In this study, metabolomic analyses of CFS patients and healthy controls revealed that the concentrations of organic acids related to the TCA cycle and energy metabolism, such as citrate, isocitrate and malate, were significantly lower in CFS patients than in healthy controls, although the concentration of glucose did not differ significantly between the two groups. As for the urea cycle, the concentration of ornithine in CFS patients was significantly higher than in healthy controls, while that of citrulline was significantly lower in CFS patients than in healthy controls. The metabolites in glutamine metabolism and the BCAAs did not show significant differences between the CFS patients and the healthy controls. The decreased concentrations of organic acids related to the TCA cycle and energy metabolism in the CFS patients suggested that they have deficiencies in adenosine triphosphate (ATP) production secondary to dysregulation of the flow from pyruvate to citrate via acetyl CoA and abnormalities in the conversion of citrate to isocitrate by aconitase. This profile was thought to reflect the pathophysiology of energy metabolism in CFS. Since more than 90% of cellular energy is produced from the electron transport chain, which is a process of ATP generation using O 2 and electron donors (such as NADH 2 +) in mitochondria22, the decreases in the concentrations of organic acids involved in the TCA cycle suggest a deficiency of ATP production in CFS patients23,24,25. Aconitase activity is closely associated with ATP production; the inhibition of aconitase reduces the cellular energy supply26,27 and induces cell death28. Aconitase includes a 4Fe–4S cluster and is vulnerable to oxidative stress29,30,31,32. In addition to the previous study33, the findings that CFS patients who were recruited from the same hospital as the present study showed higher oxidative stress relative to healthy controls34 supports the hypothesis that the predominant reduction of isocitrate may result from the inactivation of aconitase via chronic oxidative stress. Indeed, muscle pain, which is one of the major symptoms of CFS, was also reported to be induced by oxidative stress29. The decreased concentrations of metabolites in the first steps of the TCA cycle in CFS patients likely reflect the pathophysiology of fatigue, which shows the functional dysregulation of flow from pyruvate to isocitrate. Thus, the plasma pyruvate to isocitrate ratio might be an appropriate marker of this type of dysregulation.

As for the BCAAs, they did not show any significant difference in CFS relative to healthy controls. We previously reported that a fatigued animal model showed increases in BCAAs including valine, leucine and isoleucine, which could be induced by the proteolysis of skeletal muscle upon deprivation of physical rest32. Considering this, CFS might not involve sustained muscle stress.

A previous study using NMR metabolomics for CFS reported an increase of blood glucose and lactate as well as a decrease in urine pyruvate and alanine in CFS patients, suggesting a possible inhibition of glycolysis by the reduced provision of adequate amounts of acetyl-CoA required for the citric acid cycle21. There were some differences between these studies and ours, probably due to the different analytical methods used, i.e. NMR and CE-MS detect different types of metabolites based on their chemical properties35. The severity of disease in subjects with CFS is another factor that might cause different alterations of metabolites. Despite these discrepancies, the previous study indicated that dysfunctional energy metabolism through the citric acid cycle is a fatigue phenotype21. This hypothesis is in accord with our findings showing the functional dysregulation of flow in the TCA cycle might reflect the pathophysiology of fatigue.

Our findings can be attributed to a distinctive metabolic pathway in the fatigued condition: the metabolic flow from ornithine and glutamine to succinate of the TCA cycle via gamma-aminobutyric acid (GABA)36,37. Increased ornithine in the urea cycle in the fatigued condition may cause upregulation of the flow of glutamate into the TCA cycle via GABA and the succinate pathway. In the present study, the concentrations of citrate and isocitrate in the first steps of the TCA cycle related to the energy metabolism in CFS patients were reduced relative to those in healthy controls. Meanwhile, the concentration of succinate, which follows these metabolites in the TCA cycle, did not differ significantly between CFS patients and healthy controls. This suggested that activation of the fatigue metabolic pathway contributes to the supply of energy that has been reduced under the fatigued condition.

