Neutrophil ATP related parameters in chronic fatigue syndrome Norman Booth, Mansfield College, University of Oxford 28 February 2011 Norman E Booth, Mansfield College, University of Oxford Sarah Myhill, Sarah Myhill Ltd John McLaren-Howard, Acumen Medical Ltd It is gratifying to see experts in the biomedical field turn their attention to the devastating illness Chronic Fatigue Syndrome (CFS/ME), as exemplified in the above paper by Vermeulen et al . Further studies of the postexertional malaise following exercise, either physical or mental, are extremely important because this malaise is the most characteristic and disabling symptom of CFS/ME. Also, in spite of many studies, there is inadequate understanding of the biochemical processes which lead to this malaise. In 2009, we published our findings from an audit where we compared deficiencies in the provision of ATP in neutrophils with the degree of disability of patients with CFS . Here we use data from 53 controls and 61 CFS patients (in the same age range 18-65 years as the controls). The patient group spanned a wide range of CFS Ability from 0 to 7 on the Bell CFS Ability scale [2-3]. The paper by Vermeulen et al  compares exercise performance in CFS patients and controls. These authors conclude that the decrease in mitochondrial ATP production with work rate detected in their tests on peripheral blood mononuclear cells (PBMCs) is a secondary phenomenon. They focus attention on differences in transport capacity of oxygen. While agreeing about the importance of this and supporting the premise that more research is needed to understand oxygen transport-related issues in CFS, a number of ATP-related issues concern us greatly. Vermeulen et al quote the work by Maianski et al  on neutrophils in support of their dismissal of the importance of ATP-related parameters in CFS. The work in  concentrates on aspects restricted to apoptosis. The work of van Raam et al  further defines these issues and concludes that neutrophils retain some respiratory chain complex activity but this activity is limited to certain complexes in the respiratory chain and exists mainly for the maintenance of mitochondrial membrane potential. These papers raise critical doubt about the validity of our previous findings and we feel that it is important to explore these issues in terms of the ATP-related tests we have done and the results we have published. With regard to the exercise procedures given to the 15 CFS patients in the Vermeulen et al study , we are confident that many of the patients in our study , including all of those in the severe and very severe categories would not have been able to carry out the exercise procedures. Although the patients in both studies met the Centers for Disease Control criteria for CFS , we are well aware that there is enormous patient-to-patient variability in the degree of physical and mental incapacity. We felt it a strength of our paper that the measured biochemical parameters were compared individually and collectively with the CFS Ability [2-3] of the individual patients. This was much simpler with the higher number of patients in our study. We now discuss the issues relating to neutrophil ATP-related parameters in the three parts of the ATP profile test that we used. Whole cell ATP and ATP complexed with Mg ATP often functions as a complex with magnesium (Mg) and in many reactions Mg is an essential cofactor. Due to the fact that intracellular Mg deficiencies are common in CFS, we measured the whole cell ATP in neutrophils in the presence of excess Mg and again with endogenous Mg only. The ATP concentrations given in Figure 1 of the Maianski et al paper  and in Figure 4 of van Raam et al  lie between our results with and without added Mg. These may simply relate to methodological differences in the test procedures. We found that 87% of the patients were below the minimum value of the controls in the ATP with only endogenous Mg, i.e. the product ATP x (ATP Ratio). However, the value of the product was not a strong indicator of CFS Ability with a correlation coefficient of only r = 0.086. We did not compare baseline whole cell ATP levels in neutrophils and PBMCs. It is clear from van Raam et al  that although their ATP levels are similar to ours, there are markedly differing effects of some known inhibitors of oxidative phosphorylation. As a result, Vermeulen et al  understandably criticize our use of neutrophils to explore ATP function in CFS patients. It is appropriate that we should proceed with further studies comparing neutrophils with PBMCs and we are doing so. Until such results are available, we wish to explain further aspects of the work already done and the tests we have used. Inhibition study and ADP to ATP re-conversion - the Ox Phos parameter Following the measurement of the whole cell ATP, we used sodium azide to inhibit ATP production prior to a two-stage re-measurement of ATP. The azide ion is an inhibitor of cytochrome c oxidase, a component of Complex IV in the mitochondrial respiratory electron transfer chain. It is in the initial step that our results differ from those of Maianski et al . We saw a rapid (in less than 3 minutes) fall in measured ATP, usually to just a few percent (7.5 +/- 3.4 % for the controls) of the starting value. After removal of the inhibitor, in our control group we saw the total ATP levels return to between 60% and 90% of their original values as shown in the right-hand histogram in Figure 2C of our paper . These results appear to be inconsistent with the findings shown in Figure 1 of Maianski et al . However, they measured after 6 hours with and without inhibitor and also there may have been some compensatory process involved. For the 38% of the patients who had similar recoveries to the controls, the degree of recovery was independent of CFS Ability, while for the other