Preliminaries and scale of the problem
In this section we present the results from those patients of Cohort 2 who have had the ATP Profile test after some months of therapy which has been individually tailored according to the results of their first tests. Because these results come from an audit and not a funded research study, the time intervals between tests are highly variable. Another complicating factor is that we have no way of confirming the extent to which the recommendations of the therapeutic protocol were actually carried out by individual patients. Nevertheless, the results indicate that nearly all patients show biochemical improvement, with dramatic improvements in some patients, provided they put in place the prescribed dietary, nutritional and lifestyle regimes. It is also notable that the patients who did not adhere to the regimes did not see biochemical progress.
We have altogether 34 multiple-test patients: two who have taken the ATP Profile four times, seven who have been tested three times and 25 who have been tested twice. We briefly report the case histories of the 3- and 4-test patients. These are also included in a before and after study with the 25 patients who have had the ATP Profile twice. Unfortunately we have no follow-up biomedical test results for the other 104 (75%) of the 138 patients of Cohort 2. This is an inevitable result of audit where clinical decisions are made for the benefit of the patient, not the clinician or researcher. All we can say is that those patients who have multiple tests have a strong tendency to be among the most ill patients at the first test. This is illustrated by comparing the histogram () of the initial values of the revised Mitochondrial Energy Score (MESinh) for all 138 Cohort 2 patients with the histogram () of the initial values of the 34 patients who had multiple tests. The multiple-test patients have a lower mean value (compare the vertical dotted lines), and 38% of the multiple-test patients are in the first two bins with the lowest values of MESinh at the initial test compared with less than 7% of those with no repeat test.
Note that the majority of the patients have values of MESinh
well below the minimum value for the control group of 100% and further below the (144 ± 4)% average of the controls, where this error is the standard error of the mean (SEM). The four patients with MESinh
values of greater than 100% () all have at least two of the six ATP factors below the normal minimum values [1
]. The mean value of MESinh
for all of these 138 first test patients is (39.7 ± 2.1)% (SEM), and only (32.7 ± 4.7)% (SEM) for the initial value of the multiple-test patients. We cannot say whether or not the 104 patients who did not have a repeat test improved as much as those who did, and whose improvement (or lack of it) we have measured. However, because the feedback that we have had is so positive, and because it is mainly the most severely affected patients who have repeat tests, we think it likely that the improvements that we do measure are a lower limit, or at least an average, of those that have not been measured. A future properly funded research study could mitigate this problem.
illustrates the scale of the problem facing the patients and the clinician (in our case SM). The very ill patients who need to take subsequent tests peak at about 30 on the horizontal scale and the goal is to get them up among the control group which peaks at about 140, a very large difference. This large difference is measured with neutrophils which are the main effectors of the innate immune system. Our measurements of cell-free DNA suggest that there are also major effects in other cells [1
The treatment protocol is largely based on two experimental facts: 1) A large fraction of people with ME/CFS (as diagnosed with the 1994 CDC criteria) [2
] have mitochondrial dysfunction. We found measureable mitochondrial dysfunction in all of 61 patients in the age range 18-65 years (Cohort 1) [4
], and in all of the 138 patients of Cohort 2 [1
]. Every expenditure of energy in cells throughout the body requires the conversion of ATP into ADP and the patients we have studied have lower than normal levels of ATP. The mean value of ATPend
for the Cohort 2 patients is only 0.88 fmol/cell compared with the mean of 1.37 fmol/cell for the control group. The patients are also inefficient in recycling the ADP back into ATP. If patients push themselves with respect to increased mental and/or physical activities which both make more energy demands, they will be forced to make ATP by an alternative route. One such route is anaerobic metabolism via glycolysis where pyruvate is converted to lactate. Biochemically the result is acidosis, and clinically these patients suffer pain. Patients in Group B (BLOCKED) have the additional problem of replenishing their supply of adenine nucleotides due to the loss of one molecule of ADP for each molecule of ATP hydrolyzed. 2) People with ME/CFS often have low cardiac output [7
], and cerebral blood flow [12
]. These features could be explained by mitochondrial dysfunction - mitochondria make up 25% by weight of cardiac muscle [13
]. The low cardiac output could also contribute to mitochondrial dysfunction by decreasing the delivery of all substrates, but because they are so closely interrelated it is difficult to prove which comes first [14
In the treatment of ME/CFS by one of us (SM) there is a basic protocol that applies to all patients regardless of the cause and the underlying biochemical abnormalities [15
]. Indeed this basic protocol simply represents the lifestyles and dietary regimes all people should be following to optimise their health. The basic protocol is comprised of: 1) eating the evolutionary correct stone-age diet; 2) ensuring optimum hours of good quality sleep; 3) taking a standard package of nutritional supplements which at least meet the Recommended Daily Intakes of essential nutrients; 4) getting the right balance between work and rest.
