Background
Emerging evidence supports the therapeutic potential of the ketogenic diet (KD) for a variety of disease states, leading investigators to research methods of harnessing the benefits of nutritional ketosis without the dietary restrictions. The KD has been used as an effective non-pharmacological therapy for pediatric intractable seizures since the 1920s [
1‐
3]. In addition to epilepsy, the ketogenic diet has elicited significant therapeutic effects for weight loss and type-2 diabetes (T2D) [
4]. Several studies have shown significant weight loss on a high fat, low carbohydrate diet without significant elevations of serum cholesterol [
5‐
12]. Another study demonstrated the safety and benefits of long-term application of the KD in T2D patients. Patients exhibited significant weight loss, reduction of blood glucose, and improvement of lipid markers after eating a well-formulated KD for 56 weeks [
13]. Recently, researchers have begun to investigate the use of the KD as a treatment for acne, polycystic ovary syndrome (PCOS), cancer, amyotrophic lateral sclerosis (ALS), traumatic brain injury (TBI) and Alzheimer’s disease (AD) with promising preliminary results [
14‐
26].
The classical KD consists of a 4:1 ratio of fat to protein and carbohydrate, with 80–90 % of total calories derived from fat [
27]. The macronutrient ratio of the KD induces a metabolic shift towards fatty acid oxidation and hepatic ketogenesis, elevating the ketone bodies acetoacetate (AcAc) and β-hydroxybutyrate (βHB) in the blood. Acetone, generated by decarboxylation of AcAc, has been shown to have anticonvulsant properties [
28‐
32]. Ketone bodies are naturally elevated to serve as alternative metabolic substrates for extra-hepatic tissues during the prolonged reduction of glucose availability, suppression of insulin, and depletion of liver glycogen, such as occurs during starvation, fasting, vigorous exercise, calorie restriction, or the KD. Although the KD has clear therapeutic potential, several factors limit the efficacy and utility of this metabolic therapy for widespread clinical use. Patient compliance to the KD can be low due to the severe dietary restriction - the diet being generally perceived as unpalatable - and intolerance to high-fat ingestion. Maintaining ketosis can be difficult as consumption of even a small quantity of carbohydrates or excess protein can rapidly inhibit ketogenesis [
33,
34]. Furthermore, enhanced ketone body production and tissue utilization by the tissues can take several weeks (keto-adaptation), and patients may experience mild hypoglycemic symptoms during this transitional period [
35].
Recent studies suggest that many of the benefits of the KD are due to the effects of ketone body metabolism. Interestingly, in studies on T2D patients, improved glycemic control, improved lipid markers, and retraction of insulin and other medications occurred before weight loss became significant. Both βHB and AcAc have been shown to decrease mitochondrial reactive oxygen species (ROS) production [
36‐
39]. Veech et al. have summarized the potential therapeutic uses for ketone bodies [
28,
40]. They have demonstrated that exogenous ketones favorably alter mitochondrial bioenergetics to reduce the mitochondrial NAD couple, oxidize the co-enzyme Q, and increase the ΔG’ (free enthalpy) of ATP hydrolysis [
41]. Ketone bodies have been shown to increase the hydraulic efficiency of the heart by 28 %, simultaneously decreasing oxygen consumption while increasing ATP production [
42]. Thus, elevated ketone bodies increase metabolic efficiency and as a consequence, reduce superoxide production and increase reduced glutathione [
28]. Sullivan et al. demonstrated that mice fed a KD for 10–12 days showed increased hippocampal uncoupling proteins, indicative of decreased mitochondrial-produced ROS [
43]. Bough et al. showed an increase of mitochondrial biogenesis in rats maintained on a KD for 4–6 weeks [
44,
45]. Recently, Shimazu et al. reported that βHB is an exogenous and specific inhibitor of class I histone deacetylases (HDACs), which confers protection against oxidative stress [
38]. Ketone bodies have also been shown to suppress inflammation by decreasing the inflammatory markers TNF-a, IL-6, IL-8, MCP-1, E-selectin, I-CAM, and PAI-1 [
8,
46,
47]. Therefore, it is thought that ketone bodies themselves confer many of the benefits associated with the KD.
