Anticonvulsant profile of caprylic acid, a main constituent of the medium-chain triglyceride (MCT) ketogenic diet, in mice
Highlights
► Ketogenic diet (KD) is highly effective in drug-resistant epilepsy. ► Its mechanism of action is unknown. ► Caprylic acid (CA) is the main constituent of the medium-chain triglyceride (MCT) KD. ► Here, CA showed acute anticonvulsant properties alone and in combination with valproate in mice. ► We propose, anticonvulsant properties of CA may add to the overall clinical efficacy of the MCT KD.
Introduction
High-fat, low-carbohydrate, and adequate-protein containing ketogenic diets (KDs), regardless of the source of fatty acids (e.g., long-chain fatty acids in the classic KD designed by Wilder versus medium-chain fatty acids designed by Huttenlocher) (Wilder, 1921; Huttenlocher et al., 1971), are highly effective in acutely suppressing seizures in some patients (Freeman et al., 2007), and may offer disease-modifying benefits in epilepsy and perhaps in other neuropsychiatric conditions (Baranano and Hartman, 2008; Gasior et al., 2006; Kossoff and Rho, 2009). The fact that KDs are effective when pharmacological treatments fail in human epilepsy suggests there must be some important mechanistic difference(s) between these dietary and pharmacological treatment modalities. This opens a rare opportunity to apply the so called “back-translational” approach to research the KD in pre-clinical settings to identify pharmacological mechanisms responsible for KDs’ distinct clinical efficacy, and facilitate development of novel therapeutic approaches for drug-resistant epilepsy (Gasior and Wiegand, in press).
One approach applied to study KD experimentally has been to identify its biochemical consequences and pre-clinically model their potential contributions in the clinical efficacy of the KD (Gasior et al., 2007; Likhodii et al., 2003; Rho et al., 2002). Within this approach and because ketosis is the most immediate and paramount biochemical change when the KD is introduced, extensive studies have been performed to characterize acute anticonvulsant effects of the ketone bodies (i.e., β-hydroxybutyrate, acetoacetate, and acetone) elevated during exposure to the KD. Although the correlation between levels of ketosis and the therapeutic effect of the KD has not been definitively established (Gilbert et al., 2000; Musa-Veloso et al., 2006; van Delft et al., 2010), seizure protection is compromised when ketosis is abruptly decreased (Huttenlocher, 1976). There is growing evidence supporting that ketone bodies, and acetone in particular, have anticonvulsant properties and, to some degree, may contribute to the anticonvulsant efficacy of the KD (Likhodii et al., 2008). Specific pharmacological mechanisms responsible for these effects of ketone bodies are just starting to emerge (Bough and Rho, 2007; Juge et al., 2010). KD-related research also identified several novel therapeutic approaches that interfere with the body’s glucose and ketone metabolism for consideration in treating epilepsy (note that these mechanisms are not targeted by the current antiepileptic drugs, AEDs) (Gasior et al., 2010; Lian et al., 2007; Stafstrom et al., 2009; Willis et al., 2010).
Although the Wilder’s KD based on long-chain fatty acids is preferably used in the clinics today, the Huttenlocher’s medium-chain triglycerides (MCT) KD is different in several aspects that make it appealing to study for a better understanding of KD’s clinical benefit. The main constituent of the MCT KD is the 8-carbon caprylic acid (CA; approximately 55% content); other carboxylic acids include the 10-carbon capric acid, 6-carbon caproic acid, and 12-carbon lauric acid (Fig. 1). There is evidence that CA may have acute anticonvulsant properties in the PTZ seizure test in rodents (Liu and Pollack, 1994; Perlman and Goldstein, 1984), which is consistent with the proposed carrier-mediated transport of CA across the blood–brain barrier and ability to exert direct pharmacological effects (Spector, 1988). In contrast to the long-chain carboxylic acids, CA and other medium-chain carboxylic acids offer the biochemical advantage of fast oxidation without the need for restricting carbohydrate supply (Bach and Babayan, 1982). Thus the purported acute anticonvulsant properties of CA and fast biochemical catabolism would make the constituent(s) of the MCT KD different from those of the classical Wilder’s KD where (or which) have a limited capacity to enter the brain and, thus, exert direct and immediate pharmacological effects (Edmond et al., 1998; Rapoport, 2001). Therefore, unlike the classical Wilder’s KD, the MCT KD may provide an acute anticonvulsant effect through the direct action of its main constituent(s) in addition to the anticonvulsant effects produced by a metabolic conversion of MCT to ketone bodies and/or other physiological/biochemical changes.
