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Int J Clin Exp Med (2009) 2, 1-16 Original Article Chronic fatigue syndrome and mitochondrial dysfunction Sarah Myhill1, Norman E. Booth2, John McLaren-Howard3 1Sarah Myhil Limited, Llangunl o, Knighton, Powys, Wales LD7 1SL, UK; 2Department of Physics and Mansfield Col ege, University of Oxford, Oxford OX1 3RH, UK; 3Acumen, PO Box 129, Tiverton, Devon EX16 0AJ, UK Received December 2, 2008; accepted January 12, 2009; available online January 15, 2009 Abstract: This study aims to improve the health of patients suffering from chronic fatigue syndrome (CFS) by interventions based on the biochemistry of the il ness, specifical y the function of mitochondria in producing ATP (adenosine triphosphate), the energy currency for al body functions, and recycling ADP (adenosine diphosphate) to replenish the ATP supply as needed. Patients attending a private medical practice specializing in CFS were diagnosed using the Centers for Disease Control criteria. In consultation with each patient, an integer on the Bell Ability Scale was assigned, and a blood sample was taken for the "ATP profile" test, designed for CFS and other fatigue conditions. Each test produced 5 numerical factors which describe the availability of ATP in neutrophils, the fraction complexed with magnesium, the efficiency of oxidative phosphorylation, and the transfer efficiencies of ADP into the mitochondria and ATP into the cytosol where the energy is used. With the consent of each of 71 patients and 53 normal, healthy controls the 5 factors have been collated and compared with the Bel Ability Scale. The individual numerical factors show that patients have different combinations of biochemical lesions. When the factors are combined, a remarkable correlation is observed between the degree of mitochondrial dysfunction and the severity of il ness (P<0.001). Only 1 of the 71 patients overlaps the normal region. 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 Key Words: Chronic fatigue syndrome, myalgic encephalomyelitis, mitochondria, neutrophils, oxidative phosphorylation. fatigue is considered to be a subjective sensation characterized by lack of motivation Chronic Fatigue Syndrome (CFS) is a and of alertness [1], even though the brain is a multisystem illness that robs its victims of their major consumer of resting cellular energy. health and their dignity. Two of the most Patients may demonstrate negative illness characteristic and debilitating signs of CFS are beliefs that increase the severity of the very poor stamina and delayed post-exertional symptoms [2, 3]. However, if the metabolism is fatigue. Sometimes the fatigue is mainly functioning properly, the fatigue and related mental, and sometimes mainly physical. symptoms must be due to energy being Fatigue is the same as lack of energy and wasted by the mental and physical processes energy comes from the basic metabolic of stress, anxiety, tension and depression. process of the oxidation of food. Patients should be able to be helped, possibly cured by psychological intervention, e.g. A widely-held hypothesis (A) is that the cognitive behavioural therapy. In order to metabolism of people with CFS is normal, but explain the post-exertional malaise an ancillary the fatigue and other symptoms are due to hypothesis (A') is needed, namely psychological factors. It is acknowledged that deconditioning due to disuse of muscles. physical fatigue is lack of energy, but mental However, hypothesis A' is not supported by Chronic fatigue syndrome and mitochondrial dysfunction experiment in many cases as we will see have also found severe deletions of genes in mitochondrial DNA (mtDNA), genes that are associated with bioenergy production [9, 10]. An alternative hypothesis (B) is that there is a One consequence of mitochondrial metabolic dysfunction with the result that not dysfunction is increased production of free enough energy is being produced. The main radicals which cause oxidative damage. Such source of energy comes from the complete oxidative damage and increased activity of oxidation of glucose to carbon dioxide and antioxidant enzymes has been detected in water. The digestive system produces glucose, muscle specimens [11]. Some essential glycerol and fatty acids, and amino acids. If compounds (carnitine and N-acylcarnitine) there is a problem with the digestive system, needed for some metabolic reactions in e.g. gut fermentation, hypochlorhydria or mitochondria have been measured in serum pancreatic insufficiency, energy production will and found to be decreased in patients with be impaired and fatigue may result [4]. These CFS [12, 13]. Both studies found that the conditions can and should be tested for. carnitine levels correlated with functional Allergies and thyroid malfunction can also capacity. Reduced oxidative metabolism [14- produce fatigue. 