Effects of exogenous ketone supplementation on blood ketone, glucose, triglyceride, and lipoprotein levels in Sprague–Dawley rats

Abstract

Nutritional ketosis induced by the ketogenic diet (KD) has therapeutic applications for many disease states. We hypothesized that oral administration of exogenous ketone supplements could produce sustained nutritional ketosis (>0.5 mM) without carbohydrate restriction.
We tested the effects of 28-day administration of five ketone supplements on blood glucose, ketones, and lipids in male Sprague–Dawley rats. The supplements included: 1,3-butanediol (BD), a sodium/potassium β-hydroxybutyrate (βHB) mineral salt (BMS), medium chain triglyceride oil (MCT), BMS + MCT 1:1 mixture, and 1,3 butanediol acetoacetate diester (KE). Rats received a daily 5–10 g/kg dose of their respective ketone supplement via intragastric gavage during treatment. Weekly whole blood samples were taken for analysis of glucose and βHB at baseline and, 0.5, 1, 4, 8, and 12 h post-gavage, or until βHB returned to baseline. At 28 days, triglycerides, total cholesterol and high-density lipoprotein (HDL) were measured.
Exogenous ketone supplementation caused a rapid and sustained elevation of βHB, reduction of glucose, and little change to lipid biomarkers compared to control animals.
This study demonstrates the efficacy and tolerability of oral exogenous ketone supplementation in inducing nutritional ketosis independent of dietary restriction.

Full text

R E S E A R C H Open Access
Effects of exogenous ketone
supplementation on blood ketone, glucose,
triglyceride, and lipoprotein levels in
Sprague–Dawley rats
Shannon L. Kesl
1*
, Angela M. Poff
1
, Nathan P. Ward
1
, Tina N. Fiorelli
1
, Csilla Ari
1
, Ashley J. Van Putten
1
,
Jacob W. Sherwood
1
, Patrick Arnold
2
and Dominic P. D’Agostino
1
Abstract
Background: Nutritional ketosis induced by the ketogenic diet (KD) has therapeutic applications for many disease
states. We hypothesized that oral administration of exogenous ketone supplements could produce sustained
nutritional ketosis (>0.5 mM) without carbohydrate restriction.
Methods: We tested the effects of 28-day administration of five ketone supplements on blood glucose, ketones,
and lipids in male Sprague–Dawley rats. The supplements included: 1,3-butanediol (BD), a sodium/potassium β-
hydroxybutyrate (βHB) mineral salt (BMS), medium chain triglyceride oil (MCT), BMS + MCT 1:1 mixture, and 1,3
butanediol acetoacetate diester (KE). Rats received a daily 5–10 g/kg dose of their respective ketone supplement via
intragastric gavage during treatment. Weekly whole blood samples were taken for analysis of glucose and βHB at
baseline and, 0.5, 1, 4, 8, and 12 h post-gavage, or until βHB returned to baseline. At 28 days, triglycerides, total
cholesterol and high-density lipoprotein (HDL) were measured.
Results: Exogenous ketone supplementation caused a rapid and sustained elevation of βHB, reduction of glucose,
and little change to lipid biomarkers compared to control animals.
Conclusions: This study demonstrates the efficacy and tolerability of oral exogenous ketone supplementation in
inducing nutritional ketosis independent of dietary restriction.
Keywords: Ketogenic diet, Ketone ester, Ketone supplement, Appetite, β-hydroxybutyrate, Hyperketonemia,
Triglycerides
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 sei-
zures 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].
* Correspondence: skesl@health.usf.edu
1
Department of Molecular Pharmacology and Physiology, Morsani College of
Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC8,
Tampa, FL 33612, USA
Full list of author information is available at the end of the article
© 2016 Kesl et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Kesl et al. Nutrition & Metabolism (2016) 13:9
DOI 10.1186/s12986-016-0069-y
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

The classical KD consists of a 4:1 ratio of fat to pro-
tein and carbohydrate, with 80–90 % of total calories de-
rived 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-adap-
tation), 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 gly-
cemic 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 mitochon-
drial 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 effi-
ciency of the heart by 28 %, simultaneously decreasing
oxygen consumption while increasing ATP production
[42]. Thus, elevated ketone bodies increase metabolic effi-
ciency and as a consequence, reduce superoxide production
and increase reduced glutathione [28]. Sullivan et al. demon-
strated 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 asso-
ciated with the KD.
Considering both the broad therapeutic potential and
limitations of the KD, an oral exogenous ketone supple-
ment 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 triglycer-
ides. 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 acetoace-
tate diester/ ketone ester (KE).
Methods
Synthesis and formulation of ketone supplements
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/potas-
sium. Both KE and BMS were developed and synthesized
in collaboration with Savind Inc. Pharmaceutical grade
MCT oil (~65 % caprylic triglyceride; 45 % capric trigly-
ceride) 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 mix-
ture 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
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 2 of 15
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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 hyperketone-
mia (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.
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 imme-
diately prior to gavage. Whole blood samples (10 μL)
were taken from the saphenous vein for analysis of
glucose and βHB levels with the commercially avail-
able 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 choles-
terol, high-density lipoprotein (HDL), and triglycerides
with the commercially available CardioChek™blood
lipid analyzer (Polymer Technology Systems, Inc., In-
dianapolis, IN). Low-density lipoprotein (LDL)
cholesterol was calculated from the three measured
lipid levels using the Friedewald equation: (LDL Choles-
terol = 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 de-
capitation 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. Re-
sults were considered significant when p < 0.05. Trigly-
ceride 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 com-
parisons were carried out using Tukey’s multiple com-
parisons post-hoc test.
Results
Effect of ketone supplementation on triglycerides and
lipoproteins
Baseline measurements showed no significant changes
in triglycerides or the lipoproteins (data not shown).