The aim of the present study was to evaluate the acute anticonvulsant effects of CA in seizure and behavioral tests typically used in screening for potential AEDs (Giardina and Gasior, 2009; Smith et al., 2007; Wlaź and Löscher, 1998). Since most clinical trials for new AEDs are conducted using the add-on approach and most of the patients exposed to the KD are also taking adjunct AEDs, the pharmacokinetic/pharmacodynamic interactions of CA and VPA were also evaluated. VPA was selected for this proof-of-concept experiment because of its broad efficacy in different epilepsy types in humans and across seizure tests in animals, and experimental flexibility allowing for quantitative assessments of its interactions with treatment modalities of different pharmacological actions.
Section snippets
Animals
Experimentally naïve male Albino Swiss mice weighing 25–30 g were obtained from a commercial breeder (Laboratory Animals Breeding, Słaboszów, Poland). The animals were housed up to 10 per cage under controlled laboratory conditions (ambient temperature, 22–23 °C, relative humidity, 45–55%, 12 h light/dark cycle, light on at 6:00 a.m.). Chow pellets (Murigran, Agropol S.J., Motycz, Poland) and tap water were continuously available. The animals were allowed to acclimatize to the vivarium for at
Effects of CA in the i.v. PTZ, 6-Hz, and MEST seizure tests
CA (10–30 mmol/kg p.o.) dose-dependently and significantly (Fig. 2) increased doses of PTZ necessary to induce myoclonic twitch (F(3,52) = 15.431, p < 0.001) and clonic convulsions (F(3,52) = 24.117, p < 0.001). A post-hoc analysis revealed that the threshold for myoclonic twitch was significantly elevated by CA at 20 and 30 mmol/kg p.o. (by 15% and 37%, respectively), whereas the threshold for clonic convulsions was elevated by CA at 30 mmol/kg p.o. (by 55%). In contrast, CA (10–30 mmol/kg
Discussion
The present study provides evidence that CA may have intrinsic acute anticonvulsant properties in addition to serving as a metabolic substrate for producing ketone bodies during exposure to the MCT KD. In addition, CA at doses ineffective per se enhanced the anticonvulsive potency of VPA in two seizure tests. Finally, the observed effects of CA were produced by its p.o. doses that produced plasma concentrations of CA comparable to those reported in epileptic patients exposed therapeutically to
Disclosure
All the authors of this submission confirm that have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. S. J. Czuczwar has received support from UCB Pharma and Sanofi-Aventis as a speaker. M. Gasior is a full-time employee of Bristol-Myers Squibb, Princeton, NJ, USA at the time of this submission. However, this work was neither supported by Bristol-Myers Squibb nor has any association with M. Gasior’s current
Acknowledgments
All experiments were performed at the Department of Animal Physiology, Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, Lublin, Poland. This work was supported by the Funds for Statutory Activity of Maria Curie-Skłodowska University, Lublin and Medical University of Lublin, Poland. The authors thank Dr. Grzegorz Raszewski (Department of Physiopathology, Institute of Agricultural Medicine, Lublin, Poland) for providing pharmacokinetic data for CA and VPA.
References (69)
- et al.
Development and validation of a sensitive assay of valproic acid in human plasma by high-performance liquid chromatography without prior derivatization
J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci.
(2006) - et al.
Medium-chain triglycerides: an update
Am. J. Clin. Nutr.
(1982) - et al.
Pharmacological characterization of the 6 Hz psychomotor seizure model of partial epilepsy
Epilepsy Res.
(2001) - et al.
Progress report on new antiepileptic drugs: a summary of the tenth Eilat conference (EILAT X)
Epilepsy Res.
(2010) - et al.
KETOGENIC DIET; anticonvulsant mechanisms of a ketogenic diet
- et al.
Acute anticonvulsant activity of structural analogues of valproic acid and changes in brain GABA and aspartate content
Life Sci.
(1983) - et al.
Fructose-1,6-diphosphate inhibits seizure acquisition in fast hippocampal kindling
Neurosci. Lett.
(2010) - et al.
The neuropharmacology of the ketogenic diet
Pediatr. Neurol.
(2007) - et al.
Development and validation of a reverse phase HPLC method for the determination of caprylic acid in formulations of therapeutic immunoglobulins and its application to antivenom production
Biologicals
(2009) - et al.
Metabolic control of vesicular glutamate transport and release
Neuron
(2010)