group (62% of the patients) with recoveries below 60% (and as low as less than 10%) there was a strong correlation with CFS Ability, as shown in Figure 2C of our paper . We attribute these low recovery values to an inability of the mitochondria to reliably reconvert ADP to ATP. In contrast, Vermeulen et al  conclude that mitochondrial ATP production shows no defect. In order to make a comparison of the two studies we will have to ignore the fact that in our study the values of the Ox Phos parameter for the patients definitely fall into two groups, and we just take the averages. However we can do this for each value of CFS Ability. Vermeulen et al  made 4 measurements of ATP synthesis and 1 of CK in plasma for CPET1 and CPET2 and for patients and for controls. The only convenient way to compare the two studies is to compare ratios of the mean value of each parameter for patients with the corresponding mean for the controls, Patients/Controls. Because we are just comparing mean values we can use the standard error of the mean (SEM) for each mean value rather than the larger standard deviation (SD). The Ox Phos ratio for the moderate patients (CFS Ability = 4 to 7) is 0.851 +/- 0.065 (SEM, n=21) while there is a strong decrease for the more severely ill patients (0.62 +/- 0.09, 0.62 +/- 0.12 and 0.21 +/- 0.25 respectively for CFS Ability = 3, 2 and 1). This shows that mitochondrial ADP-ATP recycling does occur in mitochondria of neutrophils and is strongly correlated with CFS illness severity. The 8 Complex I and II ATP synthesis ratios, Patients/Controls, computed from the data of Table 4 , are all less than unity but the errors are such that they are also consistent with no decrease from unity. The weighted average ATP synthesis ratio is 0.905 +/- 0.062 which is consistent with the above Ox Phos ratio of the moderate group of patients. Thus the ATP synthesis data  cannot support the statement that mitochondrial ATP production shows no defect. ADP - ATP translocator (TL) study The third part of our ATP profile test explored the functionality of the translocator protein (TL or ANT for adenine nucleotide translocator), the electrogenic antiport which plays the essential role of transporting ADP into mitochondria for recycling. It also transports the ATP synthesized by recycling back into the cytosol where it is used. Neutrophils contain fewer mitochondria than PBMCs but there are sufficient for study purposes. We separated mitochondria from neutrophils and made 3 aliquots. The first was used to measure ATP within the mitochondria. The second aliquot was provided with excess ADP and the third aliquot deprived of ADP. The exact analysis conditions were decided experimentally and designed to maximize the production of mitochondrial ATP from ADP (parameter TL OUT), and the provision of ATP to the external artificial cytoplasm (parameter TL IN). In this way we explored the efficiency of ADP-ATP translocator sites in the mitochondrial inner membrane. In our control group the mitochondrial ATP increase was greater than 35% in the TL OUT part of the test with excess ADP present, while 60% of the CFS patients failed to exceed this minimum. In the TL IN part of the test where ADP access was restricted, the mitochondrial ATP decrease was between 50 and 75% for the control group. For 30/61 (or 50%) of the patients the decrease in the mitochondrial ATP was less than for the controls indicating a blocking of ATP transfer to the artificial cytosol. Both TL parameters show strong correlation with CFS Ability, and their product (TL OUT) x (TL IN) gives r = 0.683. One of us (JMH) has proceeded with further studies of TL function and has found chemical blocking in such cases. The results of molecular level fluorescence microscopy and the identification of the blocking agents by Micro Raman Spectroscopy and Fourier Transform Infrared Spectroscopy will be the subject of a further paper. Conclusions Our study  compared ATP-related parameters in CFS patients and in a similar number of controls. In retrospect, we can see that this work using neutrophils should have been paralleled by similar studies using PBMCs and we have already begun that task. In the interim, we commend the results of our paper as a significant contribution to understanding the energy depletion in CFS patients. The laboratory findings closely paralleled the functional ability of the patients. This in no way belittles the contribution made by Vermeulen et al  who conclude that transport capacity of oxygen is limited in CFS patients. We wish to see both of these avenues of investigation explored more fully. REFERENCES 1. Vermeulen RCW, Kurk RM, Visser FC, Sluiter W, Scholte HR: Patients with chronic fatigue syndrome performed worse than controls in a controlled repeated exercise study despite a normal oxidative phosphorylation capacity. J Transl Med 2010, 8:93. 2. Myhill S, Booth NE, McLaren-Howard J: Chronic fatigue syndrome and mitochondrial dysfunction. Int J Clin Exp Med 2009, 2:1-16. 3. Bell DS: The Doctor's Guide to Chronic Fatigue Syndrome. New York: Da Capo Press; 1994. 4. Maianski NA, Geissler J, Srinivasula SM, Alnemri ES, Roos D, Kuijpers TW: Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis. Cell Death and Differentiation 2004, 11:143-153. 5. van Raam BJ, Sluiter W, de Wit E, Roos D, Verhoeven AJ, Kuijpers TW: Mitochondrial Membrane Potential in Human Neutrophils Is Maintained by Complex III Activity in the Absence of Supercomplex Organisation. PLoS ONE 2008, 3:e2013. 6. Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A: The chronic fatigue syndrome: a comprehensive approach to its definition and study. Ann Intern Med 1994, 121:953-959. Competing interests One of us (JMH) carries out ATP profile tests ordered by medical doctors on a commercial basis, but makes detailed information on all procedures freely available.