Data represent triglyceride and lipoprotein concentra-
tions measured after 4 weeks of daily exogenous ketone
supplementation. No significant change in total choles-
terol was observed at 4 weeks for any of the ketone
treatment groups compared to control. (Fig. 1a). No
Table 1 Caloric density and dose of ketone supplements
Macronutrient Information Standard Diet Water BMS + MCT BMS MCT KE BD
% 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
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 3 of 15
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significant difference was detected in triglycerides for
any ketone supplement compared to control (Fig. 1b).
MCT supplemented animals had a significant reduction
in HDL blood levels compared to control (p < 0.001)
(Fig. 1c). LDL levels in ketone-supplemented animals did
not significantly differ from controls (Fig. 1d).
Ketone supplementation causes rapid and sustained
elevation of βHB
Over the 28-day experiment, ketone supplements ad-
ministered daily significantly elevated blood ketone
levels without dietary restriction (Fig. 2a, b). Naturally
derived ketogenic supplements including MCT (5 g/kg)
elicited a significant rapid elevation in blood βHB within
30–60 min that was sustained for 8 h. BMS + MCT (5 g/
kg) elicited a significant elevation in blood βHB at 4 h,
which was no longer significant at 8 h. BMS (5 g/kg) did
not elicit a significant elevation in blood βHB at any
time point. For days 14–28, BMS + MCT (10 g/kg) and
MCT (10 g/kg) elevated blood βHB levels within 30 min
and remained significantly elevated for up to 12 h. We
observed a delay in the peak elevation of blood βHB:
BMS + MCT peaked at 8 h instead of at 4 h and MCT at
4 h instead of at 1 h. Blood βHB levels in the BMS group
did not show significant elevation at any time point,
even after dose escalation (Fig. 2a). Synthetically derived
ketogenic supplements including KE and BD supple-
mentation rapidly elevated blood βHB within 30 min
and was sustained for 8 h. For the rats receiving ketone
supplementation in the form of BD or the KE, dosage
was kept at 5 g/kg to prevent adverse effects associated
with hyperketonemia. The Precision Xtra™ketone moni-
toring system measures βHB only; therefore, total blood
ketone levels (βHB + AcAc) would be higher than mea-
sured. For each of these groups, the blood βHB profile
remained consistent following daily ketone supplementa-
tion administration over the 4-week duration. (Fig. 2b).
Ketone supplementation causes a significant decrease of
blood glucose
Administration of ketone supplementation significantly
reduced blood glucose over the course of the study
(Fig. 3a, b). MCT (5 g/kg) decreased blood glucose com-
pared to control within 30 min which was sustained for
Fig. 1 Effects of ketone supplementation on triglycerides and lipoproteins: Ketone supplementation causes little change in triglycerides and
lipoproteins over a 4-week study. Graphs show concentrations at 4-weeks of total cholesterol (a), Triglycerides (b), LDL (c), and HDL (d).
MCT supplemented rats had signfiicantly reduced concentration of HDL blood levels compared to control (p< 0.001) (b). One-Way ANOVA
with Tukey’s post hoc test, results considered significant if p< 0.05. Error bars represent mean (SD)
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 4 of 15
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Fig. 2 Effects of ketone supplementation on blood βHB. a,bBlood βHB levels at times 0, 0.5, 1, 4, 8, and 12 h post intragastric gavage for ketone
supplements tested. aBMS + MCT and MCT supplementation rapidly elevated and sustained significant βHB elevation compared to controls for
the duration of the 4-week dose escalation study. BMS did not significantly elevate βHB at any time point tested compared to controls. bBD and
KE supplements, maintained at 5 g/kg, significantly elevated βHB levels for the duration of the 4-week study. Two-Way ANOVA with Tukey’s post
hoc test, results considered significant if p< 0.05. Error bars represent mean (SD)
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 5 of 15
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Fig. 3 Effects of ketone supplementation on blood glucose. a,bBlood glucose levels at times 0, 0.5, 1, 4, 8, and 12 h (for 10 dose) post intragastric
gavage for ketone supplements tested. aKetone supplements BMS + MCT and MCT significantly reduced blood glucose levels compared to controls
for the duration of the 4-week study. BMS significantly lowered blood glucose only at 8 h/week 1 and 12 h/week 3 (b) KE, maintained at
5 g/kg, significantly reduced blood glucose compared to controls from week 1–4. BD did not significantly affect blood glucose levels at
any time point during the 4-week study. Two-Way ANOVA with Tukey’s post hoc test, results considered significant if p< 0.05. Error bars
represent mean (SD)
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8 h at baseline and at week 1. MCT (10 g/kg) likewise
decreased blood glucose within 30 min and lasted
through the 12 h time point during weeks 2, 3, and 4.
BMS + MCT (5 g/kg) lowered blood glucose compared
to control from hours 1–8 only at week 1. BMS + MCT
(10 g/kg) lowered blood glucose compared to control
within 30 min and remained low through the 12 h time
point at weeks 2, 3, and 4. Rats supplemented with BMS
had lower blood glucose compared to control at 12 h in
week 4 (10) (Fig. 3a). Administration of BD did not sig-
nificantly change blood glucose levels at any time point
during the 4-week study. KE (5 g/kg) significantly low-
ered blood glucose levels at 30 min for week 1, 2, 3, and
4 and was sustained through 1 h at weeks 2–4 and sus-
tained to 4 h at week 3. (Fig. 3b).