Considering both the broad therapeutic potential and limitations of the KD, an oral exogenous ketone supplement capable of inducing sustained therapeutic ketosis without the need for dietary restriction would serve as a practical alternative. Several natural and synthetic ketone supplements capable of inducing nutritional ketosis have been identified. Desrochers et al. elevated ketone bodies in the blood of pigs (>0.5 mM) using exogenous ketone supplements: (R, S)-1,3 butanediol and (R, S)-1,3 butanediol-acetoacetate monoesters and diester [
48]. In 2012, Clarke et al. demonstrated the safety and efficacy of chronic oral administration of a ketone monoester of R-βHB in rats and humans [
49,
50]. Subjects maintained elevated blood ketones without dietary restriction and experienced little to no adverse side effects, demonstrating the potential to circumvent the restrictive diet typically needed to achieve therapeutic ketosis. We hypothesized that exogenous ketone supplements could produce sustained hyperketonemia (>0.5 mM) without dietary restriction and without negatively influencing metabolic biomarkers, such as blood glucose, total cholesterol, HDL, LDL, and triglycerides. Thus, we measured these biomarkers during a 28-day administration of the following ketone supplements in rats: naturally-derived ketogenic supplements included medium chain triglyceride oil (MCT), sodium/potassium -βHB mineral salt (BMS), and sodium/potassium -βHB mineral salt + medium chain triglyceride oil 1:1 mixture (BMS + MCT) and synthetically produced ketogenic supplements included 1, 3-butanediol (BD), 1, 3-butanediol acetoacetate diester/ ketone ester (KE).
Methods
KE was synthesized as previously described [
29]. BMS is a novel agent (sodium/potassium- βHB mineral salt) supplied as a 50 % solution containing approximately 375 mg/g of pure βHB and 125 mg/g of sodium/potassium. Both KE and BMS were developed and synthesized in collaboration with Savind Inc. Pharmaceutical grade MCT oil (~65 % caprylic triglyceride; 45 % capric triglyceride) was purchased from Now Foods (Bloomingdale, IL). BMS was formulated in a 1:1 ratio with MCT at the University of South Florida (USF), yielding a final mixture of 25 % water, 25 % pure βHB mineral salt and 50 % MCT. BD was purchased from Sigma-Aldrich (Prod # B84785, Milwaukee, WI).
Daily gavage to induce dietary ketosis
Animal procedures were performed in accordance with the University of South Florida Institutional Animal Care and Use Committee (IACUC) guidelines (Protocol #0006R). Juvenile male Sprague–Dawley rats (275–325 g, Harlan Laboratories) were randomly assigned to one of six study groups: control (water,
n = 11), BD (
n = 11), KE (
n = 11), MCT (
n = 10), BMS (
n = 11), or BMS + MCT (
n = 12). Caloric density of standard rodent chow and dose of ketone supplements are listed in Table
1. On days 1–14, rats received a 5 g/kg body weight dose of their respective treatments via intragastric gavage. Dosage was increased to 10 g/kg body weight for the second half of the study (days 15–28) for all groups except BD and KE to prevent excessive hyperketonemia (ketoacidosis). Each daily dose of BMS would equal ~1000–1500 mg of βHB, depending on the weight of the animal. Intragastric gavage was performed at the same time daily, and animals had
ad libitum access to standard rodent chow 2018 (Harlan Teklad) for the duration of the study. The macronutrient ratio the standard rodent chow was 62.2, 23.8 and 14 % of carbohydrates, protein and fat respectively.