Some aspects of the basic protocol tailored to mitochondrial dysfunction [16
] and to ME/CFS [18
] have already been reviewed. Details of the protocol used here are freely available [20
Additions to the above regime are tailored for each patient according to the results of their mitochondrial function tests, together with other nutritional tests and clues from the clinical history. We have found that mitochondrial function is typically impaired in two ways - either 1) substrate or co-factor deficiency, or 2) inhibition by chemicals which may be exogenous or endogenous [1
]. As well as the mitochondrial function tests, for most patients we also measure levels of minerals (magnesium, zinc, copper, selenium and manganese) and of specific co-factors such as Niacinamide (vitamin B3 status), Acetyl L-carnitine and Co-enzyme Q10. There may be several reasons why vitamin B12 by injection may be recommended [21
]. If a patient has evidence of blocking of mitochondrial function then further tests can be used to ascertain the nature of that blocking which could include DNA adducts, fat biopsies, micro-respirometry studies or translocator protein studies. We often find deficiencies in glutathione (GSH) and glutathione peroxidase (GSH-PX} which are needed to protect cells from oxidative damage and to detoxify xenobiotics. Other appropriate detoxification regimes, as determined by standard practices of ecological medicine, can also be put into place. For example, high levels of pesticides or volatile organic compounds can be treated by far-infrared saunas (FIRS) which have also been shown to have other beneficial effects in fibromyalgia and other illnesses [22
]. High levels of heavy metals can be tackled using selective chelation therapy, and evidence of immune-complex blocking may be treated by addressing issues of allergy and chronic infection. If there is evidence of mal-absorption of micro-nutrients then again there may be interventions to improve gut function or supply micronutrients parenterally [15
In carrying out this audit, all patients were given fictitious initials and their test results were blinded in the analysis and reporting. Here we give details of the two patients who have had the ATP Profile test on four occasions and the seven who have been tested three times. The initial and final test results of all 34 multiple test patients are included in the next subsection.
shows plots of the measured parameters ATPend, Ox Phos, TL OUT, TL IN and %ATPinh (% ATP inhibited) and the computed MESinh all normalized to the normal minimum of each taken as 100%. On this scale the maximum value of TL IN for the controls is 152% and the maximum possible value of %ATPinh is 119%. We absorb ATPMg and ATP Ratio into their product ATPend because the product of their normal minima is very close to the normal minimum of their products. The numbers below Test 2, Test 3 and Test 4 are the time intervals in months since Test 1. The fact that the computed MESinh appears to have a different normalization is just because none of the controls have the normal minimum value for all of the parameters making up MESinh, and the normal minimum of MESinh is 1.42 times the product of the 5 normal minimum values of ATPMg, ATP Ratio, Ox Phos, TL OUT and %ATPinh.
Figure 2 Plots of the ATP Profile test results for the 9 patients who have had the ATP profile test on 3 or 4 occasions. The numbers below Test 2, Test 3 and Test 4 are the time intervals in months since Test 1. The fact that the computed MESinh appears to have (more ...)
In we start with the Group B patients, those who comprise the ‘HI Blk’ category. However, during the treatment protocol we have found a few multiple-test patients who have switched from one category to another, so we need to introduce some transition categories. The first five patients (ZY, AG, AQ, AA and AM) started in the ‘HI Blk’ group and remained there for the following tests. We call this transition category B→B. We also indicate by (f) or (m) the sex of each patient.
Patient ZY (f) started with all factors very low. She made good progress by test 2 but with TL OUT still low. She discontinued the far-infrared saunas, showed little further improvement, but enough to resume work part time.
Patient AG (f) had very low TL IN and %ATPinh at the start and made good improvement between tests 1 and 2, but both TL values dropped between tests 2 and 3. She was surprised that the results were worse on the 3rd test.
Patient AQ (f) had very low %ATPinh and TL IN at test 1. By test 2 ATPend and Ox Phos improved, and moved into the normal region by the 3rd test, but % ATPinh and TL IN are still low.
Patient AA (m) may be considered as the “ideal patient” - steady improvement to near normal in all measured quantities in less than 18 months.
Patient AM (f) made steady improvement from being housebound at test 1 to all factors being almost at the normal minimum by test 3, but the normalised product MESinh is still well below the normal minimum.
Patient AJ (f) shows a new effect, a switch from Group B to Group A2 with a super-normal value of TL IN. We label this switch as B→A2. Between test 1 and test 2 she made good progress in all quantities, and at test 3 she shows the switch to Group A2: ‘HI TL IN’. This patient is now back at work 4 days per week.