16] and higher concentrations of xenobiotics, lactate and pyruvate [17] have been reported. When the digestive system is functioning In one group of patients a decrease of properly glucose and lipids are fed into the intracellular pH after moderate exercise was blood stream where, together with oxygen observed and a lower rate of ATP synthesis bound to hemoglobin in erythrocytes (red during recovery was measured [18]. These blood cells), they are transported to every cell findings suggest impaired recycling of ADP to in the body. In the cytosol of each cell glucose ATP in the mitochondria. is broken down in a series of chemical reactions called glycolysis into two molecules However, there are also some similar studies of pyruvate which enter the energy-producing that do not confirm mitochondrial dysfunction. organelles present in most cells of the body, This situation is likely due to the different the mitochondria. Some structural details and diagnostic criteria in use. For example, the the number of mitochondria per cel are Oxford criteria [1], a definition proposed by dictated by the typical energy requirements; psychiatrists, require only fatigue; "other cardiac and skeletal muscle cells and liver and symptoms may be present" but are not brain cells contain the highest numbers. The essential. The Centers for Disease Control mitochondria generate energy by oxidative (CDC) criteria are more selective as they metabolism in the form of ATP (adenosine require an additional four symptoms from a list triphosphate) which when hydrolysed to the of eight [19]. In England in 2007 the National diphosphate, ADP, releases energy to produce Institute for Clinical Excellence (NICE) muscle contractions, nerve impulses and all introduced yet another set of criteria, fatigue the energy-consuming processes including the plus one more symptom, for example chemical energy needed to synthesise all of "persistent sore throat" [20]. At the other end the complex molecules of the body [5, 6]. of the spectrum are criteria based on studies Thus, mitochondrial dysfunction will result in of patients with Myalgic Encephalomyelitis fatigue and can produce other symptoms of (ME) [21-23] which have culminated in the Canadian consensus criteria [24]; the Canadian criteria are unlikely to include The two hypotheses are not mutually patients satisfying only hypothesis A. Even exclusive. Some patients may satisfy both. more confusingly, both the Canadian and the However there are constraints; the basal new NICE criteria use the term ME/CFS metabolic rate (about 7000 kJ per day) must although their criteria are very different. At the be maintained and the first law of present time the CDC criteria are thermodynamics must not be violated. internationally widely used as the criteria for research purposes despite their lack of There is considerable evidence that precision [25]. This situation may change in mitochondrial dysfunction is present in some the future because the Canadian criteria are CFS patients. Muscle biopsies studied by gaining wider acceptance and one charitable electron microscopy have shown abnormal research funding agency (ME Research UK) mitochondrial degeneration [7-9]. Biopsies now requires both the CDC and Canadian Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction criteria to be used in research projects that it (JMH), is designed specifically for CFS and funds. We use the term CFS or CFS/ME for the other conditions where energy availability is CDC criteria and ME/CFS for the Canadian reduced. It was found early on that the "ATP criteria. Our study is aimed to assess the role profile" was very useful in predicting the level of mitochondrial dysfunction with the primary of disability and suggesting the most likely aim of helping patients interventions which would benefit patients. Tests have now been carried out on a number Hypothesis B is attractive because of patients and also on normal, healthy mitochondrial dysfunction in various organs subjects. When collated the test results show offers possible explanations for many of the features that were completely unexpected. other symptoms of CFS and ME. There is Before we report here on the test procedures mounting evidence that the symptoms are due and results we provide a brief summary of how to dysfunctions on the cel ular level. mitochondria produce energy. Abnormalities have been seen in immune cel s [26], and gene expression studies have Mitochondrial energy metabolism revealed abnormalities in genes associated with immune cells, brain cel s, skeletal muscle In each cell glucose is broken down to cells, the thyroid, and mitochondria [27, 28]. A pyruvate with the production of some ATP (2 further genetic study identified seven clinical molecules net per molecule of glucose). The phenotypes [29]. There seem to be three pyruvate and also fatty acids enter the distinct clusters of clinical abnormalities that mitochondria of each cell, shown define CFS [30]: (a) blood flow and vascular schematically in Figure 1, where two abnormalities such as orthostatic intolerance coordinated metabolic processes take place: (vascular system), (b) widespread pain, and the tricarboxylic acid (TCA) cycle, also known high sensitivities to foods, temperature, light, as the Krebs' citric acid cycle, which produces noise and odours (central nervous system some ATP, and the electron transport chain sensitization), and (c) fatigue, exhaustion and (ETC, also called the Respiratory Chain brain fog (impaired energy production). because it uses most of the oxygen we breathe Hypothesis B is that the lack of energy in the in) which regenerates ATP from ADP by the third cluster originates in the mitochondria of process of oxidative phosphorylation (ox-phos). individual cel s. But mitochondrial dysfunction Altogether some 30-odd molecules of ATP are can also produce abnormalities (a) and (b) produced per molecule of glucose and these because ATP produced in each cell by its constitute the main cellular energy packets mitochondria is the major source of energy for used for al life processes. As well as food and all body functions. oxygen the metabolic pathways require all the nutrients involved in the production of the These observations from biomedical research large number of enzymes which control the into CFS are very encouraging, but how long do many biochemical reactions involved and all patients have to wait before there is