Table 1
Caloric density and dose of ketone supplements
% Cal from Fat | 18.0 | 0.0 | 50.0 | N/A | 100.0 | N/A | N/A |
% Cal from Protein | 24.0 | 0.0 | N/A | N/A | 0.0 | N/A | N/A |
% Cal from Carbohydrates | 58.0 | 0.0 | N/A | N/A | 0.0 | N/A | N/A |
Total Caloric Density (Kcal/g) | 3.1 | 0.0 | 5.1 | 1.9 | 8.3 | 5.6 | 6.0 |
Dose 0–14 Days (g/kg) |
ad libitum
| N/A | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 |
Dose 15–28 Days (g/kg) |
ad libitum
| N/A | 10.0 | 10.0 | 10.0 | 5.0 | 5.0 |
Measurement and analysis of blood glucose, ketones, and lipids
Every 7 days, animals were briefly fasted (4 h, water available) prior to intragastric gavage to standardize levels of blood metabolites prior to glucose and βHB measurements at baseline. Baseline (time 0) was immediately prior to gavage. Whole blood samples (10 μL) were taken from the saphenous vein for analysis of glucose and βHB levels with the commercially available glucose and ketone monitoring system Precision Xtra™ (Abbott Laboratories, Abbott Park, IL). Blood glucose and βHB were measured at 0, 0.5, 1, 4, 8, and 12 h after test substance administration, or until βHB returned to baseline levels. Food was returned to animals after blood analysis at time 0 and gavage. At baseline and week 4, whole blood samples (10 μL) were taken from the saphenous vein immediately prior to gavage (time 0) for analysis of total cholesterol, high-density lipoprotein (HDL), and triglycerides with the commercially available CardioChek™ blood lipid analyzer (Polymer Technology Systems, Inc., Indianapolis, IN). Low-density lipoprotein (LDL) cholesterol was calculated from the three measured lipid levels using the Friedewald equation: (LDL Cholesterol = Total Cholesterol - HDL - (Triglycerides/5)) [
51,
52]. Animals were weighed once per week to track changes in body weight associated with hyperketonemia.
Organ weight and collection
On day 29, rats were sacrificed via deep isoflurane anesthesia, exsanguination by cardiac puncture, and decapitation 4–8 h after intragastric gavage, which correlated to the time range where the most significantly elevated blood βHB levels were observed. Brain, lungs, liver, kidneys, spleen and heart were harvested, weighed (AWS-1000 1 kg portable digital scale (AWS, Charleston, SC)), and flash-frozen in liquid nitrogen or preserved in 4 % paraformaldehyde for future analysis.
Statistics
All data are presented as the mean ± standard deviation (SD). Data analysis was performed using GraphPad PRISM™ version 6.0a and IBM SPSS Statistics 22.0. Results were considered significant when p < 0.05. Triglyceride and lipoprotein profile data were analyzed using One-Way ANOVA. Blood ketone and blood glucose were compared to control at the applicable time points using a Two-Way ANOVA. Correlation between blood βHB and glucose levels in ketone supplemented rats was compared to controls using ANCOVA analysis. Organ and body weights were analyzed using One-Way ANOVA. Basal blood ketone and blood glucose levels were analyzed using Two-Way ANOVA. All mean comparisons were carried out using Tukey’s multiple comparisons post-hoc test.
Competing interests
International Patent # PCT/US2014/031237, University of South Florida, D.P. D’Agostino, S. Kesl, P. Arnold, “Compositions and Methods for Producing Elevated and Sustained Ketosis”. P. Arnold (Savind) has received financial support (ONR N000140610105 and N000140910244) from D.P. D’Agostino (USF) to synthesize ketone esters. The remaining authors have no conflicts of interest.
Authors’ contributions
Conceived and designed the experiments: SK, AP, NW, TF, DP. Performed the experiments: SK, AP, NW, TF, CA, JS, AVP. Analyzed the data: SK, AP, DP. Contributed reagents/materials/analysis tools: PA. Helped draft the manuscript: SK, AP, NW, CA, DP. All authors read and approved the final manuscript.