The size of the population sampled in this study is comparable to published work such as Naviaux et al. [19
] and Armstrong et al. [17
], and is an expansion from the cohort we used in our previous effort [21
], as displayed in Table 4
. Moreover, the substantial number of metabolites quantified (832), is notably larger than the 420 compounds of Naviaux et al. [19
], the 361 in Germain et al. [21
] or the 29 in Armstrong et al. [17
]. Undoubtedly, the statistical power of our analysis was weakened by the combination of a 51-subject population and an 832-metabolites array, explaining the limited number of metabolites we establish as significantly different in Table 2
and Figure 1
, and only after super-pathway subgrouping. Nevertheless, our findings, through the extensive analysis of our dataset, shines light on an intriguing aspect of dysfunctional metabolism in ME/CFS patients, related to redox status.
Out of the four biological classes disturbed in our cohort, the “Nucleotides” and “Peptides” categories contain metabolites that potentially have broad repercussions on biological functions. For example, cAMP and IMP are compounds known to be involved in many aspects of human body function, such as purine metabolism, chemical energy storage in muscles, and intra-cellular signal transduction. It is therefore extremely difficult to pinpoint a singular pathway linked to ME/CFS status or symptoms based on such compounds or, on the contrary, using compounds of which there is little to no knowledge, such as 2’-O-methylcytidine or gamma-glutamylthreonine. The latter molecule, however, is mentioned as a potential compound of interest among many other biomarkers to determine liver toxicity of a given agent in a patent [36
]. Results from our previous work [21
] had focused attention on liver injury biomarkers.
Another metabolite of major interest is alpha-ketoglutarate because it is part of the “Energy” super-pathway and the TCA cycle sub-pathway. Indeed, the Krebs cycle is a pathway that consistently surfaces in ME/CFS metabolomics analysis across platforms and populations. Because fatigue is a major debilitating symptom of this disease, it has long been speculated that the energy metabolism of patients is dysfunctional. Several studies directly point to abnormal energy metabolism due to flawed TCA and urea cycles or directly upstream with putative impairment in pyruvate dehydrogenase [18
]. A pilot study using a patented nutraceutical treatment hypothesized to boost the activity of this enzyme, and consequently the Krebs cycle, describes substantial improvements to the health and condition of treated patients [45
]. Nevertheless, alpha-ketoglutarate is involved in numerous metabolic pathways such as carnitine metabolism, lysine metabolism and branched-chain amino acids, to name a few, so that a focus on a single pathway as the foundation of the disabling symptoms of ME/CFS is presently unjustified.
The “Cofactors and Vitamins” category encompasses metabolites with disparate properties, as exemplified by heme and gamma-CEHC. Higher levels of heme, part of the “Hemoglobin and Porphyrin metabolism”, and lower levels of gamma-CEHC, part of the “Tocopherol metabolism”, were measured in ME/CFS patients compared to controls in our cohort (Figure 1
). Heme is a vital component of many metalloproteins, the most well-known being hemoglobin, and is synthesized in the liver and the bone marrow. As the Metabolon®
sample preparation is methanol-based, protein precipitation is expected even though protein-bound heme could still be released depending on the level of heme coordination. Because we used plasma, which is a cell-free matrix, it is anticipated that there is a greater contribution from “free heme” to the measurement of heme abundance unless substantial hemolysis occurred. High concentration of free heme in plasma is a biomarker for sickle cell disease severity, in which increased levels of inflammatory biomarkers such as lactate dehydrogenase, bilirubin, high reticulocytes count, and lipids are detected [35
]. Three forms of bilirubin as well as biliverdin were assessed in our samples and were also present in higher abundance in patients vs. controls, demonstrating a general disturbance in the heme degradation pathway, with the last step occurring in the liver. All five compounds have strong deleterious effects that are linked to free radical generation and their degradation is claimed to be part of a cytoprotective feedback in response to oxidative stress [46
]. Antagonistically, gamma-CEHC, along with gamma-CEHC glucuronide and alpha-CEHC glucuronide, are metabolites of the vitamin E pathway, which has anti-inflammatory features, such as acting as a lipophilic antioxidant [47
]. Our previous work also suggested a disruption in vitamin E metabolism as a result of detection of 13’-carboxy-alpha-tocopherol [21
], which is unfortunately not present in this dataset.