Hyperketonemia suppresses blood glucose levels
At baseline, 4 h after intragastric gavage, the elevation of
blood ketones was inversely related to the reduction of blood
glucose compared to controls following the administration
of MCT (5 g/kg) (p = 0.008) and BMS + MCT (5 g/kg) (p =
0.039) . There was no significant correlation between blood
ketone levels and blood glucose levels compared to controls
for any other ketone supplemented group at baseline
(Fig.4a).Atweek4,4hafterintragastricgavage,therewasa
significant correlation between blood ketone levels and
Fig. 4 Relationship between blood ketone and glucose levels: aBMS + MCT (5 g/kg) supplemented rats demonstrated a significant inverse relationship
between elevated blood ketone levels and decreased blood ketone levels (r
2
= 0.4314, p = 0.0203). bAt week 4, BMS + MCT (10 g/kg) and MCT (10 g/kg)
showed a significant correlation between blood ketone levels and blood glucose levels (r2 = 0.8619, p< 0.0001; r
2
= 0.6365, p= 0.0057). Linear regression
analysis, results considered significant if p<0.05
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blood glucose levels compared to controls in MCT (10 g/kg)
and BMS + MCT (10 g/kg) (p< 0.0001, p< 0.0001) (Fig. 4b).
Ketone supplementation changes organ weight and
decreases body weight
At day 29 of the study, animals were euthanized and
brain, lungs, liver, kidneys, spleen and heart were har-
vested and weighed. Organ weights were normalized to
body weight. Ketone supplementation did not signifi-
cantly change brain, lung, kidney, or heart weights
compared to controls (Fig. 5a, b, d, f). MCT supple-
mented animals had significantly larger livers compared
to their body weight (p< 0.05) (Fig. 5c). Ketone supple-
ments BMS + MCT, MCT and BD caused a significant
reduction in spleen size (BMS + MCT p< 0.05, MCT p
< 0.001, BD p< 0.05) (Fig. 5e). Rats administered KE
gained significantly less weight over the entire study
compared to controls. BMS + MCT, BMS, and BD sup-
plemented rats gained significantly less weight than con-
trols during weeks 2 –4, and MCT animals gained less
Fig. 5 Effects of ketone supplementation on organ weight: Data is represented as a percentage of organ weight to body weight. a,b,d,f
Ketone supplements did not significantly affect the weight of the brain, lungs, kidneys or heart. cLiver weight was significantly increased as
compared to body weight in response to administered MCT ketone supplement compared to control at the end of the study (day 29) (p< 0.001).
eRats supplemented with BMS+ MCT, MCT, and BD had significantly smaller spleen percentage as compared to controls (p<0.05,p< 0.001, p< 0.05).
Two-Way ANOVA with Tukey’s post-hoc test; results considered significant if p< 0.05. Error bars represent mean (SD)
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weight than controls at weeks 3 –4 (Fig. 6). Increased
gastric motility (increased bowel evacuation and changes
to fecal consistency) was visually observed in rats sup-
plemented with 10 g/kg MCT, most notably at the 8 and
12-h time points. All animals remained in healthy weight
range for their age even though the rate of weight gain
changed with ketone supplementation [53–54]. Food in-
take was not measured in this study. However, there was
not a significant change in basal blood glucose or basal
blood ketone levels over the 4 week study in any of the
rats supplemented with ketones (Fig. 7).
Discussion
Nutritional ketosis induced with the KD has proven
effective for the metabolic management of seizures and
potentially other disorders [1–26]. Here we present
evidence that chronic administration of ketone supple-
ments can induce a state of nutritional ketosis without
the need for dietary carbohydrate restriction and with
little or no effect on lipid biomarkers. The notion that
we can produce the therapeutic effects of the KD with
exogenous ketone supplementation is supported by our
previous study which demonstrated that acutely admin-
istered KE supplementation delays central nervous
system (CNS) oxygen toxicity seizures without the
need for dietary restriction [29]. We propose that
exogenous ketone supplementation could provide an
alternative method of attaining the therapeutic
benefits of nutritional ketosis, and as a means to
further augment the therapeutic potential of the KD.
Ketone supplementation causes little to no change in
triglycerides and lipoproteins
One common concern regarding the KD is its purported
potential to increase the risk of atherosclerosis by elevat-
ing blood cholesterol and triglyceride levels [55, 56]. This
topic remains controversial as some, but not all, studies
have demonstrated that the KD elevates blood levels of
cholesterol and triglycerides [57–62]. Kwitervich and col-
leagues demonstrated an increase in low-density lipopro-
tein (LDL) and a decrease in high-density lipoprotein
(HDL) in epileptic children fed the classical KD for
two years [27]. In this study, total cholesterol in-
creased by ~130 %, and stabilized at the elevated level
over the 2-year period. A similar study demonstrated that
the lipid profile returned to baseline in children who
remained on the KD for six years [63]. Children typically
remain on the diet for approximately two years then re-
turn to a diet of common fat and carbohydrate ingestion
[64]. The implications of these findings are unclear, since
the influence of cholesterol on cardiovascular health is
controversial and macronutrient sources of the diet vary
per study. In contrast to these studies, the majority of re-
cent studies have suggested that the KD can actually lead
to significant benefits in biomarkers of metabolic health,
including blood lipid profiles [65–72]. In these studies, the
Fig. 6 Effects of ketone supplementation on body weight: Rats administered ketone supplements gained less weight over the 4-week period;
however, did not lose weight and maintained healthy range for age. KE supplemented rats gained significantly less weight during the entire
4-week study compared to controls. BMS +MCT, BMS, and BD supplemented rats gained significantly less weight than controls over weeks 2–4.MCT
supplemented rats gained significantly less weight than controls over weeks 3–4, Two-Way ANOVA with Tukey’s post hoc test, results considered
significant if p< 0.05. Error bars represent mean (SD)
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KD positively altered blood lipids, decreasing total triglyc-
erides and cholesterol while increasing the ratio of HDL
to LDL [68–77]. Although, the KD is well-established in
children, it has only recently been utilized as a strategy to
control seizures in adults. In 2014, Schoeler and col-
leagues reported on the feasibility of the KD for adults,
concluding that 39 % of individuals achieved > 50 % reduc-
tion in seizure frequency, similar to the results reported in
pediatric studies. Patients experienced similar gastrointes-
tinal adverse advents that have been previously described
in pediatric patients, but they did not lead to discontinu-
ation of the diet in any patient [78].
