Metabolic fingerprinting of dogs with idiopathic epilepsy receiving a ketogenic medium-chain triglyceride (MCT) oil

Abstract

Consumption of medium-chain triglycerides (MCT) has been shown to improve seizure control, reduce behavioural comorbidities and improve cognitive function in epileptic dogs. However, the exact metabolic pathways affected by dietary MCT remain poorly understood. In this study, we aimed to identify changes in the metabolome and neurotransmitters levels relevant to epilepsy and behavioural comorbidities associated with the consuming of an MCT supplement (MCT-DS) in dogs with idiopathic epilepsy (IE). Metabolic alterations induced by a commercial MCT-DS in a population of 28 dogs with IE were evaluated in a 6-month multi-centre, prospective, randomised, double-blinded, controlled cross-over trial design. A metabolic energy requirement-based amount of 9% MCT or control oil was supplemented to the dogs' stable base diet for 3 months, followed by the alternative oil for another 3 months. A validated, quantitative nuclear magnetic resonance (NMR) spectroscopy platform was applied to pre- and postprandially collected serum samples to compare the metabolic profile between both DS and baseline. Furthermore, alterations in urinary neurotransmitter levels were explored. Five dogs (30%) had an overall reduction in seizure frequency of ≥50%, and were classified as MCT-responders, while 23 dogs showed a ≤50% reduction, and were defined as MCT non-responders. Amino-acid metabolism was significantly influenced by MCT consumption compared to the control oil. While the serum concentrations of total fatty acids appeared similar during both supplements, the relative concentrations of individual fatty acids differed. During MCT supplementation, the concentrations of polyunsaturated fatty acids and arachidonic acid were significantly higher than under the control oil. β-Hydroxybutyric acid levels were significantly higher under MCT supplementation. In total, four out of nine neurotransmitters were significantly altered: a significantly increased γ-aminobutyric acid (GABA) concentration was detected during the MCT-phase accompanied by a significant shift of the GABA-glutamate balance. MCT-Responders had significantly lowered urinary concentrations of histamine, glutamate, and serotonin under MCT consumption. In conclusion, these novel data highlight metabolic changes in lipid, amino-acid and ketone metabolism due to MCT supplementation. Understanding the metabolic response to MCT provides new avenues to develop better nutritional management with improved anti-seizure and neuroprotective effects for dogs with epilepsy, and other behavioural disorders.

Keywords: biomarker; canine; epilepsy; medium-chain triglyceride; metabolome; neurotransmitter.

Figure 1

CONSORT flow diagram—Flow diagram of the progress through the phases of this randomized clinical trial of two groups (that is, as shown, enrolment, intervention allocation, follow-up, and data analysis).

Figure 2

Urinary neurotransmitter profile: Effect of the medium-chain TAG dietary supplement (MCT-DS, light pointed) on the γ-Aminobutyric acid (GABA)—glutamate balance in urine compared with the control dietary supplement (Control-DS, dark-grey). Significant (A) increased γ-aminobutyric acid (GABA) concentrations (P = 0.0044) and a shift in the GABA/glutamate ratio (B) during the MCT phase was found (P = 0.0025). A predominant shift to the GABAeric side in the GABA/glutamate ratio (C) was observed under MCT-DS consumption (light grey) compared to the Control DS (dark-grey). Data are shown as box-and-whisker plots (central lines of the box represent the median, lower and upper limits of the box represent the 25th and 75th percentiles and whiskers represent the minimum and maximum). Two-sided Wilcoxon's matched-pairs rank tests were used to compare control and MCT-DS groups. *P < 0.05.