Patient AL (m) is an example of Group A2: ‘HI TL IN’ who had a super-normal value of TL IN at the first test which stayed high in spite of the facts that ATPend, Ox Phos, TL OUT and MESinh all moved into the normal region. We label this transition category A2→A2. Test 3 was not really necessary but the patient requested it. The super-normal values of TL IN suggest lack of substrate, but in spite of this all parameters are in the normal region for tests 2 and 3.
Patient ZZ (f) also started in Group A2: ‘HI TL IN’, but switched to Group A1: ‘no HIs’ by test 4 (we label this switch as A2→A1). The biomedical results of tests 2-4 may reflect the common situation where a patient improves and then overdoes things and suffers a relapse which takes some time to recover from. However, by test 4 all parameters and MESinh are in the normal region but most just barely.
Patient AO (m) started in Group A1: ‘no HIs’, and made some progress between the first and second tests, but was not pacing and discontinued the detoxifying saunas after 7 months. The test results illustrate the importance of these parts of the treatment protocol. We label this patient as category F: ‘poor regime’.
These multi-test results help us to understand the immediate sources of dysfunction and, when supplemented by other appropriate measurements, enable one of us (SM) to fine-tune the treatment protocol for individual patients. They also show that 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. These transitions indicate biochemical improvements. Some of these cases show that the treatment protocol needs to be maintained as time goes on. However, this may not be necessary in all cases.
Patients with 2 test results
Here we summarize the test results of all 34 multiple test patients including the 25 patients who have had the ATP profile test on just two occasions. For the patients with more than two tests we take the initial and final tests. The time interval between the first and final tests ranges from 5 to 67 months with an average of 30 months. We have considered removing the results at the long time intervals, e.g. beyond 48 months, but this just reduces the sample size without changing any conclusions. Because there are a number of cases where a patient makes a transition from one Group to another, we order them according to the relevant transition category ().
Transition categories of patients with two or more ATP Profile tests.
Besides the patients in transition category F: ‘poor regime’, there are also a number of patients who were lax in just one aspect of the treatment protocol. In view of the fact that they still improved they have been included in the other transition categories where appropriate. Below we will refer to patients in the first five transition categories combined (B→B to A1→A1) as ‘good regime’. Patients of category F: ‘poor regime’ constitute a small, but important, internal control group. They show that there is little or no improvement (and even deterioration) when the treatment regime is not fully followed.
The results () are further sorted in each transition category by increasing value of % ATPinh.
Figure 3 Plots of ATPend, % ATPinh, Ox Phos, TL OUT, TL IN and MESinh for the 34 of 138 patients of Cohort 2 who have had two or more ATP Profile tests. We show results of the initial test (Test 1) and the last test (Test f). The transition categories are defined (more ...)
In interpreting these results we first consider % ATPinh
for transition category B→B, the first 16 entries in the second row. All these patients increased in %ATPinh
to 80% or more of the normal minimum, with the largest increases (as much as a factor of 3) observed for the lowest initial values. We can conclude that the treatment protocol is very effective in minimising and even removing the blocking of the normal mitochondrial pathway of the Krebs cycle and oxidative phosphorylation via the electron transfer chain [1
]. Looking down we see that these increases are associated with increases in TL IN
except for patient ZD, but as a possible compensation this patient’s value of TL OUT
increased into the normal region. All patients showed increased ATPend
(first row) up to 80% or more of the normal minimum except ZG (and ZD who was already in the normal region). For Ox Phos
(third row) all 16 patients improved with 8 (50%) ending up in the normal region and correlating with increases in ATPend
. The changes in TL OUT
are variable but most patients show small increases. For TL IN
, 15 of 16 show increases, some by more than a factor of 2. Finally the MESinh
values all increase, some dramatically, by on average a factor of 3.5.
All five B→A2 patients moved into the normal region of %ATPinh and all show increases in ATPend and especially in Ox Phos. The TL IN changes are dramatic, but changes in TL OUT are mixed and mainly small. All these five patients improved in MESinh by on average a factor of 7.9 but all still well below the normal minimum. This makes biochemical sense if blocking problems have been corrected but substrate deficiency has not been adequately addressed.
The two patients in Group A2: ‘HI TL IN’ for both tests (transition category A2→A2) show increases in ATPend, Ox Phos, TL OUT, and MESinh. Of the two patients who moved to Group A1: ‘no HIs’ (transition category A2→A1) ZA’s value of TL IN became normal but AW’s value became sub-normal, possibly because of no increase in TL OUT. MESinh shows ZZ moving into the normal region, but AW barely improving in any of the parameters.
