen test is the one that is not offered outside of the UK.

This research on energy production is really interesting, but impossible for a non-scientist to understand, so thanks to whoever might be able to translate a bit.
 
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That would certainly be my impression. I tried very hard to see what her studies meant and they did not add up to anything I could make sense of. It is very easy to spin what appear to be scientific stories but they need to fit in with basic information from other sources to be credible. My memory is that the studies were on white cells under laboratory conditions and I doubt you can draw any conclusions about body metabolism from that.

We had several presentations relating to mitochondrial metabolism at the last IiME research colloquium from people from three continents and nobody mentioned anything about Myhill's paper (my memory is that there is only one).
Another red flag lies in the fact that they tested neutrophils. @Jonathan Edwards maybe you could be of some help here because I am really not very knowledgeable about any of this but after looking at mitochondrial function and measurement in neutrophils I am totally confused as to why and how Myhill et al came up with their results.

The patients and 53 normal, healthy controls had the ATP Profile test carried out on neutrophils from a 3-ml venous blood sample.
From what I have read:

- the mitochondria in neutrophils are functionally different in that they preserve mainly cell death-mediating abilities as opposed to mitochondria, for example, in the muscle cells which are all about energy production
- neutrophil mitochondria hardly participate in ATP synthesis and have a low enzymatic activity
- mitochondria are limited in number in neutrophils
- they are loaded with proapopotic proteins
- cytochrome c is strongly reduced and almost completely absent -- isn't cytochrome c necessary for ATP production

Myhill states:

The most significant test in the Mitochondrial function profile is called "ATP profiles". It was developed by Dr John McLaren-Howard. The result is made up of three elements. First of all, it measures the rate at which ATP is recycled in cells. Because production of ATP is highly dependent on magnesium status so the first part of the test studies this aspect.

The second part of the test measures the efficiency with which ATP is made from ADP. If this is abnormal, then this could be as a result of magnesium deficiency, and/or low levels of Co-enzyme Q10, and/or low levels of vitamin B3 (NAD) and/or low levels of acetyl L-carnitine.

The third possibility is that the protein which transports ATP and ADP across mitochondrial membranes is impaired and this is also measured.
From HERE:

ATP assays are extremely sensitive but they are not an ideal metric of mitochondrial function as cells strive to maintain a particular ATP budget and will adjust metabolism accordingly. Thus, alterations in ATP levels are usually only detectable during pathophysiological changes.

Artifact has been reported from residual ATP present in dying or dead cells. ATP assays are also destructive and lack kinetic information. Perhaps the two greatest deficiencies of ATP assays are that they do not measure ATP turnover and they cannot determine the relative contribution of energy yielding pathways to total ATP yield.
ATP assays are extremely sensitive but they are not an ideal metric of mitochondrial function as cells strive to maintain a particular ATP budget and will adjust metabolism accordingly. Thus, alterations in ATP levels are usually only detectable during pathophysiological changes.

Artifact has been reported from residual ATP present in dying or dead cells. ATP assays are also destructive and lack kinetic information. Perhaps the two greatest deficiencies of ATP assays are that they do not measure ATP turnover and they cannot determine the relative contribution of energy yielding pathways to total ATP yield.
It just seems to me that this is at odds with what Myhill is claiming because an ATP test doesn't appear to measure ATP turnover. In neutrophils, mitochondrial function appears NOT to be related to energy production, so how can they be making the claims they are making and then extrapolating extra mg, Co-enzyme Q10, vitamin B3 and acetyl L-carnitine could help with mitochondrial dysfunction when research indicates that ATP production in neutrophils is very low. It is also true that research indicates that ATP production in neutrophils is not indicative of ATP in other cell types. I hope this isn't too muddled as i am having difficulty concentrating.

The research by Myhill is here

From the abstract:

The “ATP profile” test is a powerful diagnostic tool and can differentiate patients who have fatigue and other symptoms as a result of energy wastage by stress and psychological factors from those who have insufficient energy due to cellular respiration dysfunction. The individual factors indicate which remedial actions, in the form of dietary supplements, drugs and detoxification, are most likely to be of benefit, and what further tests should be carried
How is it that they can claim all this? The research seems highly questionable in light of all the red flags.
 

Hip

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cytochrome c is strongly reduced and almost completely absent
That is an interesting finding. In the paper this comes from, they say:
Neutrophil mitochondria lacking cytochrome c and having no complex IV activity do, however, create Δψm (Figure 3), although the underlying mechanism remains unclear.
Δψm = the mitochondrial membrane potential = the electrical gradient (voltage) across the mitochondrial membrane.

So it seems that in spite of having no having no complex IV activity, these neutrophil mitochondria do produce a membrane voltage, which is necessary in order to produce ATP. Just how these neutrophil mitochondria produce this Δψm membrane voltage, the authors are unclear, but they ruled out various possibilities, and concluded that:
More likely, here is again a role for the remaining respiratory complexes, whose proton-pumping activity may control Δψm.
In other words, they think that that other mitochondrial complexes (I, II and III) may make up for the lack of complex IV activity, such that the neutrophil mitochondria are still able to supply energy.