Figure 3

Serum metabolite profile: Effects of the medium-chain TAG dietary supplement (MCT-DS, light-grey) on serum metabolites compared with the control dietary supplement (Control-DS, dark-grey) in (A) pre-prandial (light-orange) and post-prandial (dark-orange) state (N = 28). If significant between both dietary supplements (A), altered parameters are labelled according to their change related to baseline (red = decreased, blue = stable, green = increased, x = not significant). MCT-R (B) showed significant abundances in four different metabolites. MCT-R (C) show a shift in their fatty acid profiles to increased serum levels of arachidonic acid (P = 0.0283) and Ω 6 fatty acids (P = 0.0171), but decreased levels of triglycerides (P = 0.0034) and pyruvate (P = 0.0386). Data are shown as box-and-whisker plots (central lines of the box represent the median, lower and upper limits of the box represent the 25th and 75th percentiles and whiskers represent the minimum and maximum). Two-sided Wilcoxon's matched-pairs rank tests were used to compare control and MCT-DS groups. *P < 0.05.

Figure 4

BHB-TAG Ratio: The BHB-TAG ratio was evaluated on its clinical relevance as a monitoring tool. (A) When MCT was consumed, a significant association between BHB-TAG ratio and seizure frequency per month (N = 28, p = 0.0150) was found. (B) The relative change in seizure frequency correlated negatively with BHB-TAG ratio (N = 28, r: −0.455, p = 0.015) under MCT intake. The lower the BHB-TAG ratio was, the higher the seizure frequency per month, or lower the change in seizure frequency under MCT. The higher the reduction in seizures (–%), the higher the BHB-TAG ratio. (C) Independent of diet or dietary supplementation, a significant negative correlation was found between the occurring seizure frequency per month and the BHB-TAG ratio (N = 84; r: −0.387; p = 0.0016).

Figure 5

The hypothetical metabolic response to MCT oil supplementation: The dietary supplementation of MCT oil induces significant metabolic changes. Substantial changes in the lipid, energy and neurotransmitter metabolism have been detected in dogs with idiopathic epilepsy after 3 months under MCT DS as a nutritional, therapeutic approach. In brief, after oral intake, [1] MCT can be degraded in the intestinal lumen by lipases into MCFAs. MCFAs can be directly absorbed, transported into the liver and metabolized into C4 ketone bodies, such as beta-hydroxybutyrate (BHB). BHB levels [2] were significantly increased in all dogs during MCT consumption compared to the control oil phase. Based on these findings, MCT might lead to a significantly increased anaplerotic influx into the TCA cycle [3] and thereby evokes an additional production of adenosine triphosphatase (ATP) via the mitochondrial respiratory chain resulting in a compensation of epilepsy associated energy deficits [4]. Moreover, Pyruvate was found being especially decreased in MCT responders [3]. The compensation of energy deficits may thus also be given via (a) an increase in the enzymatic activity of the pyruvate dehydrogenase (PDH) activity, or/and (b) the promoted entry of oxaloacetate into the TCA by increased pyruvate carboxylase activity (PC). PDH and PC may be here both the key metabolic enzymes relevant for overcoming energy shortage under MCT consumption via the use of pyruvate. Furthermore, an additional de-novo lipogenesis from MCT into other MCFA via ß-oxidation leading to anaplerotic and neuroprotective effects has been hypothesized in the past [5]. Some prostaglandins deriving from arachidonic acid (AA) play significant roles in the processes of neurodegeneration and neuroinflammation, but also neuroprotection and regeneration. Prostaglandine can induce both depending on its concentration ratios of different types of prostaglandin [6]. The energy and neurotransmitter metabolism in neurons and astrocytes is tightly coupled [7]. Astrocytes take up GABA and glutamate from the synapse, and in turn, provide neurons with glutamine, an essential substrate for the re-synthesis of glutamate and GABA in the neurons. Based on our findings and previous research findings, we speculated that astrocytes are the main cellular compartment of MCFA metabolism and result in promoted astrocyte glutamine synthesis. The promoted glutamine supply from astrocyte metabolism of C8 and C10 MCFAs may lead then to elevated neuronal GABA synthesis [8], and thus aid in maintaining the inhibitory tone of the brain and provide another anticonvulsant mechanism of MCT oil supplementation. In summary, anticonvulsive effects may thus be provoked by I. compensating the energy shortage [2–5], II. influence on GABA/ glutamate balance via astrocyte metabolism [7–8], III. Reduction of antioxidative stress [9] via ATP increase and neuroprotective effects on the brain by MCT influencing metabolic pathways [10].