A previous study reported that the ornithine/citrulline ratio is a marker that can be used to evaluate the activity of the urea cycle38. An increase in the ornithine/citrulline ratio indicates a relative slowdown at the entry point of the urea cycle where ornithine is combined with carbamyl phosphate by ornithine transcarbamylase to make citrulline, which is the first intermediate in this cycle after the rate-limiting enzyme carbamyl phosphate synthetase I39. Our results also showed that the ornithine/citrulline ratio was significantly higher in CFS patients than in healthy controls. This might reflect the enzymatic dysfunction of either carbamyl phosphate synthetase I, which makes carbamyl phosphate from ammonia and HCO 3 , or ornithine transcarbamylase38 in CFS. Ornithine and citrulline, which is produced from ornithine and carbamyl phosphate by ornithine transcarbamylase, are involved in the major function of detoxification in the liver40. A previous study reported that citrulline is also produced from arginine by the NO production pathway and is catalysed by nitric oxide synthase, which is upregulated by AMP-activated protein kinase in response to ADP/ATP imbalance41. The metabolic balance of citrulline and arginine is reported to influence intracellular and extracellular lipid peroxidation levels42,43. In fatigued animals, a previous study showed that plasma NO X levels were increased, and plasma oxidative levels were also elevated19. Another study reported that thiobarbituric acid-reactive lipoperoxide was increased in the liver tissue of fatigued animals44. Taking these findings together, the higher oxidative stress levels in the plasma and serum in CFS33,34 may cause metabolic dysregulation, such as the inactivation of aconitase, which likely results in the reduction of isocitrate in the TCA cycle and the inhibition of metabolic flow from ornithine to citrulline in the urea cycle.

With AUC values of 0.801 (95% CI: 0.711–0.890, P < 0.0001) and 0.750 (95% CI: 0.584–0.916, P = 0.0069) for the training and validation datasets, respectively, the two ratios of pyruvate/isocitrate and ornithine/citrulline levels in plasma could be used to distinguish between CFS patients and healthy controls. Both CV and resampling validations showed narrow 95% CIs, indicating the low dependence of the predictive performance on the given data and confirming the model’s versatility. AUC values of a single index (i.e. pyruvate/isocitrate or ornithine/citrulline) were 0.709 (95% CI: 0.601–0.816, P = 0.00053) and 0.758 (95% CI: 0.660–0.856, P < 0.0001) for training data, respectively, and 0.705 (95% CI: 0.526–0.884, P = 0.027) and 0.695 (95% CI: 0.521–0.870, P = 0.035) for validation data, respectively, which were less than the AUC values using the two indexes combined. Therefore, we suggest that these two ratios represent objective index markers that can facilitate rapid screening for CFS.

In the present study, metabolomic analysis revealed decreased activity in the TCA cycle and the urea cycle in CFS patients. Application of the two ratios, pyruvate/isocitrate and ornithine/citrulline, which might reflect such dysregulation mainly in these two cycles in CFS patients, specifically discriminated between CFS patients and healthy controls. Because our findings may reflect the pathophysiological state of CFS, they might contribute not only to the objective diagnosis but also to the treatment of CFS patients by indicating appropriate nutrients to be ingested in food or supplements.

There were several limitations in this study. Additional studies with a larger patient population should be performed to confirm the metabolic dysregulation in the TCA cycle and the urea cycle, which are thought to reflect the pathophysiological state in CFS. The developed discrimination model should be further validated using a larger population. In addition, longitudinal measurements with detailed clinical investigations are needed to prove its generalizability. To discriminate CFS patients from healthy controls, it is possible that other combinations of quantified metabolites can also be used. However, the use of the ratio of two metabolites as an index, rather than an individual metabolite concentration, would eliminate the inconsistency in overall concentration derived from diurnal variation. In this study, we only utilized the data identified by our standard library whereas TOF-MS provides non-targeted data. The peaks without assignment, but which showed potential discriminating ability, should also be analysed in future studies to identify other markers and provide a more accurate discrimination. The number of peaks showing S/N≥10 was different between the training and validation data (Supplementary Fig. S1). Although the metabolites were quantified by eliminating the bias of MS sensitivity, other factors, such as sprayer setting and splay condition, might also affect the sensitivity of the observed data. More rigorous quality control should be implemented in future studies.

Time-course analyses of metabolomic profile in individual patients for short and long periods might help to realize the diagnosis with higher accuracy.

In summary, our study has demonstrated a profile of abnormal energy metabolism resulting from deficiencies in aconitase activity in the TCA cycle and dysregulation in the urea cycle in CFS patients. Two ratios, pyruvate/isocitrate and ornithine/citrulline, the changes of which may reflect inactivity of the two above-mentioned cycles in CFS, could be useful index markers to discriminate CFS patients from healthy controls. Although a further large-scale investigation is needed, the metabolite index markers identified in this study provide compelling evidence that a clinical diagnostic tool could be developed for CFS based on the ratios of small molecules in plasma.