With oral ketone supplementation, we observed a sig-
nificant elevation in blood βHB without dietary restric-
tion and with little change in lipid biomarkers (Fig. 1).
Over the 4 week study, MCT-supplemented rats demon-
strated decreased HDL compared to controls. No signifi-
cant changes were observed in any of the triglycerides or
lipoproteins (HDL, LDL) with any of the remaining ex-
ogenously applied ketone supplements. It should be
noted that the rats used for this study had not yet
reached full adult body size [79]. Their normal growth
rate and maturation was likely responsible for the
changes in triglyceride and lipoprotein levels observed in
the control animals over the 4 week study (baseline data
not shown, no significant differences) [80, 81]. Future
studies are needed to investigate the effect of ketone
supplementation on fully mature and aged animals.
Overall, our study suggests that oral ketone supplemen-
tation has little effect on the triglyceride or lipoprotein
profile after 4 weeks. However, it is currently unknown if
ketone supplementation would affect lipid biomarkers
after a longer duration of consumption. Further studies
are needed to determine the effects of ketone supplements
on blood triglyceride and lipoproteins after chronic
administration and as a means to further enhance the
hyperketonemia and improve the lipid profile of the clinic-
ally implemented (4:1) KD.
LDL is the lipoprotein particle that is most often associ-
ated with atherosclerosis. LDL particles exist in different
sizes: large molecules (Pattern A) or small molecules
(Pattern B). Recent studies have investigated the im-
portance of LDL-particle type and size rather than
total concentration as being the source for cardiovascular
risk [56]. Patients whose LDL particles are predominantly
small and dense (Pattern B) have a greater risk of
Fig. 7 Effects of ketone supplementation on basal blood ketone and basal blood glucose levels: Rats administered ketone supplements did not
have a significant change in basal blood ketone levels (a) or basal blood glucose levels (b) for the four week study. Two-Way ANOVA with Tukey’s
post-hoc test, results considered significant if p< 0.05. Error bars represent mean (SD)
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 10 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

cardiovascular disease (CVD). It is thought that small,
dense LDL particles are more able to penetrate the endo-
thelium and cause in damage and inflammation [82–85].
Volek et al. reported that the KD increased the pattern
and volume of LDL particles, which is considered to re-
duce cardiovascular risk [73]. Though we did not show a
significant effect on LDL levels for ketone supplements,
future chronic feeding studies will investigate the effects
of ketone supplementation on lipidomic profile and LDL
particle type and size.
Therapeutic levels of hyperketonemia suppress blood
glucose levels
We demonstrated that therapeutic ketosis could be
induced without dietary (calorie or carbohydrate) restric-
tion and that this acute elevation in blood ketones was
significantly correlated with a reduction in blood glucose
(Figs. 2, 3 and 4). The BMS ketone supplement did not
significantly induce blood hyperketonemia or reduced
glucose in the rats. The KE supplemented rats trended
towards reduced glucose levels; however, the lower dose
of this agent did not lower glucose significantly, as re-
ported previously in acute response of mice [59]. MCTs
have previously been shown to elicit a slight hypoglycemic
effect by enhancing glucose utilization in both diabetic
and non-diabetic patients [86–88]. Kashiwaya et al. dem-
onstrated that both blood glucose and blood insulin
decreased by approximately 50 % in rats fed a diet where
30 % of calories from starch were replaced with ketone es-
ters for 14 days, suggesting that ketone supplementation
increases insulin sensitivity or reduced hepatic glucose
output [89]. This ketone-induced hypoglycemic effect has
been previously reported in humans with IV infusions of
ketone bodies [90, 91]. Recently, Mikkelsen et al. showed
that a small increase in βHB concentration decreases glu-
cose production by 14 % in post-absorptive health males
[92]. However, this has not been previously reported with
any of the oral exogenous ketone supplements we studied.
Ketones are an efficient and sufficient energy substrate for
the brain, and will therefore prevent side effects of
hypoglycemia when blood levels are elevated and the pa-
tient is keto-adapted. This was most famously demon-
strated by Owen et al. in 1967 wherein keto-adapted
patients (starvation induced therapeutic ketosis) were
given 20 IU of insulin. The blood glucose of fasted pa-
tients dropped to 1–2 mM, but they exhibited no
hypoglycemic symptoms due to brain utilization of ke-
tones for energy [93]. Therefore, ketones maintain brain
metabolism and are neuroprotective during severe
hypoglycemia. The rats in the MCT group had a correl-
ation of blood ketone and glucose levels at week 4,
whereas the combination of BMS + MCT produced a sig-
nificant hypoglycemic correlation both at baseline and at
week 4. No hypoglycemic symptoms were observed in the
rats during this study. Insulin levels were not measured in
this study; however, future ketone supplementation stud-
ies should measure the effects of exogenous ketones on
insulin sensitivity with a glucose tolerance test. An
increase in insulin sensitivity in combination with our
observed hypoglycemic effect has potential therapy impli-
cations for glycemic control in T2D [40]. Furthermore, it
should be noted that the KE metabolizes to both AcAc
and βHB in 1:1 ratio [29]. The ketone monitor used in this
study only measures βHB as levels of AcAc are more diffi-
cult to measure due to spontaneous decarboxylation to
acetone; therefore, the total ketone levels (βHB + AcAc)
measured were likely higher, specifically for the KE [14].
Interestingly, the 10 g/kg dose produced a delayed blood
βHB peak for ketone supplements MCT and BMS + MCT.