some real the cofactors needed to activate the enzymes progress in ameliorating their symptoms? In a [31-33]. Most of the enzymes are coded by private medical practice which specializes in nuclear DNA (nDNA) in the cell's nucleus and a CFS the primary goal is to make the patients few are coded by mtDNA. Some of the feel and function better. Treatment is started enzymes rely on other organs. For example, by making use of the existing biomedical thyroid hormone is needed in the TCA cycle. On knowledge to provide a basis of nutrition, the other hand hyperthyroidism can uncouple lifestyle management and pacing. Thyroid, the ox-phos process [34], so a thyroid problem adrenal and allergy problems are also can lead to fatigue and this can be tested for. addressed if they occur. Most patients improve The human body contains typically less than with these interventions. However, in many 100 g of ATP at any instant, but can consume cases the improvement is not as great as the up to 100 kg per day. Thus the recycling ox- patient and doctor would like. When one of us phos process is extremely important and it (SM) became aware of the commercial "ATP produces more than 90% of our cellular profile" testing package it was thought that energy. The main features and processes are this might be useful in predicting the level of illustrated in a simplified form in Figure 1 disability and identifying any biochemical (further details can be found in all college-level lesions that were at fault. The "ATP profile" textbooks on biochemistry, e.g. [6], and in testing package, developed by one of us secondary school advanced-level biology Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction Figure 1. Main stages and location of energy metabolism in a human cel (left), and simplified details of a mitochondrion showing the main metabolic cycles and the oxidative phosphorylation respiratory chain (right). The outer mitochondrial membrane is highly permeable whereas the inner membrane is permeable only to water and gases. Special carrier and Translocator proteins pass reactants through it. At the top are the proteins involved in the respiratory electron transfer chain (ETC) and in the transfer of ATP and ADP between the cytosol and mitochondrion. ADP and Pi are combined by ATP synthase to make ATP. The ADP/ATP Translocator opens OUT to transfer ADP into the matrix and opens IN to transfer ATP to the cytosol. Nicotinamide adenine dinucleotide plays a key role in its oxidised form NAD+ and its reduced form NADH + H+ in carrying and transferring protons (H+) and electrons (e-). Adapted from: [35] and [5]. textbooks, e.g. [35]). The ETC culminates with transfer outwards [37], and there is the the protein complex ATP synthase which is possibility that there may be other molecules effectively a reversible stepping motor in including environmental contaminants which which 3 ATP molecules are produced from can block transfers. ADP and inorganic phosphate (Pi) every revolution [36]. Because of evolutionary What happens if some part of these cellular history ATP is made inside the mitochondrial metabolic pathways goes wrong? If the inner membrane but used outside in the mitochondrial source of energy is cytosol where it releases energy by dysfunctional many disease symptoms may converting to ADP and Pi. The Pi as a appear [38] including the symptoms of CFS. negative ion is co-transported back inwards together with H+, while ADP3- is transported Suppose that the demand for ATP is higher inwards through the Translocator protein than the rate at which it can be recycled. This adenosine nucleotide translocase (TL or ANT) happens to athletes during the 100 meters in exchange for ATP4- moving out into the sprint. The muscle cells go into anaerobic cytosol. There are potential problems here metabolism where each glucose molecule is because it is known that some specific converted into 2 molecules of lactic acid. This molecules (e.g. atractyloside) block the process is very inefficient (5.2% energy transfer inwards and certain others can block production compared to the 100% of complete Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction oxidation) and can last for only a few minutes. be performed. The laboratory carrying out the The increased acidity leads to muscle pain. tests (Biolab Medical Unit, Also, when the concentration of ADP in the was blinded to the Ability associated with any cytosol increases and the ADP cannot be blood sample. As tests were carried out on recycled quickly enough to ATP, another more patients, it became clear that the "ATP chemical reaction takes place. This becomes profile" results were providing helpful important if there is any mitochondrial information, and patients were asked to give dysfunction. Two molecules of ADP interact to written, informed permission for their test data produce one of ATP and one of AMP to be used anonymously. All patients have (adenosine monophosphate). The AMP cannot be recycled [6] and thus half of the potential ATP is lost. This takes some days to replenish Blood samples from fifty-three normal, healthy and may account for the post-exertional volunteers were obtained by one of us (JMH) malaise symptom experienced by patients [39- as Laboratory Director of Biolab until retirement from that position in 2007. Biolab obtained written permission with informed Thus, mitochondrial dysfunction resulting in consent from each volunteer. The samples impaired ATP production and recycling is a from the patient group and the normal biologically plausible hypothesis, and there is (control) group were processed in the same considerable evidence that it is a contributory