Response to the paper 'Assessing cellular energy dysfunction in CFS/ME using a commercially available laboratory test' by Cara Tomas et al

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Background

On 7 August 2019, Cara Tomas et al published Assessing cellular energy dysfunction in CFS/ME using a commercially available laboratory test.

This paper commented on the ATP Profiles test included within Mitochondrial Function Profile Test

Below are the responses of myself and Dr John McLaren Howard to this paper.

Response of me, Dr Myhill

The essence of this paper appears to be that tests of mitochondrial function are not relevant in the assessment of patients with CFS/ME. However, it is clear from the above paper that the authors lack understanding of how mitochondria are relevant in these conditions and we hope that the following comments will be helpful.

Background Information:

What is CFS/ME?

It is important to recognize that neither CFS/ME are diagnoses – they are simply clinical pictures that may have many causes. The central symptom common to both clinical pictures is that of devasting mental and physical fatigue. Dr John McLaren Howard, Dr Norman Booth and I hypothesised that this is partly due to poor energy delivery mechanisms and that poor mitochondrial function may be part of these poor energy delivery aspects. Indeed, it is biologically plausible that poor mitochondrial function forms part of the clinical picture and a great many other studies confirm such.

With such a hypothesis and in response to clinical demand, John McLaren Howard started to investigate mitochondrial function whilst working at Biolab in the 1990s. Initially he was measuring activity of mitochondrial enzyme complexes one to five, but he found poor correlation with clinical disability. He went on to use ATP profiles – this test was already in regular use in the research sector and John developed this further for clinical use.

The ATP profile test

This was the first test available to clinicians to allow them to assess mitochondrial function in patients with fatigue. This test has been very useful clinically. It tells us if mitochondria are going slow because they lack the raw materials to function or because they are inhibited (‘blocked’). Often, we can go on to determine the nature of such blocking. As importantly we were able to quantify the degree of malfunction by a simple calculation namely the mitochondrial energy score (MES).

The mitochondrial energy score is simply a way of providing an objective measure of the efficiency of ATP production, transport to cells, energy release and then ADP recycling. What we found in our first paper Chronic fatigue syndrome and mitochondrial dysfunction is that this correlated well with the clinical levels of fatigue experience by the patient. This was so important because for those patients for whom other causes of fatigue had already been addressed, this constituted an objective measurement of the degree of illness severity. This has been very helpful to patients applying for state and private sector welfare benefits and also for compensation for injury since clinicians have an objective measure of disability.

Our first paper was done under blinded clinical conditions

Dr Norman Booth, Dr John McLaren Howard and I immediately appreciated that for any study to be relevant it had to be blinded so that no one person could influence the result either consciously or subconsciously. I, Dr Myhill, assessed patients and, with the patient, we agreed a clinical score using the Bell disability scale. Bloods were sent to Dr McLaren Howard who performed the ATP profile test. At no time did he know the clinical score. Dr Booth calculated the mitochondrial energy score, analysed the ATP profiles and wrote the scientific papers.

Our first paper, 2009 , showed a clear correlation between the clinical disability of 71 patients and their mitochondrial energy score when compared to 53 normal healthy controls - see Chronic fatigue syndrome and mitochondrial dysfunction

Our second paper 2012, was an audit of 138 patients. This showed that mitochondria were going slow either because they lacked the raw materials to function or because they had been inhibited by a toxin. See Mitochondrial dysfunction and the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Our third paper, 2012, was an audit of 34 patients who had undergone follow up mitochondria tests. The 30 who had done the necessary dietary and supplement regimes and detox all improved their mitochondrial energy scores. The 4 patients who had not done the regimes did not improve their energy scores. This told us that the regimes were effective. See Targeting mitochondrial dysfunction in the treatment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) – a clinical audit

Availability of the ATP profile test

Since these studies, we have been actively looking for other laboratories to develop this test. The problem is that it is technically very difficult. Indeed, John McLaren Howard routinely tests in quadruplicate to ensure consistency. Potentially this test is extremely expensive. It is a measure of Dr McLaren-Howard’s dedication and generosity that the test is affordable at all! By contrast the Tomas test used 23 patients and was funded to the tune of, I believe, £21,305 (2015 figure - ME Association Press Release 2015 ) making each test cost around £1,000.

Since 2005 I have audited 986 ATP profiles and the results continue to be consistent. There are many other clinical doctors whose experience is similar.

Many people are unable to access the mitochondrial tests. So, I have made available on my website details of all the regimes necessary to correct mitochondrial function. These are freely available at Dr Sarah Myhill website Those that prefer a book can see them at Diagnosis and Treatment of Chronic Fatigue Syndrome and Myalgic Encephalitis

Why does the Tomas Paper fail to replicate our findings?

Dr Norman Booth suggested that before the Tomas group proceeded with their test, they should do paired samples to compare with the Acumen test. The idea was that Dr Myhill would take blood from one patient and send samples to both laboratories to be tested on the next day. Dr Myhill offered to fund the cost of such a comparison. This offer was never taken up. What this means is that we can have little confidence in the ability of the Tomas group to replicate the Acumen test.