Many ME/CFS patients self-report specialized diets as well as supplements as management tools to mitigate their symptoms. Such behavior is widespread within the patient communities of many diseases, including ME/CFS, fibromyalgia and cancer to only name a few [48
]. Excluding prescription pharmaceuticals, such nutrition approaches can either be defined by dietary restrictions or associated with specific supplements thought to exhibit beneficial effects against inflammation, cardiovascular problems or aging. The adoption of restrictive diets to ameliorate a potential wheat sensitivity in some patients [51
] or ketogenic diets and fasting, have been reported to be helpful by other patients [48
]. Many patients also consider some supplements beneficial, including NADH, coenzyme Q10 or polyphenols, although systematic review of study outcomes does not lead to clear recommendations to the patient community [52
]. However, a commonality between all the work on nutrition in ME/CFS cited above can be found in redox metabolism. Clearly, our current work as well as other reports suggests that nutritional alterations might be of assistance to patients, though further research is necessary before any recommendations can be made.
An ambitious aim in applying untargeted metabolomics to ME/CFS samples is to probe for a pattern that can further our limited understanding of this disease. The enrichment analysis unit of MetaboAnalyst revealed a potential imbalance in the redox state of patients, as their metabolic profiles matched several conditions unrelated to each other, but which all involved redox enzymatic reactions (Table 3
). Our hypothesis is that a disturbance in the redox status influences the status of chemical reaction donors and acceptors as well as their coenzymes such as NAD+/NADH, FAD+/FADH for dehydrogenases. Oxidases would obviously also be affected as catalyzers of redox reactions.
Many transferase catalytic activities could be influenced by the redox state of their environment and many illnesses are caused by transferase deficiencies. For instance, succinyl CoA: 3-ketoacid CoA transferase deficiency leads to a buildup of ketones and diabetic ketoacidosis [54
], as reported in Figure S1b and S1c
and Table 3
respectively. Carnitine palmitoyl transferase deficiency II (CPT II) is another example in which fatty acid metabolism is disrupted by the lack of transport of long chain fatty acids into the mitochondria, where they are used as a fuel source [55
Anoxia is one of the disease-associated metabolite sets shown in Table 3
, a condition that also appeared using the Armstrong et al. datasets [17
] (data not shown), and along with asphyxia in the Germain et al. dataset [21
] (data not shown). The ME/CFS metabolite profile also has similarities to those of infants who develop hypoxic-ischemic encephalopathy (HIE) due to oxygen deprivation. Anoxia and asphyxia are both linked to the lack of oxygen, which can obviously have severe repercussions on muscle and body activity. Inability to deliver oxygen to muscles adequately is evident in studies of response to exercise in ME/CFS patients [15
]. Furthermore, prefrontal cortex oxygenation of the brain is reduced in exercising ME/CFS patients [58
]. Many individuals with ME/CFS exhibit reduced blood volume, thus affecting oxygenation of many tissues [59
]. Disturbances in circulation and provision of oxygen to tissues could underlie many symptoms of ME/CFS. Hypoxia results in generation of reactive oxygen species by mitochondria, resulting in activation of protective systems [61
An association of oxidative stress and ME/CFS has been reported in a number of prior ME/CFS studies [24
]. Of note, when measuring known oxidative stress markers, Richards et al. [25
] found that methaemoglobin was one of the principal components that differentiated their ME/CFS patients and control cohorts. This hemoglobin carries the oxidized form of the iron ion, namely, the ferric state instead of the ferrous state necessary for the hemoglobin to bind oxygen. Even though methaemoglobin measurements are not part of our dataset, it is intriguing to relate its oxidation state to a disturbed redox environment while the effect of the inability to bind oxygen could translate into anoxia and asphyxia.
ME/CFS biomarkers, as a mean of unambiguous diagnosis and monitoring of efficacy of therapies, are one of the urgently needed developments in this field. Figure 2
as well as Table S2
were generated from univariate ROC analysis with this goal in mind. This method used metabolite to accomplish the highest prediction rates with over 0.75 AUC. Future work in which a larger and independent cohort is analyzed and compared to other fatiguing illnesses will likely increase the prediction confidence and will reveal whether plasma metabolomics may serve as a reliable tool for objective identification and monitoring of ME/CFS patients.