Here we report targeted and untargeted analyses of 562 molecules representing primary metabolites, biogenic amines, lipid complexes and oxylipins in plasma of ME/CFS patients and controls. We also describe linkage of the resulting metabolomic data to a fecal metagenomic dataset and clinical data. As in our previously reported metagenomic analyses, we found that
metabolomic profiles differ not only between ME/CFS patients and controls but also between ME/CFS patients who do or do not have IBS.
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Subjects included
50 ME/CFS cases who met the criteria15,16 for ME/CFS and 50 matched healthy controls recruited at four sites across the United States (New York, NY; Salt Lake City, UT; Incline Village, NV; and Miami, FL). Subject demographics are shown in Table
1. The same subjects were enrolled as in our previous study
14. Cases included 41 female and 9 male ME/CFS patients (mean age 51.1 years; standard error of the mean [SEM] 1.6). Controls included 41 female and 9 male subjects (mean age 51.3 years; SEM 1.6). All case and control samples were collected between June 22, 2014 and October 27, 2014. Irritable bowel syndrome (IBS) was diagnosed in 24 of the 50 ME/CFS patients (48%). One of the 50 control subjects reported a diagnosis of IBS.
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Among the top plasma biomarkers differentiating ME/CFS patients from controls were decreased levels of
betaine, complex lipids (lysophosphatidylcholine [LPC], phosphatidylcholine [PC]) and sphingomyelin (SM), and increased levels of triglycerides (TG), α-N-phenylacetyl-glutamine, ε-caprolactam and urobilin (Table
S2). Set enrichment analysis of the results of logistic regression models revealed that ME/CFS subjects had reduced levels of PCs and dysregulation of the choline-carnitine pathway.
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In ME/CFS with IBS,
5-methylthioadenosine (a metabolite derived from S-adenosylmethionine as a by-product of polyamine biosynthesis that can be toxic to mammalian cells21) was associated with functional impairment.
Ceramides were associated with increased physical fatigue (Table
S4). Decreased γ-butyrobetaine correlated with increased
Faecalibacterium, a bacterium known to play an important role in the production of colonic butyrate, a by-product of fermentation with known beneficial impacts on intestinal barrier function and anti-inflammatory effects
22,
23.
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The Naviaux group previously reported decreased plasma levels of SM, ceramides and PLs with the exception of PC 18:1 and PC 22:6, which were increased
8. Tomic
et al. reported increased plasma levels of TGs
26. Germain
et al. reported metabolomic biomarkers of disturbed amino acid, energy, sugar and fatty acid metabolism in ME/CFS patients
13. Neither group examined the impact of IBS on metabolomic profiles in ME/CFS.
Our analysis confirmed decreased levels of phospholipids and SM, and increased levels of TGs, but differed with respect to specific compounds. We did not find case-control differences in levels of PC 18:1 and PC 22:6. Nor did we find a consistent decrease in ceramide levels. Whereas ME/CFS without IBS had decreased levels of ceramides d43:1 and d42:1, ME/CFS with IBS had increased levels of six ceramide species: d36:1, d40:0, d42:0, d 34:1, d38:1 and d40:1.
Ceramide is a waxy lipid implicated in suppression of electron transport, insulin and leptin resistance and apoptosis
27,
28. Previous studies reported that ceramides might be involved in the pathology of IBS and metabolic disorders
29,
30. Increased levels of lipopolysaccharides (LPS) associated with an altered gut microbiome
31 may trigger the activation of sphingomyelinases (SMAse) and the hydrolysis of SM to produce ceramides. Ceramides are toxic to many cell subtypes via the production of reactive oxygen species and may play a role in gut barrier dysfunction and increased gut permeability. Increased levels of ceramides were reported in mucosal samples from IBS patients
29 as well as in plasma and tissue samples in diabetes, cardiomyopathy, insulin resistance, atherosclerosis and steatohepatitis. Blocking SMAse to decrease ceramide levels may be therapeutic in reducing inflammation
32. Patients with ME/CFS and IBS also had higher plasma mannitol levels.
We speculate that mannitol may increase permeability of both the gut mucosa and the blood-brain barrier resulting in trafficking of molecules such as cytokines and neurotransmitters that contribute to disease.
Similar to Armstrong and colleagues11, we found alterations in metabolites associated with mitochondrial energy metabolism. Previous study reported significant decreases in TCA cycle metabolites related to energy metabolism in ME/CFS patients
9. The reported dysfunction impacted carnitine metabolism and ATP/energy metabolism in the muscle of ME/CFS patients
11. Carnitine is an important supplement that transfers acyl-CoA group into the mitochondrial matrix and participates in fatty acid β-oxidation (TCA cycle, ATP production and energy metabolism). Studies of carnitine levels in serum and plasma in ME/CFS have been inconclusive, with some groups reporting reductions whereas others find normal levels
33,
34. In an open label study of 30 patients, acetyl-carnitine supplements were reported to improve fatigue and cognitive function in up to 59% of patients with ME/CFS
35. In our study (Table
2), compounds in the choline-carnitine pathway were decreased in ME/CFS patients regardless of their IBS status.
Our results are consistent with earlier reports that suggest that metabolites linked to lipid and energy metabolism are affected in ME/CFS. They extend earlier work by demonstrating that ME/CFS subjects with IBS co-morbidity have a distinct metabolomic profile compared to subjects without IBS and controls.
Many ME/CFS patients in our cohort take vitamin B supplements (26/50, 52%) that have the potential to increase levels of pantothenic acid.
Use of vitamin B supplements was associated with higher levels of pantothenic acid and lower fatigue scores (data not shown). However, the numbers of samples were not sufficient to test for a significant relationship.