Therapeutic Ketosis With Ketone Ester Delays Central Nervous System Oxygen Toxicity Seizures in Rats

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epilepsy, acetoacetate, acetone, ketogenic diet, hyperbaric oxygen

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Central nervous system oxygen toxicity (CNS-OT) seizures occur with little or no warning, and no effective mitigation strategy has been identified. Ketogenic diets (KD) elevate blood ketones and have successfully treated drug-resistant epilepsy. We hypothesized that a ketone ester given orally as R,S-1,3-butanediol acetoacetate diester (BD-AcAc2) would delay CNS-OT seizures in rats breathing hyperbaric oxygen (HBO2). Adult male rats (n = 60) were implanted with radiotelemetry units to measure electroencephalogram (EEG). One week postsurgery, rats were administered a single oral dose of BD-AcAc2, 1,3-butanediol (BD), or water 30 min before being placed into a hyperbaric chamber and pressurized to 5 atmospheres absolute (ATA) O2. Latency to seizure (LS) was measured from the time maximum pressure was reached until the onset of increased EEG activity and tonic-clonic contractions. Blood was drawn at room pressure from an arterial catheter in an additional 18 animals that were administered the same compounds, and levels of glucose, pH, Po2, Pco2, β-hydroxybutyrate (BHB), acetoacetate (AcAc), and acetone were analyzed. BD-AcAc2 caused a rapid (30 min) and sustained (>4 h) elevation of BHB (>3 mM) and AcAc (>3 mM), which exceeded values reported with a KD or starvation. BD-AcAc2 increased LS by 574 ± 116% compared with control (water) and was due to the effect of AcAc and acetone but not BHB. BD produced ketosis in rats by elevating BHB (>5 mM), but AcAc and acetone remained low or undetectable. BD did not increase LS. In conclusion, acute oral administration of BD-AcAc2 produced sustained ketosis and significantly delayed CNS-OT seizures by elevating AcAc and acetone.

seizures from hyperbaric oxygen (HBO2), also known as central nervous system oxygen toxicity (CNS-OT), compromise the safety of undersea divers using rebreathers and patients undergoing HBO2 therapy (HBOT) (13). Breathing 100% O2 at barometeric pressure (Pb) > 2.4 atmospheres absolute (ATA) increases the likelihood of seizures in patients, and current applications of HBOT routinely use up to 3 ATA HBO2 (48). The potential for CNS-OT is the primary limiting factor in HBOT. HBO2 provides a unique, reversible, and reproducible stimulus for generalized tonic-clonic seizures in animal models and is thus an effective model for assessing anti-seizure strategies.

Previous studies in rats show that fasting delays the onset of CNS-OT (4), presumably by fundamentally shifting brain energy metabolism. Fasting (24–36 h) delays the latency to seizure from HBO2 by up to 300%, which is comparable to high doses of anti-epileptic drugs (AEDs) (3, 50) and experimental anticonvulsants that block excitatory glutamatergic neurotransmission (10). During periods of fasting or ketogenic diet (KD) use, the body utilizes energy obtained from free fatty acids (FFA) released from adipose tissue; however, the brain is unable to derive significant energy from FFA (8). Hepatic ketogenesis converts FFAs into the ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc), and a small percentage of AcAc spontaneously decarboxylates to acetone. During prolonged fasting or KD, large quantities of ketone bodies accumulate in the blood (>5 mM) and are transported across the blood-brain barrier (BBB) by monocarboxylic acid transporters (MCT1–4) to fuel brain function, and this ketone transport is enhanced under oxidative stress or limited glucose availability (40). The brain derives >60% of its energy from ketones when glucose availability is limited (8). The metabolic adaptations associated with fasting-induced ketosis improve mitochondrial function, decrease reactive oxygen species (ROS) production, reduce inflammation, and increase the activity of neurotrophic factors (34).

KD mimics the metabolic state of fasting (i.e., therapeutic ketosis) and is efficacious in treating drug-resistant seizure disorders (22). This therapeutic method is well established in children and adults (30). The anticonvulsant effects of the KD correlate with an elevation of blood ketones, especially AcAc and acetone (6, 36). The KD requires extreme dietary carbohydrate restriction and only modestly increases blood ketones compared with levels associated with prolonged fasting (8). Elevating blood ketones with ketogenic medical foods or exogenous ketones is largely ineffective or problematic for a variety of reasons. Ketogenic fats, like medium chain triglyceride oil (MCT oil), are generally not well tolerated by the gastrointestinal system, and supplementation produces only low levels of ketones (<0.5 mM) (27). Oral administration of BHB and AcAc in their free acid form is expensive and ineffective at producing sustained ketosis. One idea has been to buffer the free acid form of BHB with sodium salts, but this is largely ineffective at preventing seizures in animal models and causes a potentially harmful sodium overload at therapeutic levels of ketosis (6). However, esters of BHB or AcAc can effectively induce a rapid and sustained ketosis (7, 21) that mimics the sustained ketosis achieved with a strict KD or prolonged fasting without dietary restriction. Recent studies have demonstrated that orally administered esters of BHB are safe and well tolerated in rats (14) and humans (15). Producing esters of BHB or AcAc is expensive and technically challenging but offers great therapeutic potential (52). Orally administered ketone esters have the potential to induce ketosis and circumvent the problems associated with fasting-induced or diet-induced ketosis (15). The ketone ester that we synthesized and tested [R,S-1,3-butanediol acetoacetate diester (BD-AcAc2)] has been shown to induce therapeutic ketosis in dogs (12, 41) and pigs (21) and was proposed as a metabolic therapy for parenteral and enteral nutrition (7). We were interested in esters of AcAc because precursors to BHB do not prevent CNS-OT (9), and animal studies suggest that AcAc and acetone have the greatest anticonvulsant potential (6, 23, 33, 36).

In this study, we explored the potential of ketone ester-induced therapeutic ketosis as a mitigation strategy against CNS-OT seizures. We hypothesized that oral administration of BD-AcAc2 mimics the anticonvulsant effect of fasting-induced ketosis and delays the onset of CNS-OT.

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Citation / Publisher Attribution

American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, v. 304, issue 10, p. R829-R836