The higher dose of the ketogenic supplements elevated
blood levels more substantially, and thus reached their
maximum blood concentration later due to prolonged
metabolic clearance. It must be noted that the dosage used
in this study does not translate to human patients, since
the metabolic physiology of rats is considerably higher.
Future studies will be needed to determine optimal dosing
for human patients.
Effects of ketone supplementation on organ weight and
body weight percentage
Ketone supplementation did not affect the size of the
brain, lungs, kidneys or heart of rats. As previously men-
tioned, the rats were still growing during the experimen-
tal time frame; therefore, organ weights were normalized
to body weight to determine if organ weight changed in-
dependently to growth. There could be several reasons
why ketones influenced liver and spleen weight. The ra-
tio of liver to body weight was significantly higher in the
MCT supplemented animals (Fig. 5). MCTs are readily
absorbed in the intestinal lumen and transported directly
to the liver via hepatic portal circulation. When given a
large bolus, such as in this study, the amount of MCTs
in the liver will likely exceed the β-oxidation rate, caus-
ing the MCTs to be deposited in the liver as fat droplets
[94]. The accumulated MCT droplets in the liver could
explain the higher liver weight to body weight percentage
observed with MCT supplemented rats. Future toxicology
and histological studies will be needed to determine the
cause of the observed hepatomegaly. It should be empha-
sized that the dose in this study is not optimized in
humans. We speculate that an optimized human dose
would be lower and may not cause hepatomegaly or po-
tential fat accumulation. Nutritional ketosis achieved with
the KD has been shown to decrease inflammatory markers
such as TNF-α, IL-6, IL-8, MCP-1, E-selectin, I-CAM, and
PAI-1 [8, 46], which may account for the observed
decrease in spleen weight. As previously mentioned,
Veech and colleagues demonstrated that exogenous
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 11 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

supplementation of 5 mM βHB resulted in a 28 % increase
in hydraulic work in the working perfused rat heart and a
significant decrease in oxygen consumption [28, 41, 42].
Ketone bodies have been shown to increase cerebral blood
flow and perfusion [95]. Also, ketone bodies have been
shown to increase ATP synthesis and enhance the effi-
ciency of ATP production [14, 28, 40]. It is possible that
sustained ketosis results in enhanced cardiac efficiency
and O
2
consumption. Even though the size of the
heart did not change for any of the ketone supple-
ments, further analysis of tissues harvested from the
ketone-supplemented rats will be needed to determine
any morphological changes and to understand changes in
organ size. It should be noted that the Harlan standard
rodent chow 2018 is nutritionally complete and formu-
lated with high-quality ingredients to optimize gestation,
lactation, growth, and overall health of the animals. The
same cannot be said for the standard American diet
(SAD). Therefore, we plan to investigate the effects of
ketone supplements administered with the SAD to deter-
mine if similar effects will be seen when the micronutrient
deficiencies and macronutrient profile mimics what most
Americans consume.
MCT oil has recently been used to induce nutritional
ketosis although it produces dose-dependent gastrointes-
tinal (GI) side effects in humans that limit the potential
for its use to significantly elevate ketones (>0.5 mM).
Despite these limitations, Azzam and colleagues pub-
lished a case report in which a 43-year-old-man had a
significant decrease in seizure frequency after supple-
menting his diet with 4 tablespoons of MCT oil twice
daily [96]. An attempt to increase his dosage to 5 table-
spoons twice daily was halted by severe GI intolerance.
Henderson et al. observed that 20 % of patients re-
ported GI side effects with a 20 g dose of ketogenic
agent AC-1202 in a double blind trial in mild to moderate
Alzheimer’s patients [24]. We visually observed similar
gastrointestinal side effects (loose stools) in the rats
treated with MCT oil in our study. Rats were closely mon-
itored to avoid dehydration, and gastric motility returned
to normal between 12–24 h. Interestingly, the BMS +
MCT supplement elevated βHB similarly to MCT oil
alone, without causing the adverse gastrointestinal effects
seen in MCT-supplemented rats. However, this could be
due to the fact in a 10 g/kg dose of BMS + MCT, only 5 g/
kg is MCT alone, which is less than the 10 g/kg dose that
elicits the GI side effects. This suggests that this novel
combination may provide a more useful therapeutic
option than MCT oil alone, which is limited in its ability
to elevate ketones in humans.
Exogenously delivered ketone supplements signifi-
cantly altered rat weight gain for the duration of the
study (Fig. 6). However, rats did not lose weight and
maintained a healthy range for their age. Rats have been
shown to effectively balance their caloric intake to prevent
weight loss/gain [97–99]. Due to the caloric density of the
exogenous ketone supplements (Table 1) it is possible for
the rats to eat less of the standard rodent chow and there-
fore less carbohydrates while maintaining their caloric
intake. Food intake was not measured for this study.
However, if there was a significant carbohydrate restric-
tion there would be a signifcant change in basal blood ke-
tone and blood glucose levels. As the hallmark to the KD,
carbohydrate restriction increases blood ketone levels and
reduces blood glucose levels. Neither an increase in basal
blood ketone levels nor a decrease in basal blood glucose
levels was observed in this study (Fig. 7). Additionally, if
there were an overall blood glucose decrease due to a
change in food intake, this would not explain the
rapid reduction (within 30 min) in blood glucose cor-
related with an elevation of blood ketone levels after
an intragastric bolus of ketone supplement (Figs. 2, 3
and 4).