way. The control group consisted of 40 factor in CFS, at least for a subset of patients. females of average age 36 (range 18 to 63) Our study may be considered to be a test of and 13 males of average age 35 (range 18 to this hypothesis. For both groups al procedures were consistent with the Declaration of Helsinki (2000) of the Participants World Medical Associationnd this report follows the guidelines of the Seventy-one patients, 54 female of average International Committee of Medical Journal age 47 (range 14 to 75) and 17 male of Editors (icmje.pdf available at average age 52 (range 20 to 86), were selected from a total of 116 consecutive Procedures patients attending a private medical clinic specializing in CFS/ME. Patients were ATP is present in cel s mainly as a complex excluded only if they did not meet the CDC with magnesium and is hydrolysed to the diagnostic criteria for CFS [19] or if the "ATP diphosphate (ADP) as the major energy source profile" test had been made before they had for muscle and other tissues. ADP conversion been seen clinically. Evaluations, tests and to ATP within mitochondria can be blocked or interventions, where appropriate, were carried partially blocked by some environmental out for diet and sleep problems, al ergies, and contaminants. Specifically, the TL in the thyroid and adrenal problems. Advice on mitochondrial membrane that controls the pacing was also given. After this stage a transfer of ADP from the cytosol and ATP to the meeting was held with each patient at which cytosol may be chemically inhibited and its an agreed numerical Ability was assigned and efficiency is also pH dependent. Changes in recorded in the clinical notes. The integral CFS acid:base balance, magnesium status, and the Ability Scale [44] runs from 0 to 10 and is presence of abnormal metabolic products can given in Appendix A. It was proposed to those have similar effects to xenobiotic inhibition of patients who had not improved to an acceptable clinical level after these interventions that they have the "ATP profile" A number of methods have been developed for test done. All the participating patients had assaying ATP. Methods such as magnetic scores of 7 or less on the CFS Ability Scale. resonance spectroscopy (MRS) of 31P require the patient to be at a facility which is available The nature of the test was explained and each only at major hospitals or research institutes. patient agreed (and paid) for the "ATP profile" Biopsies of skeletal muscles can be taken, but test (needing a 3-ml venous blood sample) to not of vital organs such as the heart, brain or Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction liver. Methods using blood samples The mitochondria should then rapidly replete (specifically neutrophils) are relatively non- the ATP from ADP and restore the ATP invasive and are amenable to routine testing. concentration. The overall result gives Ox In addition the blood stream reaches almost Phos, which is the ADP to ATP recycling every cell in the body and carries much efficiency that makes more energy available as information concerning what is going on. The method of measuring ATP used in the "ATP profile" dates from 1947 when McElroy (C). The TL switches a single binding site poured a solution of ATP onto ground-up firefly between two states. In the first state ADP is tails and observed bright luminescence and recovered from the cytosol for re-conversion to found that the amount of light produced was ATP, and in the second state ATP produced in proportional to the ATP concentration [45]. the mitochondria is passed into the cytosol to Thus he showed that the energy contained in release its energy. Measurements are made by ATP can produce light and this led the way to trapping the mitochondria on an affinity the development of bioluminescent chromatography medium. First the measurements which can be carried out mitochondrial ATP is measured. Next, an ADP- routinely and reproducibly with commercially containing buffer is added at a pH that available biochemical assay kits and strongly biases the TL towards scavenging ADP bioluminescence equipment [46-49]. Light is for conversion to ATP. After 10 minutes the produced when ATP reacts with D-luciferin and ATP in the mitochondria is measured. This oxygen in the presence of Mg2+ and the yields the number TL OUT. This is a measure of enzyme luciferase. When ATP is the limiting the efficiency for transfer of ADP out of the reagent, the light emitted is proportional to the cytosol for reconversion to ATP in the mitochondria. In the next measurement a buffer is added at a pH that strongly biases the The "ATP profile" test TL in the direction to return ATP to the cytosol. After 10 minutes the mitochondria are washed The "ATP profile" test yields 5 independent free of the buffer and the ATP remaining in the numerical factors from 3 series of mitochondria is measured and this gives the measurements, (A), (B), and (C) on blood number TL IN. This is a measure of the samples (neutrophils). Details of the efficiency for the transfer of ATP from the measurements made and how the numerical mitochondria into the cytosol where it can factors were calculated are given in Appendix release its energy as needed. B. The 3 series are: (A). ATP concentration in the neutrophils is measured in the presence of excess The individual numerical factors magnesium which is needed for ATP reactions. This gives the factor ATP in units of nmol per Figure 2 shows scatter plots (a point for each mil ion cells (or fmol/cell), the measure of how patient) of each of the 5 factors vs. CFS Ability. much ATP is present. Then a second As we will