Table 2. List of metabolites found to be significantly different between controls and patients according to the Wilcoxon rank-sum test.
Cofactors and Vitamins
Heme HMDB03178 0.002 0.06
Gamma-CEHC HMDB01931 0.005 0.08
Alpha-CEHC glucuronide HMDB62445 0.018 0.13
Gamma-CEHC glucuronide N/A 0.019 0.13
Energy Alpha-ketoglutarate HMDB00208 0.003 0.03
Nucleotide
Inosine 5’-monophosphate (IMP) HMDB00175 0.003 0.11
2’-O-methylcytidine N/A 0.009 0.13
Adenosine 3’-5’-cyclic monophosphate (cAMP) HMDB00058 0.012 0.13
Peptide
Gamma-glutamylthreonine HMDB29159 0.003 0.11
All metabolites with a q < 0.15 are included. N/A stands for Not Assigned. HMDB stands for Human Metabolome Database.
Figure 1. Box plot distribution of logged values for metabolites scored as being statistically different between controls (red) and patients (blue) at p < 0.05 and q < 0.15 by the Wilcoxon test. HMDB identity and test values can be found in
Table 2. Y-axis scale is log10 transformed data.
"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,
20]. 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]. "