The five Group A1: ‘no HIs’ patients (transition category A1→A1) all show increases in most parameters except TL IN which is mixed. One (AN) moves well into the normal region of MESinh and ZJ just to the edge, most likely limited by how the low value of TL IN affects other parameters.
Finally transition category F: ‘poor regime’ shows the four patients who were lax in two or more aspects of the treatment protocol. Most of their parameters stayed about the same or decreased.
In order to summarize these before and after results in a straightforward way we will consider just the main quantities ATPend, Ox Phos and MESinh and in particular how much they change between the initial and final tests. We do this for the 30 patients who have followed most of the demanding treatment regime (‘good regime’) and separately for the four ‘poor regime’ patients. The results are shown in .
Figure 4 Histograms of the changes in ATPend, Ox Phos and MESinh between the initial and final ATP Profile tests. The bins labelled ‘good regime’ are for the 30 patients who carefully followed the treatment protocol, and those labelled ‘poor (more ...)
The ATPend histograms show that 28 out of the 30 ‘good regime’ patients improved in this quantity and there is a clear separation between the 95% confidence intervals (CIs) for the means of the ‘good regime’ and ‘poor regime’ categories. The Ox Phos and MESinh plots show that all 30 ‘good regime’ patients improved, and for MESinh by large values especially compared with the ‘poor regime’ patients. If we prefer, we can compute the mean increase in MESinhfor the ‘good regime’ patients relative to that for the ‘poor regime’ patients. The result is 64% (95% CI 50% to 78%).
The means and standard errors of the differences and ratios of the initial and final test measurements are summarized in also for the other quantities: % ATPinh, TL OUT and TL IN.
Summary of differences and ratios of initial and final ATP profile test results
Comparison of 2-test nutrient status
In addition to the ATP Profile and Cell-free DNA tests, the majority of Cohort 2 patients have other tests relevant to cellular metabolism and mitochondrial dysfunction such as red cell Niacinamide (NAD, a marker of vitamin B3 status), L-carnitine (L-C) and coenzyme Q10 (CoQ10 or Ubiquinone). These auxiliary tests use red cells and plasma and are independent of the ATP Profile which uses neutrophils and their mitochondria. NAD and CoQ10 are important electron carriers in the ETC, and CoQ10 is also an important antioxidant. One important function of L-C is to transport fatty acids across mitochondrial membranes where the beta-oxidation process performs a vitally important part of the energy supply chain, particularly in cardiac muscle. Most of the L-C is in muscle but numerous studies have shown that the 1% that is in plasma correlates well with the whole body status.
We use the results of these and other auxiliary tests in order to find out where the biochemical lesions are so that the nutritional supplement part of the treatment regime can be tailored for each patient. Most patients have deficits in more than one of these co-factors. As an example, we show in a summary of the results of the initial and final measurements of NAD, L-C and CoQ10 for those multi-test patients who have had these tests.
Figure 5 Chart showing percentage of patients with normal values of three of the essential co-factors involved in cellular ATP production: Niacinamide, L-Carnitine and CoQ10. For each co-factor we show the results (with binomial errors) from the initial test, (more ...)
We do not have a control group for these tests but we can use the well-established reference ranges (also, we use Biolab to perform the CoQ10 tests). In the initial test, only 40-50% of the patients have a value within the reference range of NAD or L-C or CoQ10, and between 1/5 and 1/3 of them are within the reference ranges for two of these quantities, but only 11% are within the reference ranges for all three quantities. For the final test, 44% have all three within the reference ranges, a substantial improvement.
We obtain similar results and conclusions for the important superoxide dismutase (SOD) antioxidants (4 measured quantities), and GSH and GSH-PX.
Comparison of 2-test ATP Profile results with Cell-free DNA
Of the 34 patients with two or more ATP Profile results there are 28 (including the four in transition category F) who have also had at least two tests of Cell-free DNA
in plasma. As we showed in our paper on the pathophysiology [1
], there are strong negative correlations of Cell-free DNA
and with Ox Phos
and with MESinh
and this suggests that the dysfunctions we observe in neutrophils also occur in other cells, because cell-free DNA reflects damage to cells of all types, not just neutrophils [5
We find that for the ‘good regime’ patients, the mean increases in the ATP quantities on the scale shown in are accompanied by a decrease of Cell-free DNA. We illustrate this in where we show the mean changes in Cell-free DNA compared with the mean changes in Ox Phos for both ‘good regime’ and ‘poor regime’ patients. This shows that the therapeutic interventions provide marked improvement in both mitochondrial function and level of tissue damage.
Figure 6 Plot of the mean value of the decrease in Cell-free DNA vs the mean value of the increase in Ox Phos for the 30 patients who had both Test 1 and Test f and followed the treatment regime, ‘good regime’, and for the 4 patients who did not (more ...)