Metabolomics defines the characterization of all metabolite composition within the sample of interest together with quantitative information (176). In the current study, we performed metabolomics analysis using urine samples collected from 43 epilepsy patients and aiming to identify different pathways profiles between epilepsy subgroups.
By employing two different statistical approaches, different pathways were enriched in KEGG pathway database. Firstly, ten unique pathways were retrieved from the IE and Epi+ groups comparison. Secondly, samples of drug-R and drug-S subgroups were examined, and separation was more visible in this classification. Particularly, the pathway analyses revealed eleven unique pathways. Comparing the results of the two groups, there were overlapping pathways: the “ABC transporters”, “Aminoacyl tRNA biosynthesis pathway”, “Metabolic pathways”, “Alanine, aspartate, and glutamate metabolism”, “Lysine degradation”, “Lysine biosynthesis”, “Phenylalanine metabolism” and “Glycine, serine, and threonine metabolism” pathways.
The ABC transporter superfamily comprises different types of proteins and regulates the balance of substances within cells via ATP hydrolysis-coupled efflux activity.
Therefore, it plays a role in several key functions involving drug metabolism, like drug efflux activity at the blood-brain barrier (177). Elevated protein levels and increased efflux activity of these proteins have been linked to the drug resistance mechanism in epilepsy (178). Therefore, it is of pivotal importance to obtain this pathway when comparing drug-S versus drug-R subgroups of patients and the drug resistance could just arise from the efflux activity of the ABC transporters. Besides,
there is some evidence showing a possible relationship between a gut microbiota member, the Fusobacterium nucleatum, and the development of drug resistance in colorectal cancer cells through gain of cancer stem cell properties and increased ABC transport activity (179). To this end, it would be informative to examine the gut microbiome profile of these patients about their metabolite levels to be able to capture the holistic view of the resistance mechanism. Moreover, one recent study (180) has indicated a gut dysbiosis in paediatric intractable epilepsy and, when compared to the control group, the ABC transporter pathway was one of the most powerful indicators in classification. Similarly, in our study, urine metabolite changes between IE and Epi+ group pointed out ABC transporters and may be important in the subclassification of epilepsy and regulation of different mechanisms underlying this difference.
Aminoacyl-tRNA biosynthesis is another significant pathway determined to be important in both IE and Epi+, and drug-R and drug-S group comparisons.
Aminoacyl-tRNA synthetases are crucial players in protein translation procedure via regulating conjugation of charged amino acids to their cognate tRNAs (181).
Previous reports have presented mutations in several aminoacyl-tRNA synthetase genes in epileptic patients (182, 183). Nevertheless, further investigations are necessary whether the findings in this study are reflecting differences in potential mutations and consequent perturbations in aminoacyl-tRNA biosynthesis pathways between subgroups of epilepsy patients.
Urine metabolite signatures between IE and Epi+ and between drugR and drugS subgroup revealed different metabolic pathways of broad range of amino acids.
Amino acids, as building blocks of proteins, are pivotal in cell physiology functions, and most inborn errors are seen in amino acid metabolism. Seizures are one of the most common neurological symptoms in children having amino acid metabolism deficiencies, as they are key components of neurotransmitters (184). Within the last two decades, the ketogenic diet has been proposed in the treatment of epilepsy, being directly effective on brain amino acid metabolism (185, 186). In a study on seizure-induced mice models, a mechanism of the protective impact of the ketogenic diet on seizures through the gut microbiota has been elucidated. Accordingly, a synergetic action between two gut bacteria, namely A. muciniphila and Parabacteroides spp., in response to ketogenic diet-induced changes in gamma-glutamylation of amino acids resulted in seizure protection through elevated GABA/glutamate levels in the brain (173). Hence, it would be important to further investigate the gut microbiota composition of the IE and Epi+ groups through metagenomics profiling, in future.
Another pathway specifically enriched in the IE/Epi+ subgroup is pyrimidine metabolism. Pyrimidines, cytosine, thymine, and uracil are important players in most biological functions, being part of DNA and RNA together with adenine and guanine.
They are obtained via two different ways, de novo synthesis or re-use from degradation pathways (187). Up to date, several studies have revealed mutations in enzymes associated with pyrimidine metabolism behind the development of different CNS pathologies, including DEE (188). As one example, mutations in CAD,
encoding CAD protein with enzyme activities, glutamine amidotransferase, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase, involved in pyrimidine de novo synthesis has been reported in children with EE (189, 190). Moreover, in a study carried on 450 children, an investigation of the products of pyrimidine metabolism has been suggested as a possible marker for the diagnosis of children having epileptic seizures and additional neurological symptoms (191).
