Alteration of metabolites in plasma of EAE mice are linked to various metabolic pathways
In search of circulating metabolites as biomarkers of EAE disease, we profiled the global metabolome using liquid and gas chromatography coupled with mass spectrometry to identify the relative levels of metabolites in plasma of EAE diseased mice versus healthy control mice. Evaluation of untargeted metabolomic profiling of plasma from EAE and healthy SJL mice detected 282 known metabolites (Supplementary Table 1
). One control sample was found to be an outlier dominating the first principal component in a PCA (Supplementary Figure 1
) and was excluded from further analysis. PLS-DA revealed a clear separation between EAE and healthy groups, indicating presence of unique metabolite profiles for the EAE and healthy mice group (). We have depicted the EAE profile (red) relative to that of healthy mice (blue) in a Z score plot (), where each point represents a metabolite intensity measure (rows) normalized by the mean and standard deviation of the healthy samples. Positive points represent up-regulation whereas negative points represent down regulation of the particular metabolite in the EAE mice. Metabolites are arranged by class and then by subclass. Two-sample t-tests per metabolite identified biochemicals with significantly different average intensity between the experimental groups. We found that 44 out of the 282 (15%) metabolites were differentially altered (P<0.05 with FDR<0.23), indicating a robust alteration in the circulating metabolomic profile during disease. Among the perturbed metabolites, 32 were up regulated in EAE plasma whereas 12 were down regulated.
Metabolomics profiling of plasma distinguishes EAE from healthy group
Of these 44 altered compounds, ~47% belonged to the lipid class including lysolipids, bile acids, sterols, medium/long chain fatty acids, fatty acid and fatty acid metabolism, and inositol metabolism. The rest of the compounds represent amino acids, xenobiotics, cofactor and vitamins, and carbohydrates with one nucleotide and one energy class molecule (, and ). To visualize the relationship between the 44 altered metabolites, a heatmap was generated with the metabolites arranged on the basis of relative change (up/down) and then by super pathway and samples ordered by hierarchical clustering (). Though the metabolites in this heatmap were selected based on average intensity differences, it is reassuring to see that these metabolites show enough distinct changes to separate the two diagnostic groups.
Up-regulated metabolites in plasma of EAE compared to healthy group .
Down-regulated metabolites in plasma of EAE compared to healthy group.
The most striking alteration was observed in a system-wide increase in plasma free fatty acid (FFA) levels, predominantly in the medium-chain and long-chain fatty acids (LCFAs) in the plasma of EAE group (). The increase in FFAs could be due to higher lipolysis or break down of membrane lipids. Lipolysis from adipose tissue and liver could also potentially be a significant source for the observed increase in plasma FFA levels. Our interpretation of higher lipolysis due to adipose tissue is supported by a recent metabolic flexibility study in MS patients, where higher lipolytic activity in adipose tissue was observed in MS patients compared to healthy [35
]. Although no significant change was found in choline levels in plasma of the EAE group compared to healthy, a decreasing trend was observed. Presently, we do not have an explanation for this observation as choline serves several cellular functions including being the major head group for membrane lipid (phosphatidylcholine) especially the myelin sheath. The levels of many detected lysolipids increased with EAE disease, suggesting a possible increase in membrane breakdown and offering one possible scenario for the observed increase in the plasma FFA. The role of phospholipases in MS and EAE is well documented and they have been shown to be elevated during disease [36
]. An elevated level of myo-inositol (MI) was found in EAE which is an organic osmolyte and a purported glial marker [38
]. Changes in MI levels have been associated with onset of cognitive decline in neuroinflammatory conditions including MS [39
]. Disease induction was also associated with changes in glycolysis (3-phosphoglycerate), TCA (alpha-ketogluterate) and glycemic control (higher levels of 1,2-AG) and histidine metabolism ( and ). Primarily, EAE disease induction brought a system-wide increase in FFA levels. The increase in plasma FFA levels could result from multiple processes including but not limited to lipolysis, membrane breakdown and dietary uptake. Without supporting information from other tissues, it is difficult to pinpoint the source(s) of this observed increase in plasma FFAs.