Response of Dr John McLaren Howard

The following are Dr McLaren Howard’s comments on ‘Assessing cellular energy dysfunction in CFS/ME using a commercially available test’ and refer more to the technical aspects of testing.

Page 1 Abstract.

General Comments
This test has never been presented as a diagnostic test for CFS/ME by me or by Dr Myhill. It was designed to explore ATP-related energy pathways in patients with chronic fatigue.

The MES protocol referred to was not a design feature of the test and has never been used by me. Dr Myhill developed this as a way of conveying complex information to patients.

Technical details

  • When initially setting up the ATP-profile I explored heparinised whole blood storage times in relation to patients and controls. Samples were processed within minutes of venepuncture and re-tested 6, 12, 24, 48 and 72 hours later. Provided the samples were kept in the original ‘vacutainer’ tubes and not subjected to extremes of temperature there were only minor changes in test results up to the 48 hour point. Some samples were unsuitable at the 72 hour point as judged by the ATP-profile results and the observation of white cell damage by phase-contrast microscopy. Every sample for these tests whatever the time scale since venepunture has a cell preparation examined by phase contrast microscopy. This has applied, and continues to do so, to every sample presented for these tests. When evidence of cell damage is observed, the ATP-profile is not done. No test in this category has ever been charged for.
  • Test situations regarding time since venepunture and storage/delivery methods continue to be explored.
  • The vast majority of samples arrive by Post Office special delivery and, because we collect our special delivery packages from a local sorting office every working morning at 8:30 am, almost all samples are into the analytical process within 24 hours of venepunture. Regardless, they are still subjected to phase contrast microscopy examination in an attempt to identify storage changes.
  • Moving to page 3, I am very surprised by the quoted finding that there were no significant differences between the findings between fresh and frozen cells. That is not in accord with my findings or those of several scientists from other labs who have spent time in my lab exploring these tests.
  • My own concerns about possible differences in test parameters on fresh and frozen cells goes back as far as approx 1990 when speaking with Linus Pauling who was finding lower ascorbate levels in frozen white cells than in fresh cells. That made me cautious and introduced great care in exploring this possible difficulty when introducing newer tests. While Prof Pauling’s findings for vitamin C might not have relevance to the issue at hand there is another test that involves mitochondria and is subject to big differences in findings between fresh and frozen cells. This tests the Mitochondrial Permeability Transition. Mitochondria have half lives of only a few minutes. The demise of a mitochondrion is triggered by a permeability event that allows transit of substances across the mt-membrane that destabilize the membrane and lead on to the death of the mitochondrion. In performing the test three parameters are usually reported: the mean time to permeability transition, the initial mt-membrane potential and the mt-membrane potential 1 minute post permeability transition. If the latter potential goes below 100mv the mitochondrion is lost.
  • The time to permeability transition is about 25% lower in cells that have been frozen and the proportion of mitochondria prematurely lost after this event is around 45% higher than in fresh cells.
  • On page 5, we see that in this work no significant differences were found between CFS patients and controls for the ATP concentration in the presence of excess magnesium or the ATP concentration with only endogenous magnesium present. This does not accord with the bulk of the results from my lab. Had this been the case for the several hundred investigations performed before this test was made available to clinicians then the test would never have been suggested for ATP-related energy parameters in fatigue patients.
  • On page 6, I take the point that calling the calculated ADP to ATP efficiency ‘OXPHOS’ is open to dispute. Dr Norman Booth strongly favoured the use of this term although mainly as a convenience in his statistical evaluation. It persisted in the three papers we published and I can quite see that a better description would have been more appropriate. What is at odds with our findings is that in this present work no differences were seen between this parameter in CFS/ME patients and controls!
  • If this is so then it becomes very difficult to explain the extreme fatigue suffered by a good proportion of CFS/ME patients on increased energy demand. As almost all energy for such activity is ATP-derived, one would expect to see the kind of differences that we so frequently find in our test. While not a part of the our ATP test procedure, I should point out that on increased energy demand many CFS/ME patients move to anaerobic metabolism with lowering of intracellular pH and increasing lactate concentrations. Why would that occur if their ADP to ATP reconversion worked as well as that of the controls?
  • On page 7 it is concluded that there were differing values for azide inhibition neutrophils and PBMCs. Up until February 2016 all of our ATP profiles were performed using neutrophils.
  • Since that date and in response to a number of published queries about the suitability of neutrophils we changed the test so that mixed leucocytes are used. This did necessitate the change of some reference ranges and, although I am not seeing much practical difference from a clinical perspective I was pleased to be able to respond to those who doubted the use of neutrophils.
  • More importantly from my perspective, the change gave me the opportunity to re-explore all of the fundamentals of the test and to do extensive comparisons that involved almost every ATP-profile performed over a 4 month period. The untimely death of Dr Norman Booth meant that his statistical analysis of these comparisons has not been published.
  • In their conclusions, the authors advise that the MES test should not be used as a diagnostic tool for CFS/ME. It never has been!

John McLaren-Howard.


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