Conclusions
Several studies have investigated the safety and efficacy
of ketone supplements for disease states such as AD and
Parkinson’s disease, and well as for parenteral nutrition
[40, 48–50, 100–103]. Our research demonstrates that
several forms of dietary ketone supplementation can ef-
fectively elevate blood ketone levels and achieve deleted:
therapeutic nutritional ketosis without the need for diet-
ary carbohydrate restriction. We also demonstrated that
ketosis achieved with exogenous ketone supplementation
can reduce blood glucose, and this is inversely associated
with the blood ketone levels. Although preliminary results
are encouraging, further studies are needed to determine
if oral ketone supplementation can produce the same
therapeutic benefits as the classic KD in the broad-
spectrum of KD-responsive disease states . Additionally,
further experiments need to be conducted to see if the ex-
ogenous ketone supplementation affects the same physio-
logical features as the KD (i.e. ROS, inflammation, ATP
production). Ketone supplementation could be used as an
alternative method for inducing ketosis in patients
uninterested in attempting the KD or those who have pre-
viously had difficulty implementing the KD because of
palatability issues, gall bladder removal, liver abnormal-
ities, or intolerance to fat. Additional experiments should
be conducted to see if ketone supplementation could be
used in conjunction with the KD to assist and ease the
transition to nutrition ketosis and enhance the speed of
keto-adaptation. In this study we have demonstrated the
ability of several ketone supplements to elevate blood
ketone levels, providing multiple options to induce thera-
peutic ketosis based on patient need. Though additional
studies are needed to determine the therapeutic potential
of ketone supplementation, many patients that previously
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 12 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

were unable to benefit from the KD may now have an
alternate method of achieving therapeutic ketosis. Ketone
supplementation may also represent a means to further
augment ketonemia in those responsive to therapeutic
ketosis, especially in those individuals where maintaining
low glucose is important.
Abbreviations
AcAc: acetoacetate; ALS: amyotrophic lateral sclerosis; AD: Alzheimer’s;
βHB: beta-hydroxybutyrate; BMS: sodium/potassium βHB mineral salt; BMS +
MCT: BMS + MCT 1:1 mixture; BD: 1,3-butanediol; CNS: central nervous
system; HDL: high density lipoprotein; HDACs: histone deacetylases; LDL: low
density lipoprotein; KD: ketogenic diet; KE: 1, 3-butanediol acetoacetate dies-
ter/ketone ester; MCT: medium chain triglyceride oil; PCOS: polycystic ovary
syndrome; ROS: reactive oxygen species; SAD: standard American diet;
TBI: traumatic brain injury; T2D: type-2 diabetes.
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.
Acknowledgements
The authors would like to thank Savind Inc for manufacturing some of the
ketone supplements, Jay Dean for the use of his Hyperbaric Research
Laboratory, and the ONR and Scivation Inc. for funding the project.
Grants
This study was supported by the Office of Naval Research (ONR) Grant
N000140610105 (DPD); and a Morsani College of Medicine Department of
Molecular Pharmacology and Physiology departmental grant.
Author details
1
Department of Molecular Pharmacology and Physiology, Morsani College of
Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC8,
Tampa, FL 33612, USA.
2
Savind Inc, 205 South Main Street, Seymore, IL
61875, USA.
Received: 10 September 2015 Accepted: 28 January 2016
References
1. Sirven J, Whedon B, Caplan D, Liporace J, Glosser D, O’Dwyer J, et al. The
ketogenic diet for intractable epilepsy in adults: preliminary results.
Epilepsia. 1999;40(12):1721–6. doi:10.1111/j.1528-1157.1999.tb01589.x.
2. Wilder R. The effect of ketonemia on the course of epilepsy. Mayo Clin
Bulletin. 1921;2:307–8.
3. Thiele E. Assessing the efficacy of antiepileptic treatments: the ketogenic
diet. Epilepsia. 2003;44 Suppl 7:26–9. doi:10.1046/j.1528-1157.44.s7.4.x.
4. Paoli A, Rubini A, Volek JS, Grimaldi KA. Beyond weight loss: a review of the
therapeutic uses of very-low-carbohydrate (ketogenic) diets. Eur J Clin Nutr.
2013;67(8):789–96. doi:10.1038/ejcn.2013.116.
5. Foster GD, Wyatt HR, Hill JO, McGuckin BG, Brill C, Mohemmed BS, et al. A
randomized trial of a low-carbohydrate diet for obesity. N Engl J Med. 2003;
348:2082–90.
6. Westman EC, Feinman RD, Mavropoulos JC, Vernon MC, Volek JS, Wortman
JA, et al. Low-carbohydrate nutrition and metabolism. Am J Clin Nutr. 2007;
86:276–84.
7. Westman EC, Yancy WS, Edman JS, Tomlin KF, Perkins CE. Effect of 6-month
adherence to a very low carbohydrate diet program. Am J Med. 2002;
113(1):30–6.
8. Forsythe C, Phinney S, Fernandez M, Quann E, Wood R, Bibus D, et al.
Comparison of low fat and low carbohydrate diets on circulating fatty acid
composition and markers of inflammation. Lipids. 2008;43(1):65–77. doi:10.
1007/s11745-007-3132-7.
9. Boden G, Sargrad K, Homko C, Mozzoli M, Stein T. Effect of a low-
carbohydrate diet on appetite, blood glucose levels, and insulin resistance
in obese patients with type 2 diabetes. Ann Intern Med. 2005;142(6):403–11.
10. Gumbiner B, Wendel J, McDermott M. Effects of diet composition and
ketosis on gly cemia during very-low-energy-diet therapy in obese
patients with non-insulin-dependent diabetes mellitus. Am J Clin Nutr.
1996;63(1):110–5.
11. Nielsen J, Joensson E. Low-carbohydrate diet in type 2 diabetes: stable
improvement of bodyweight and glycemic control during 44 months
follow-up. Nutr Metab. 2008;5:14. doi:10.1186/1743-7075-5-14.