see later it is convenient to divide measurement is made with just endogenous the data from the 71 patients into 3 magnesium present. The ratio of this to the categories, "very severe", "severe", and one with excess magnesium is the ATP Ratio. "moderate", which have about the same This tells us what fraction of the ATP is number of entries (25, 21, and 25). To the available for energy supply. right of each scatter plot we show a stacked projection histogram for the 3 categories of (B). The efficiency of the oxidative Ability, and at the far right a histogram for the phosphorylation process is measured by first normal controls. Looking first at the ATP inhibiting the ADP to ATP conversion in the histogram for the normal controls we see a laboratory with sodium azide. This chemical well defined minimum value with a long tail up inhibits both the mitochondrial protein to a maximum value of 2.89 fmol/cell. The cytochrome a3 (last step in the ETC) and ATP average value is 2.00 ± 0.05 (SEM (Standard synthase [50]. ATP should then be rapidly used Error of the Mean), n=53) which can be up and have a low measured concentration. compared with the measurement, 1.9 ± 0.1 Next, the inhibitor is removed by washing and (SEM, n=12), made some 25 years ago by the re-suspending the cells in a buffer solution. same technique in a study of the energetics of Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction Figure 2. Scatter plots of the 5 factors (A to E) measured in the "ATP profile" test vs. CFS Ability. In the middle are stacked projection histograms of the 3 categories of the patient group, and on the right projection histograms of the control group. The heavy horizontal dashed lines correspond to the minimum value of each factor measured for the control group. Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction phagocytosis in neutrophils [51]. The stacked control group. Many patients, particularly the histogram for the patients and the Ability plot "very severe", are far below the normal clearly show that some patients are in the normal region and some are below and they split into two groups with very little overlap. The TL IN plot of Figure 2E also has a peak in Rather than comparing the patients with the the normal region. However, some patients average of the normal control group which is have very low values, including the patient with customary, we prefer to compare with the CFS Ability = 0. The product TL OUT × TL IN is minimum value of the control group which is only 0.012 for this patient who is very severely more cleanly defined. Also, this method ill whereas this product is 0.17 at the normal permits us to classify patients as being in the minima, a factor of 14 larger. If just ATP or Ox normal region or being below the normal Phos had been measured the very severe region. Clearly this can be changed easily and mitochondrial dysfunction of this patient would al the numbers are given in Figure 2. We not have been detected. Note the strong therefore show as a heavy horizontal dashed positive correlation for TL IN. line the minimum value of each factor measured for the controls. Most patients are below normal in more than one factor (average [range] is 3.7 [2 to 5] for Figure 2A, ATP vs. CFS Ability, shows that the "very severe", 3.5 [2 to 5] for "severe" and 2.2 majority of the "very severe" and "severe" [1 to 4] for "moderate"). Some of the features patients are below the normal minimum but are summarised numerical y in Table 1. very few are below 75% of this minimum. Note that 3 of the "very severe" patients are well For most of the factors the percentage of into the normal region; they have problems patients who are in the normal region with one or more of the 4 other factors. Just increases in going from "very severe" to over 50% of the "moderate" patients are in the "severe" and to "moderate". The exception is normal region. There is a small positive the ATP Ratio which gently decreases, but correlation which is indicated by the "trend" within the statistical errors is constant. Both TL crosses. There is not a gentle increase in ATP IN and the product TL OUT × TL IN increase by with Ability, but an increase in the fraction of large factors. For patients in the "moderate" patients above the normal minimum line. category the main influence on their il ness appears to be the ATP Ratio. Figure 2B shows ATP Ratio vs. CFS Ability. The majority of patients in all 3 categories are Table 1 also illustrates the importance of below the normal minimum, and about 1/3 of measuring more than one factor. For example, "moderate" patients are below 75% of the if only ATP had been measured, 28% of al the normal minimum. The correlation with Ability is patients would be classified as normal, and if slightly negative. Values for the normal only Ox Phos had been measured, 32% of the controls are rather tightly grouped with a "very severe" patients would be classified as minimum of 0.65 and average of 0.69. The Ox Phos plot in Figure 2C shows a wide Correlations between numerical factors range of values and a strong positive correlation for this factor for the patient group. It is also helpful to look at correlations The stacked projection clearly shows that between pairs of numerical factors. The five there are two groups – above and below the most relevant examples are shown in Figure 3. normal minimum and the upper group spans a similar range to the controls. Note the high In the scatter plots of Figure 3 the normal value for the sole patient with CFS Ability = 0. region is the rectangular region in the upper This patient also has ATP = 1.26 and ATP Ratio right corner defined by