Just how this situation might affect Myhill et al's results and interpretation, I am not sure.



after looking at mitochondrial function and measurement in neutrophils I am totally confused as to why and how Myhill et al came up with their results.
I think Myhill, Booth and McLaren-Howard may have chosen neutrophils (in lieu of muscle cells) to study, because muscle biopsies are invasive, painful, and leave a scar. That's not ideal for routine testing, or for repeat testing, when you may want to measure mitochondrial function in a patient several times, through the course of a treatment.

Though certainly it would be great if some research team could try to replicate Myhill et al's results on actual muscle cells.

They do include caveats in their papers about the use of neutrophils in lieu of muscle cells; for example in Myhill 2012 they say:
Our experimental results are all obtained from neutrophils. Neutrophils are similar to skeletal muscle cells and most other cells (but not cardiac muscle cells) in that the proton gradient across the mitochondrial inner membrane is about 50 % electrical and 50 % chemical. However, at this stage we cannot claim that the mitochondria in other cell types behave similarly, even though mitochondria are systemic. However, some of the features that we observe are very similar to some of the effects seen in exercise studies of patients with ME/CFS.
Nevertheless, if you look at Figure 4a from Myhiil 2009, with these tests on neutrophils, they achieved almost complete separation of ME/CFS patients from healthy controls; and moreover, the degree of mitochondrial dysfunction in neutrophils strongly correlated to the severity of ME/CFS (they tested moderate, severe and very severe patients, though did not test mild ME/CFS patients).



From HERE:
ATP assays are extremely sensitive but they are not an ideal metric of mitochondrial function as cells strive to maintain a particular ATP budget and will adjust metabolism accordingly. Thus, alterations in ATP levels are usually only detectable during pathophysiological changes.
This is actually what I found the most instructive about the Myhill, Booth and McLaren-Howard papers: they examine the whole picture of energy production, and look in detail at the way cells metabolically adjust in order to try to compensate for ATP shortages resulting from mitochondrial dysfunction.

In some ME/CFS patients Myhill et al found that patients' cells try to make up for the energy shortfall by boosting anaerobic glycolysis, which can produce ATP independently of the mitochondria (but results in the problem of lactate build-up).

For me, this ambitious analysis of the whole picture is one of the most interesting aspects of the papers. They have not just reported their empirical findings of mitochondrial dysfunction in ME/CFS, but have striven to provide a wider framework of understanding of the energy metabolism in ME/CFS patients, which looks at the way that cells metabolically adjust to try to compensate for the mitochondrial dysfunction.



Huge red flags related to all three of the research papers = Huge conflicts of interest.
In terms of what you suggest are conflicts of interest, in Myhill 2013 they write:
None of the authors have conflict of interest in the measurements carried out on blood samples and any other aspects of this audit. Dr Myhill’s income arises from treating patients and full details of the treatment and management regime are freely available on the website www.doctormyhill.co.uk. Dr Booth is a retired academic physicist and contributes on a fully voluntary basis. Since his retirement from Biolab Medical Unit, Dr McLaren-Howard continues to carry out the ATP Profile and other biomedical tests at Acumen.
The struck out text was a proof-reading error that should not have been there, according to Dr Norman E. Booth in this post.
 
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Hip

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neutrophil mitochondria hardly participate in ATP synthesis and have a low enzymatic activity
The study you found says:
These organelles [mitochondria] hardly display any marker mitochondrial enzymatic activity, do not synthesize much ATP, but preserve their transmembrane potential (Δψm)
The study explains how it arrived at the conclusion that neutrophil mitochondria do not produce much ATP:
However, in neutrophils, these organelles [mitochondria] hardly play a role in energy metabolism as they do in other cells. This conclusion was derived from the experiment shown in Figure 1, where ATP concentrations in neutrophils were measured under various conditions.
...

The inhibitors of mitochondrial respiration rotenone, TTFA, and sodium azide caused no or only little additional reduction in the neutrophil ATP levels (Figure 1), whereas in the myeloid HL-60 cell line, for instance, rotenone induced a pronounced (approx 70%) ATP depletion.
...

This can be explained by the fact that neutrophils mainly use glycolysis rather than mitochondrial oxidative phosphorylation for their energy supply.
So I can see that this unusual situation with neutrophil mitochondria might have the potential to impact the results of Myhill et al, but it is beyond my abilities to come to any definite conclusion on this.
 

Hip

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I think Myhill, Booth and McLaren-Howard will be aware of this unusual situation with neutrophil mitochondria, because one of the studies that they cite in their first paper (Myhill 2009) is this 1982 study on the energy metabolism of human neutrophils, which states:
The results demonstrate that the ATP concentration in the human neutrophil is not dependent on mitochondrial activity, whereas addition of glycolytic inhibitors reduces intracellular ATP. This strongly indicates that the human neutrophil derives energy mainly from glycolysis. This conclusion has been drawn before by other investigators working with neutrophils from different sources [guinea pig, rabbit, rat, man].
 