12. Yancy W, Foy M, Chalecki A, Vernon M, Westman E. A low-carbohydrate,
ketogenic diet to treat type 2 diabetes. Nutr Metab. 2005;2:34. doi:10.1186/
1743-7075-2-34.
13. Dashti HM, Al-Zaid NS, Mathew TC, Al-Mousawi M, Talib H, Asfar SK, et al.
Long term effects of ketogenic diet in obese subjects with high cholesterol
level. Mol Cell Biochem. 2006;286(1–2):1–9. doi:10.1007/s11010-005-9001-x.
14. Maalouf M, Rho JM, Mattson MP. The neuroprotective properties of calorie
restriction, the ketogenic diet, and ketone bodies. Brain Res Rev. 2009;59(2):
293–315. doi:10.1016/j.brainresrev.2008.09.002.
15. Mavropoulos JC, Yancy WS, Hepburn J, Westman EC. The effects of a
low-carbohydrate, ketogenic diet on the polycystic ovary syndrome: a pilot
study. Nutr Metab. 2004;2:35. doi:10.1186/1743-7075-2-35.
16. Seyfried T, Flores R, Poff A, D’Agostino D. Cancer as a metabolic disease:
implications for novel therapeutics. Carcinogenesis. 2014;35:515–27. doi:10.
1093/carcin/bgt480.
17. Poff AM, Ari C, Arnold P, Seyfried TN, D’Agostino DP. Ketone supplementation
decreases tumor cell viability and prolongs survival of mice with metastatic
cancer. Int J Cancer. 2014;135:1711–20. doi:10.1002/ijc.28809.
18. Poff A, Ari C, Seyfried T, D’Agostino D. The ketogenic diet and hyperbaric
oxygen therapy prolong survival in mice with systemic metastatic cancer.
PLoS ONE. 2013;8(6), e65522. doi:10.1371/journal.pone.0065522.
19. Seyfried T, Shelton L. Cancer as a metabolic disease. Nutr Metab. 2010;7:7.
doi:10.1186/1743-7075-7-7.
20. Fine E, Segal-Isaacson C, Feinman R, Herszkopf S, Romano M, Tomuta N,
et al. Targeting insulin inhibition as a metabolic therapy in advanced
cancer: a pilot safety and feasibility dietary trial in 10 patients. Nutrition.
2012;28(10):1028–35. doi:10.1016/j.nut.2012.05.001.
21. Zhao Z, Lange D, Voustianiouk A, MacGrogan D, Ho L, Suh J, et al. A
ketogenic diet as a potential novel therapeutic intervention in amyotrophic
lateral sclerosis. BMC Neurosci. 2006;7:29. doi:10.1186/1471-2202-7-29.
22. White H, Venkatesh B. Clinical review: ketones and brain injury. Crit Care.
2011;15(2):219. doi:10.1186/cc10020.
23. Prins M. Cerebral metabolic adaptation and ketone metabolism after brain injury.
J Cereb Blood Flow Metab. 2008;28(1):1–16. doi:10.1038/sj.jcbfm.9600543.
24. Henderson S, Vogel J, Barr L, Garvin F, Jones J, Costantini L. Study of the
ketogenic agent AC-1202 in mild to moderate Alzheimer’s disease: a
randomized, double-blind, placebo-controlled, multicenter trial. Nutr Metab.
2009;6:31. doi:10.1186/1743-7075-6-31.
25. Brownlow M, Benner L, D’Agostino D, Gordon M, Morgan D. Ketogenic diet
improves motor performance but not cognition in two mouse models of
Alzheimer’s pathology. PLoS ONE. 2013;8(9), e75713. doi:10.1371/journal.
pone.0075713.g008.
26. Kossoff EH, Hartman AL. Ketogenic diets: new advances for metabolism-based
therapies. Curr Opin Neurol. 2012;25:173–8. doi:10.1097/WCO.0b013e3283515e4a.
27. Kwiterovich P, Vining E, Pyzik P, Skolasky R, Freeman J. Effect of a high-fat
ketogenic diet on plasma levels of lipids, lipoproteins, and apolipoproteins
in children. JAMA. 2003;290(7):912–20. doi:10.1001/jama.290.7.912.
28. Veech RL, Chance B, Kashiwaya Y, Lardy HA, Cahill Jr GF. Ketone bodies,
potential therapeutic uses. IUBMB Life. 2001;51(4):241–7. doi:10.1080/
152165401753311780.
29. D’Agostino D, Pilla R, Held H, Landon C, Puchowicz M, Brunengraber H,
et al. Therapeutic ketosis with ketone ester delays central nervous system
oxygen toxicity seizures in rats. Am J Physiol Regul Integr Comp Physiol.
2013;304(10):R829–36. doi:10.1152/ajpregu.00506.2012.
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 13 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

30. Gasior M, French A, Joy M, Tang R, Hartman A, Rogawski M. The anticonvulsant
activity of acetone, the major ketone body in the ketogenic diet, is not
dependent on its metabolites acetol, 1,2-propanediol, methylglyoxal, or pyruvic
acid. Epilepsia. 2007;48(4):793–800. doi:10.1111/j.1528-1167.2007.01026.x.
31. Likhodii S, Nylen K, Burnham W. Acetone as an anticonvulsant. Epilepsia.
2008;49 Suppl 8:83–6. doi:10.1111/j.1528-1167.2008.01844.x.
32. Seymour K, Bluml S, Sutherling J, Sutherling W, Ross B. Identification of
cerebral acetone by 1H-MRS in patients with epilepsy controlled by
ketogenic diet. Magma. 1999;8(1):33–42.
33. Halevy A, Peleg-Weiss L, Cohen R, Shuper A. An update on the ketogenic
diet, 2012. Rambam Maimonides Med J. 2012;3(1), e0005. doi:10.5041/RMMJ.
10072.