the normal minima = 0.59 which are not very far below the normal dashed lines. In the ATP Ratio vs. ATP plot (Figure 3A) most patients are fairly close to the normal region apart from the small cluster at The TL OUT plot of Figure 2D also shows two groups with a rather sharp peak in the stacked projections just above the normal minimum In the Ox Phos vs. ATP plot (Figure 3B) there and this closely matches the projection of the are only a few patients, all "moderate", in the Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction Table 1. Some features of the factors measured in the "ATP profile" tests Percentage of patients in normal region The errors shown are ±1 SD (Standard Deviation), computed with the binomial distribution. Figure 3. Scatter plots of correlations between pairs of factors measured in the "ATP profile". normal region for both factors. Some of the and vice versa. This is expected because the "very severe" and "severe" patients are in the ATP concentration is a major factor in the normal region for Ox Phos and some are far control of the rate of the ox-phos process and below. Note the apparent negative correlation the energy supply is adjusted to meet the for the normal controls. This shows that for energy demand. There is no obvious evidence normal subjects there is a compensatory for this effect in the patient group. mechanism, i.e. if ATP is high Ox Phos is low Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction In the Ox Phos vs. TL OUT plot (Figure 3C) only supply of ATP. Ox Phos is the efficiency of the 6 (all "moderate") patients are in the normal ETC which converts ADP into ATP. However, for region of both factors. Note that as a function the recycling of ADP to make more energy of TL OUT there are 2 groups, a distinct narrow available the Translocator protein must band to the right of the vertical dashed line efficiently have its binding site facing out to and a spread-out group to the left of this line. collect ADP (TL OUT) and alternately facing in Looking vertically, note that for the first group (TL IN) to efficiently transmit ATP from the the Ability of patients (indicated by the 3 mitochondria into the cytosol where its energy categories) is correlated with their value of Ox Phos. There is a large spread in this factor and again there appears to be two groups roughly We have found it useful to calculate the divided by the horizontal normal minimum line. product of the five factors, the overall Some of the patients in all 3 categories are mitochondrial energy-producing relative above this line, but they have problems with efficiency, and call it the Mitochondrial Energy one or more of the other factors. Some of the Score. We just multiply the 5 factors together "very severe" patients have Ox Phos lower for each patient and each control. The than the normal minimum by an order-of- minimum value for the controls is 0.182 fmol/cell. We have chosen this as our normalisation point so we divide all the Energy In the Ox Phos vs. TL IN plot (Figure 3D) there Scores (for both patients and controls) by this are many more patients in the normal region value. Thus all controls have Mitochondrial for both factors, but also many with very low Energy Score ≥ 1.00. values of one or both factors. A scatter plot of the Energy Score for each In the TL IN vs. TL OUT plot (Figure 3E) there patient at each value of CFS Ability and each are two clusters, one in the upper right corner control is shown in Figure 4A. The horizontal which is the normal region for both factors, dashed line indicates the minimum value for and another well below it at TL IN 0.1. the normal controls and this is our Several patients are far below the normal normalization value of 1.00. Only one of the minimum for both factors. 71 patients has an Energy Score > 1 (namely 1.25 for one of the patients with Ability = 7). In the biochemical methods used we might However this patient has 2 of the 5 factors expect some correlation between the TL below the normal minima. factors and Ox Phos because they are closely coupled and interacting parts of the ADP to Note the high degree of correlation between ATP reconversion cycle. However, the plots Energy Score and CFS Ability and this is indicate that the biochemical methods used independent of where the mean or minimum can separate the Ox Phos and TL factors and of normal subjects is. It is natural to believe measure them individually. that the CFS Ability of patients is more likely to depend upon mitochondrial dysfunction than To our knowledge this is the first time that vice versa, so we should really plot CFS Ability such detailed effects have been observed. vs. Energy Score. However, the Ability was measured first, and Figure 4A shows The Mitochondrial Energy Score convincingly that mitochondrial dysfunction is a major risk factor, and this has not been The biochemical measurements in the "ATP demonstrated before. Also shown in Figure 4A profile" separate the energy generation and is the best straight line fit to all 71 entries. The recycling processes into 5 steps. As in any fit is good, but there is no reason that the multistep process, for example electrical relationship should be a straight line. Table 2 power production or an assembly line, the gives the parameters of the fit. The Standard efficiency of the overal process is the product Error in the slope of the fitted straight line is so of the efficiencies of the individual steps. Any small that the probability P of the null suggestion of relative weighting is irrelevant; it hypothesis (i.e. that the slope is zero) is only results in an overall normalization factor. extremely small, P <0.001, when computed The product of ATP and ATP Ratio is the from the Student's t-distribution [52]. The cellular concentration of ATP complexed with 99.9% confidence interval is 0.092 < β < magnesium and this is the available energy 0.174 where β is the true slope and this lower Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction Figure 4. The Mitochondrial Energy Score. A. The Energy Score plotted against CFS Ability with a point for each patient. A point for each control is plotted at CFS Ability = 10. The horizontal dashed line at Energy Score = 1.00 is our normalisation at the minimum Energy Score for controls. Also shown is the best straight line fit to the patient data. B. The Energy Score plotted vs. Age of patients and controls. Table 2. Parameters of straight line fit to Mitochondrial Energy Score data Straight line fit results Observed t-test Degrees of freedom t-test probability, P *R2 (cal ed the "coefficient of determination" or the "explained variation") is the square of the product moment correlation coefficient. † There are 71 data points and 2 parameters, slope and intercept. limit is still several Standard Errors above zero. control group as compared to the patient group may influence our results. We have In Figure 4B the Energy Scores are plotted as a looked at the age dependence of al 5 factors function of the age of each participant. It is and see no effect, and this is not surprising in believed that mitochondria play a major role in view of the wide spread in values of each the aging process [31, 33] so there is the factor. The Energy Score is a more reliable possibility that the younger mean age of the measure of mitochondrial dysfunction. In Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction Figure 4B there is no evidence for age dependence in the control group but the maximum age is only 65 years (points for three The ways that the individual factors in the controls have been omitted because their Mitochondrial Energy Score behave show that Energy Score is more than 2.00 with a not all patients are affected in the same way. maximum of 2.83). There are six patients of This may be due to the heterogeneous nature age ≥ 70, one "very severe" with Ability = 2, of the precipitating agents or to variations in one "severe" with Ability = 3, and four the way patients react to them. The results "moderate" who al have Ability = 4. These four indicate specific biochemical lesions and some patients have Energy Scores which are below of these may be amenable to ameliorative the average (0.42) for this Ability so there may intervention. Mitochondria need all of their well be a decrease with increasing age. On the essential vitamins, minerals, essential fatty other hand there is a 33-year old patient who acids and amino acids to function properly also has Ability = 4 and is well below the [31-33]. From the clinical point-of-view of average. Excluding the six patients of age ≥ 70 helping patients this is very important; the slightly improves the straight line fit (R2 = typical time-consuming hit-or-miss protocol 0.677) but has negligible effect on the other can be replaced by interventions based on parameters or our conclusions. biochemical information and understanding An analysis of the outcomes of interventions The "ATP profile" results indicate being carried out (se mitochondrial dysfunction of the neutrophils in will be the subject of a future publication. the patients in our cohort, and moreover the degree of dysfunction is strongly correlated with the severity of their illness. Neutrophils are the major effector cells of the immune We have demonstrated the power and system and the observed mitochondrial usefulness of the "ATP profile" test in dysfunction is bound to have a deleterious confirming and pin-pointing biochemical effect on this system. We note that increased dysfunctions in people with CFS. apoptosis of neutrophils has been observed previously in people with CFS [26]. Our observations strongly implicate Mitochondria are important functional parts of mitochondrial dysfunction as the immediate almost all human cells but we cannot assert cause of CFS symptoms. However, we cannot from the present study that the mitochondria tel whether the damage to mitochondrial in other cells are dysfunctional to the same function is a primary effect, or a secondary degree; human biology provides energy to vital effect to one or more of a number of primary organs at the expense of less important parts. conditions, for example cel ular hypoxia [30], However, dysfunction in heart muscle cells or oxidative stress including excessive and in central nervous system cells could [54-58]. Mitochondrial explain respectively the vascular and central dysfunction is also associated with several sensitization clusters of clinical abnormalities other diseases and this is not surprising in mentioned in the Introduction. Thus, our view of the important role of mitochondria in results strongly suggest that the immediate almost every cell of the body, but this fact cause of the symptoms of CFS/ME is appears to have been recognised only in mitochondrial dysfunction. recent years [34, 38, 59, 60]. We cannot overemphasize the importance of a The observations presented here should be careful diagnosis using the CDC criteria [19], confirmed in a properly planned and funded or even better the Canadian criteria which study. The biochemical tests should be done more precisely describe the symptoms [24]. on CFS patients after, as well as before, (An abridged version designed for health appropriate interventions and possibly on professionals, patients and carers is available patients