Abstracts

Rationale: Elucidation of metabolic pathways altered by ketogenic diets (KDs) can offer valuable insights into mechanisms that underlie their neuroprotective and/or anticonvulsant effects. The goal of this study was to identify key metabolites and pathways altered in hippocampus and plasma of rats fed a KD using an untargeted mass spectrometry-based metabolomics approach.Methods: Adolescent male Sprague Dawley rats (P28) were fed a KD or control diet (CD) either ad libitum (AL) or isocalorically, calorie-restricted (CR) to 90% of the recommended daily requirement for a duration of 3 weeks (N = 6 per group). Tissue and plasma samples were extracted using the Bligh-Dyer extraction method. Extracts were analyzed by High Performance Liquid Chromatography-Mass Spectrometry (Agilent Technologies 6550 Q-TOF LC/MS and 6210 TOF LC/MS). Statistical analysis (p<0.05) was performed using Agilent Technologies Mass Profiler Professional. Metabolites were tentatively identified using exact mass and isotope ratios.Results: In the hippocampus multiple acylcarnitines were changed (p<0.05) in the KD vs. CD as well as KDAL vs. CDAL groups. For example, hydroxyglutarylcarnitine was downregulated 2.3-fold while hydroxybutyrylcarnitine was upregulated 5.7-fold in KD vs. CD animals, respectively. Besides their role in fatty acid transport carnitines and acylcarnitines are suggested to be involved in neuroprotection such as improvement of mitochondrial function and enhancement of antioxidant activity. Compounds related to the common metabolic redox pairs GSH/GSSG and glutathione (ox/red), which have been shown to play an important role in chemoconvulsant models, were significantly altered in plasma of KDAL vs. CDAL animals. Downregulation of homocysteine sulfinic acid (2.0-fold change), S-formyglutathione (1.4-foled change), and S-(3-oxo-3-carboxy-n-propyl)cysteine (1.7-fold change) occurred while homocysteic acid (2.4-fold change) was upregulated in the plasma of the KDAL vs. CDAL group. Changes in metabolites of or linked to the kynurenine pathway were observed such as in 3-hydroxykynurenine (downregulated 2.4-fold in KDAL vs. CDAL), vanilpyruvic acid (upregulated 16-fold in KDAL vs. CDAL), picolinic acid (downregulated 7.5-fold in CDAL vs. CDCR), and nicotinic acid (upregulated 2.0-fold in KDAL vs. CDAL). Kynurenic acid influences neurotransmitter concentrations and has been shown to be involved in epilepsy. Other metabolites with potential relation to epilepsy and seizure frequency/threshold were changed in KD vs. CD and AL vs. CR groups such as phospholipids as well as compounds linked to inflammation.Conclusions: A metabolomics approach can uncover anti-epileptic and potentially neuroprotective mechanisms of the KD and/or calorically restricted food intake. Our data demonstrates that we can assess changes in metabolites in rat hippocampus and plasma relevant to the identification of target pathways of the KD.