Box plots of various metabolite classes altered in EAE
Perturbation of metabolic pathways in EAE compared to healthy group
Acylcarnitines, a marker of incomplete fatty acid β-oxidation, have been reported in metabolic disorders including diabetes, cardiovascular and mitochondrial diseases, were also elevated in EAE plasma [41
]. In animal models of metabolic disorders, its increased levels have been linked to mitochondrial overload under metabolic stress [43
]. Recent studies are implicating mitochondrial dysfunction as one of the causes of axonal dysfunction in MS [45
We found elevated levels of various metabolites categorized under xenobiotics class (), which are normally not synthesized in the body, but can be metabolized by the micro-biome of the distal gut, and their elevated levels can be observed in biological fluids under normal or pathological state. The most dramatically increased xenobiotic metabolites observed in the plasma of EAE were equolsulphate and homostachydrine. While, no biological role has been identified for these two metabolites, a recent study reported that equolsulphate was observed in the plasma of conventional mice but not in the plasma of germ-free mice [47
], suggesting a significant interplay between resident bacterial and mammalian metabolism. Homostachydrine has been reported in citrus genus plants [48
], but with no biological activity defined. Stachydrine was significantly elevated in plasma of EAE, which was recently shown to activate Th17/Th1 by reducing Th2/Treg in RU486 induced mouse model [49
]. This observation agrees with the identified higher levels of stachydrine, increased inflammation and severity in SJL mice with EAE (). Benzoate (benzoic acid) a monocarboxylate, is used as a food preservative and is also used for the treatment of hyperammonemia [50
]. It has been shown to have anti-inflammatory properties, and reduced microglial and astroglial inflammatory responses and EAE disease progression [51
]. Its metabolism occurs exclusively by conjugation with glycine to form hippurate. It is a mammalian-microbial co-metabolite and a normal constituent of the endogenous urinary metabolite profile. Its excretion has been reported in various disease conditions including normal constituent of the endogenous urinary metabolite profile. Its excretion has been reported in various disease conditions including obesity, diabetes, gastrointestinal diseases, impaired renal function and psychological disorders [53
]. We observed significantly higher level of both benzoate and hippurate in the plasma of EAE afflicted mice. Strong evidence for the pivotal role of the gut microbiota in the generation of hippurate [54
] further suggesting the alteration of gut microbiome during disease, which may be responsible for altered levels of xenobiotics in plasma of EAE group. Indoleacrylate (indoleacrylic acid; IAcrA), a metabolite of tryptophan pathway, is highly reactive and conjugates with glycine to form indolylacryloylglycine (IAG), which is excreted in urine. The only significantly decreased xenobiotic found in EAE plasma was glycolate (glycolic acid), which is considered a major precursor of oxalate in hepatocytes.
We observed elevated levels of two metabolites, alpha-tocopherol (vitamin E) and salicylate in plasma (), known to have anti-inflammatory and protective effect against inflammation. Alpha-tocopherol exhibits anti-oxidant and anti-inflammatory properties. Alpha-tocopherol was shown to be significantly increased during clinical attack in EAE [55
], however, its levels were found to be reduced in MS patients [56
], which is dissimilar from our finding. Salicylate is a well known inhibitor of cyclooxygenase and has been shown to attenuate EAE disease progression and inflammation [57
]. Trigoneline, an alkaloid and coffee constituent, was elevated in the plasma of EAE (). It has been reported to have anti-diabetic property [58
]. Higher levels of these compounds found in the plasma of EAE could be a self-protective mechanism up regulated during disease against the inflammatory cascades.
To get a holistic view of the metabolic alterations occurring in the plasma of EAE mice we conducted pathway analysis of the biochemical pathways of the Kyoto Encyclopedia of Genes and Genomics (KEGG, http://genome.jp/kegg
). We considered both concerted changes in metabolite intensity within the pathway (GlobalTest) [31
] and alterations of high impact (i.e. at major junctions in the pathway), and found that a number of pathways were significantly altered (). We selected six pathways based on p values and high impact factor (). Pathways found to be altered based on the Global Test included primary bile acid biosynthesis, taurine and hypotaurine metabolism, tryptophan and histidine metabolism. Linoleic acid and D-arginine and D-orinithine metabolism pathways had altered metabolites with high impact. These pathways are highly integrated as shown in , suggesting that perturbation of certain central metabolites could have impact on multiple metabolic pathways. While some of these metabolite changes could easily be developed as biomarkers of the disease, the key to translating metabolomics into therapeutics would require figuring out the central altered metabolic pathway(s).
Metabolites altered in EAE compared to healthy group map to multiple biosynthetic pathways