with other disabling fatigue conditions. It would also be good to confirm . It is doubtful that the biochemical test results in a second patient selection with less selective criteria (perhaps government-supported) laboratory. would yield the high degree of correlation Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction Acknowledgements exertion. Able to work full-time with difficulty. We acknowledge helpful comments from Dr. 8. Mild symptoms at rest. Symptoms worsened by Derek Pheby and Dr. Neil Abbot. exertion. Minimal activity restriction noted for activities requiring exertion only. Able to work full Address correspondence to: Norman E. Booth, PhD, time with difficulty in jobs requiring exertion. ‘Applegate', Orchard Lane, East Hendred, Wantage OX12 8JW, UK, Telephone: +44 (0)1235 833486 9. No symptoms at rest; mild symptoms with activity; normal overall activity level; able to work full-time without difficulty. Appendix A - The Bell CFS Ability scale 10. No symptoms at rest or with exercise; normal This scale is a useful and sensitive measure of the overall activity level; able to work or do house/home level of activity and ability to function of patients work full time without difficulty. with CFS/ME [44]. It is similar to the Energy Index Point Score (EIPS™, Appendix B - The "ATP profile" tests [61]. It runs from 0 to 10 with: The "ATP profile" tests were developed and carried 0. Severe symptoms on a continuous basis; out at the Biolab Medical Unit, London, UK bedridden constantly; unable to care for self. where one of us (JMH) was Laboratory Director until retirement in 2007. Blood 1. Severe symptoms at rest; bedridden the majority samples in 3-ml heparin tubes were normally of the time. No travel outside of the house. Marked received, tested and processed within 72 hours of cognitive symptoms preventing concentration. venepuncture. We briefly describe here the 3 series of measurements, (A), (B) and (C) and how the 5 2. Moderate to severe symptoms at rest. Unable to numerical factors are calculated. (Step-by-step perform strenuous activity. Overall activity 30-50% details can be obtained by contacting JMH at of expected. Unable to leave house except rarely. Confined to bed most of day. Unable to concentrate for more than 1 hour per day. Neutrophil cells are separated by HistopaqueTM density gradient centrifugation according to Sigma® 3. Moderate to severe symptoms at rest. Severe Procedure No. 1119 (1119.pdf available at symptoms with any exercise; overall activity level . Cell purity is checked using reduced to 50% of expected. Usual y confined to optical microscopy and cel concentration is house. Unable to perform any strenuous tasks. Able assessed using an automated cel counter. to perform desk work 2-3 hours per day, but Quantitative bioluminescent measurement of ATP is requires rest periods. made using the Sigma® Adenosine 5'-triphosphate (ATP) Bioluminescent Somatic Cel Assay Kit 4. Moderate symptoms at rest. Moderate to severe (FLASC) according to the Sigma® Technical Bulletin symptoms with exercise or activity; overall activity No. BSCA-1 (FLASCBUL.pdf). In this method ATP is level reduced to 50-70% of expected. Able to go out consumed and light is emitted when firefly once or twice per week. Unable to perform luciferase catalyses the oxidation of D-luciferin. The strenuous duties. Able to work sitting down at home light emitted is proportional to the ATP present, and 3-4 hours per day, but requires rest periods. is measured with a Perkin-Elmer LS 5B Fluorescence Spectrometer equipped with a flow- 5. Moderate symptoms at rest. Moderate to severe through micro cell. Sigma® ATP Standard (FLAA.pdf) symptoms with exercise or activity; overall activity is used as a control and as an addition-standard for level reduced to 70% of expected. Unable to checking recovery. Similar kits are available from perform strenuous duties, but able to perform light other providers, e.g. the ENLITENTM ATP Assay duty or desk work 4-5 hours per day, but requires System (Technical Bulletin at , and dedicated instruments are now available, e.g. Modulus Luminescence Modules (see Application 6. Mild to moderate symptoms at rest. Daily activity limitation clearly noted. Overall functioning 70% to 90%. Unable to work full time in jobs requiring physical labour (including just standing), but able to (A). ATP is first measured with excess magnesium work full time in light activity (sitting) if hours added via Sigma® ATP Assay Mix giving result a. This is the first factor, the concentration of ATP in whole cel s, ATP = a in units of nmol/106 cells (or 7. Mild symptoms at rest; some daily activity limitation clearly noted. Overall functioning close to 90% of expected except for activities requiring The measurement is repeated with just the endogenous magnesium present by using Int J Clin Exp Med (2009) 2, 1-16 Chronic fatigue syndrome and mitochondrial dysfunction analogous reagents produced in-house without with chronic fatigue syndrome. Br J Health added magnesium, giving result b in the same Psychol 2003; 8: 195-208. units. The ratio, c = b/a, is the second factor, the [4] Howard JM. Intestinal dysbiosis. Complement Ther Med 1993; 1: 153-157. [5] Alberts B, Johnson A, Lewis J, Raff M, Roberts (B). In order to measure the ADP to ATP conversion K and Walter P. Molecular Biology of the Cell. efficiency via the ox-phos process, the ATP (with New York: Garland Science, 2002. excess magnesium) result, a, is used and then the [6] Voet D, Voet JG and Pratt CW. Fundamentals of conversion is inhibited in the laboratory with sodium Biochemistry. 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