Urinary biomarkers analysis in children with epilepsy revealed increased vitamin B6 metabolism and the resultant increase in 4-pyridoxic acid concentrations due to the catabolism of B6. Moreover, increased 4-pyridoxic acid/kynurenine ratio can be used as a measurement of the number of seizures experienced by patients (192). In our analysis, urinary 4-pyridoxic acid levels were significantly increased (p<0.05) in Epi+ patients, and together with the finding of vitamin B6 metabolism in pathway analysis this may indicate elevated B6 metabolism selectively in Epi+ patients.
Accordingly, the Epi+ subgroup did include patients with early-onset seizures classically featuring ID or ASD and, generally, their seizures are less well controlled by ASMs.
Nicotinamide adenine dinucleotide (NAD+) is involved in many important cellular functions as a co-enzyme in oxidation-reduction reactions or as an ADP-ribose group donator, and tryptophan, nicotinate, and nicotinamide can be metabolized to produce NAD+ (193, 194). Beneficial role of NAD+ in neurons has been described, and in one study, administration of NAD+ to pilocarpine-induced status epilepticus (SE) model mice has been shown to decrease epileptogenesis (195).
Butyrate (butanoate) is one of the SCFAs, produced by the members of gut microbiota through digestion of dietary fibres, and strongly associated with beneficial effects on several biological processes, including roles such as long-term memory formation, microglia and astrocyte regulation through inhibition of histone deacetylases, and neuronal plasticity (196, 197). Hence, it may be an important clue to obtain butyrate metabolism in epilepsy subgroup classifications to highlight potential impact of gut microbiome in epilepsy.
Metabolomics analysis carried on brain samples obtained from deceased patients diagnosed with epilepsy has revealed disturbed pathways, and butyrate metabolism has been listed at the top among others (198).
The lysine degradation pathway was obtained only in drug-R/drug-S group comparison. L-lysine is an important amino acid in humans, and catabolism is mainly driven by two different routes, pipecolic acid or saccharopine pathway (199). An in-vitro study has found that lysine degradation is predominant following the saccharopine pathway in cultured human brain cells (200). A common end-product of the pipecolic acid and saccharopine pathways is the α-aminoadipic-d-semialdehyde (AASA) that is then converted to α-aminoadipic acid (AAA) through an enzyme called antiquitin. Perturbations in AASA to AAA conversion due to mutations in the antiquitin-coding gene yield elevated levels of both AASA and piperideine-6-carboxylate (199). This accumulation results in the inactivation of a co-enzyme in the vitamin B6-metabolism pathway and has been associated with pyridoxine-dependent seizures (PDS) (200). It should also be noted that, for IE/Epi+ comparison, the FDR
score of this pathway was very close to the threshold (0.255) from Mann Whitney U test analysis. Together with the finding of vitamin-B6 metabolism pathway in IE/Epi+ comparison, it would be interesting to focus on these pathways to check whether any dysregulations in the lysine pathway and its potential impact on vitamin-B6 metabolism are behind the development of different clinical outcomes.
Finally, caffeine metabolism also emerged when comparing IE vs Epi+ groups.
Caffeine has an impact on neural activities via interactions with several neurotransmitters, and thereby, enhances motor activities. Animal studies have shown controversial effects of caffeine, which might be arisen from dosage, exposure, duration or even the age of the subject. However, because of the different metabolisms between mice and humans, such effects have not yet been reported in humans (201).
Recently, the role of inflammation in epilepsy and other neuropsychiatric disorders has gained light (202, 203). Neuromodulated stimuli, pro-inflammatory cytokines, SCFAs, amino acids, endocrine neurotransmitters, reactive oxygen species (ROS), and many other pathogenetic metabolites produced in the GI tract increase the leakiness of the Intestine Mucous and the BBB allowing to grant access to the CNS compartment, and thus triggering a neuroinflammatory chronic state. Nowadays, it is well known that patients affected by chronic inflammatory diseases such as immunological, rheumatic, or inflammatory bowel diseases (IBD) have strong dysbiosis with an increased risk of developing neurological comorbidities (204, 205).
In conclusion, our study supports the relevance of investigating the MGB axis through a clinical and metabolomic fingerprint. Targeting the MGB axis could be an additional approach not only for the treatment of CNS-related disorders but also for all those affections in which dysbiosis is involved.