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Another red flag lies in the fact that they tested neutrophils. @Jonathan Edwards maybe you could be of some help here because I am really not very knowledgeable about any of this but after looking at mitochondrial function and measurement in neutrophils I am totally confused as to why and how Myhill et al came up with their results.

How is it that they can claim all this? The research seems highly questionable in light of all the red flags.
I quite agree, Kina. I cannot make any sense of any of it. That may be partly because I am not an expert on mitochondrial function but my main concern is that it does not seem to explain anything sensible anyway. I have looked after lots of people who cannot make ATP over the years, with lots of different conditions. By and large they were in intensive care, often needed ventilating and did not look the least like PWME. This was more or less exactly the comment made by the mitochondrial expert Mike murphy who we had come and talk at the IiME colloquium. He could not work out why he was there. We may have all got this wrong and mitochondria may turn out to be involved in some signalling function - which is the straw Murphy was clutching to. But neutrophils seem entirely the wrong thing to look at. They are little suicide bomber packages that respire in a very usual way tailored to a short burst of maximal activity primed by cytokines and other signals.

If energy metabolism was out of line in ME I think the best test would be MRI spectroscopy of muscle, perhaps at rest and after exercise. I am pretty sure that people like David Jones have done that sort of thing and not found much.
 
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Well it has to start somewhere ! They though Fleming was crazy....penicillin and mould ??????
I am not sure anyone thought Fleming was crazy. The problem was that he did not even take much notice of his own finding. When Florey and Chain thought it was worth following up I am not aware that anyone thought they were mad. They probably thought the idea was impractical - which it very nearly was. It only became viable when someone discovered a rogue strain of penicillium that chucked out far more penicillin than the original strain, if I remember rightly.
 

wdb

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Well it has to start somewhere ! They though Fleming was crazy....penicillin and mould ??????

The fact that some geniuses were laughed at does not imply that all who are laughed at are geniuses. They laughed at Columbus, they laughed at Fulton, they laughed at the Wright brothers. But they also laughed at Bozo the Clown. - Carl Sagan
 

Hip

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I have looked after lots of people who cannot make ATP over the years, with lots of different conditions. By and large they were in intensive care, often needed ventilating and did not look the least like PWME.
Well that certainly seems a valid point; though perhaps ME/CFS patients are not quite as bad as people with classic mitochondrial diseases (although maybe they might be if ME/CFS patients they were not resting at home or bed, conserving energy).


But what about hypothyroidism, whose symptom are very similar to those of ME/CFS?

Hypothyroidism involves a mitochondrial down-regulation (since triiodothyronine is a major regulator of mitochondria), and I would think a shortage of ATP. This study found ATP synthesis lower in hypothyroid rats' livers.

So we know people who struggle with reduced energy metabolism and presumably reduced ATP due to having low triiodothyronine (T3) have symptoms near identical to ME/CFS. That is suggestive that low ATP output can produce ME/CFS-type symptoms.



The other thing of great interest is the mitochondrial dysfunction that exists in chronic coxsackievirus B infection of the heart muscle (chronic myocarditis). Prof Nora Chapman and Prof Steve Tracy think that coxsackievirus B infections of the heart muscle may be a good model for studying the coxsackievirus B infections in ME/CFS patients' skeletal muscles (lots of studies found CVB infection in ME/CFS patients' skeletal muscles).

Now this study found that energy metabolism in coxsackievirus B myocarditis and dilated cardiomyopathy is running at low efficiency, due to mitochondrial translocator protein dysfunction. (Dilated cardiomyopathy is a further progression of myocarditis).

And this study found autoantibodies which target and disable mitochondrial translocator protein in patients with myocarditis and dilated cardiomyopathy, which they then reproduced in a murine model infected with coxsackievirus B3.

So in these chronic coxsackievirus B infections of the heart, they are finding exactly the same mitochondrial translocator protein dysfunction that Myhill et al found in ME/CFS patients. (Remember that 82% of ME/CFS patients have a chronic enterovirus infection, according to Dr Chia's research).

For me, this data on energy metabolism dysfunction in coxsackievirus B myocarditis is too much of a smoking gun to ignore.

And the fact that mitochondrial translocator protein dysfunction has also been found in coxsackievirus B heart muscle infections corroborates Myhill et al's work to an extent, and suggests that Myhill et al are onto something.
 
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ash0787

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I have looked after lots of people who cannot make ATP over the years, with lots of different conditions.
This reminded me of something I was thinking about and also sort of answered it - I keep hearing from various sources that there are people that are born with known mitochondrial defects, like recently I read " a baby can take X amount times more magnesium without issues " and I was wondering how much symptom overlap there was with that group of people, this was not mentioned in the Naviaux study.

You say there are significant differences between us and them, also considering the effectiveness of immune suppressing drugs, so its not looking good for the Dauer state theory ?

The other thing that makes me reconsider the metabolism as a root cause idea is that often the symptoms are very localized, like when we get mental crashes it can come on very quickly in the space of a few hours and then disappear entirely after 2 or 3 days, likewise I might be mentally clear completely but have acute POTS etc.