34. Amari A, Grace N, Fisher W. Achieving and maintaining compliance with
the ketogenic diet. J Appl Behav Anal. 1995;28(3):341–2. doi:10.1901/jaba.
1995.28-341.
35. Zhang Y, Kuang Y, LaManna J, Puchowicz M. Contribution of brain glucose
and ketone bodies to oxidative metabolism. Adv Exp Med Biol. 2013;765:
365–70. doi:10.1007/978-1-4614-4989-8_51.
36. Maalouf M, Sullivan P, Davis L, Kim D, Rho J. Ketones inhibit mitochondrial
production of reactive oxygen species production following glutamate
excitotoxicity by increasing NADH oxidation. Neuroscience. 2007;145(1):
256–64. doi:10.1016/j.neuroscience.2006.11.065.
37. Milder J, Patel M. Modulation of oxidative stress and mitochondrial function
by the ketogenic diet. Epilepsy Res. 2012;100(3):295–303. doi:10.1016/j.
eplepsyres.2011.09.021.
38. Shimazu T, Hirschey M, Newman J, He W, Shirakawa K, Le Moan N, et al.
Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone
deacetylase inhibitor. Science. 2013;339:211–4. doi:10.1126/science.1227166.
39. Kim DY, Davis L, Sullivan P, Maalouf M, Simeone T, van Brederode J, et al.
Ketone bodies are protective against oxidative stress in neocortical neurons.
J Neurochem. 2007;101(5):1316–26. doi:10.1111/j.1471-4159.2007.04483.x.
40. Veech R. The therapeutic implications of ketone bodies: the effects of
ketone bodies in pathological conditions: ketosis, ketogenic diet, redox
states, insulin resistance, and mitochondrial metabolism. Prostaglandins
Leukot Essent Fat Acids. 2004;70(3):309–19. doi:10.1016/j.plefa.2003.09.007.
41. Sato K, Kashiwaya Y, Keon C, Tsuchiya N, King M, Radda G, et al. Insulin, ketone
bodies, and mitochondrial energy transduction. FASEB J. 1995;9(8):651–8.
42. Kashiwaya Y, Sato K, Tsuchiya N, Thomas S, Fell D, Veech R, et al. Control of
glucose utilization in working perfused rat heart. J Biol Chem. 1994;269(41):
25502–14.
43. Sullivan P, Rippy N, Dorenbos K, Concepcion R, Agarwal A, Rho J. The
ketogenic diet increases mitochondrial uncoupling protein levels and
activity. Ann Neurol. 2004;55(4):576–80. doi:10.1002/ana.20062.
44. Bough K, Rho J. Anticonvulsant mechanisms of the ketogenic diet. Epilepsia.
2007;48(1):43–58. doi:10.1111/j.1528-1167.2007.00915.x.
45. Bough K, Wetherington J, Hassel B, Pare J, Gawryluk J, Greene J, et al.
Mitochondrial biogenesis in the anticonvulsant mechanism of the ketogenic
diet. Ann Neurol. 2006;60(2):223–35. doi:10.1002/ana.20899.
46. Ruskin D, Kawamura M, Masino S. Reduced pain and inflammation in
juvenile and adult rats fed a ketogenic diet. PLoS ONE. 2009;4(12), e8349.
doi:10.1371/journal.pone.0008349.
47. Paoli A, Moro T, Bosco G, Bianco A, Grimaldi KA, Camporesi E, et al. Effects
of n-3 polyunsaturated fatty acids (ω-3) supplementation on some
cardiovascular risk factors with a ketogenic Mediterranean diet. Marine
drugs. 2015;13(2):996–1009. doi:10.3390/md13020996.
48. Desrochers S, Dubreuil P, Brunet J, Jetté M, David F, Landau BR, et al.
Metabolism of (R, S)-1,3-butanediol acetoacetate esters, potential
parenteral and enteral nutrients in conscious pigs. Am J Physiol. 1995;
268(4 Pt 1):E660–7.
49. ClarkeK,TchabanenkoK,PawloskyR,CarterE,KnightN,MurrayA,etal.
Oral 28-day and developmental toxicity studies of (R)-3-hydroxybutyl (R)-3-
hydroxybutyrate. Regul Toxicol Pharmacol. 2012;63(2):196–208. doi:10.1016/j.
yrtph.2012.04.001.
50. Clarke K, Tchabanenko K, Pawlosky R, Carter E, Todd King M, Musa-Veloso K,
et al. Kinetics, safety and tolerability of (R)-3-hydroxybutyl (R)-3-
hydroxybutyrate in healthy adult subjects. Regul Toxicol Pharmacol. 2012;
63(3):401–8. doi:10.1016/j.yrtph.2012.04.008.
51. Warnick G, Knopp R, Fitzpatrick V, Branson L. Estimating low-density
lipoprotein cholesterol by the Friedewald equation is adequate for
classifying patients on the basis of nationally recommended cutpoints. Clin
Chem. 1990;36(1):15–9.
52. Friedewald W, Levy R, Fredrickson D. Estimation of the concentration of
low-density lipoprotein cholesterol in plasma, without use of the
preparative ultracentrifuge. Clin Chem. 1972;18(6):499–502.
53. Harlan Laboratories Inc U. Sprague Dawley Outbred Rat. In: http://www.
harlan.com/products_and_services/research_models_and_services/
research_models/sprague_dawley_outbred_rat.hl. 2008. http://www.harlan.
com/products_and_services/research_models_and_services/research_
models/sprague_dawley_outbred_rat.hl. Accessed date January 30, 2014.
54. Inc TB. Sprague Dawley Rat. In: http://www.taconic.com/sd. 2014. http://
www.taconic.com/user-assets/documents/spraguedawley_booklet.pdf.
Accessed date January 30, 2014.
55. McPherson P, McEneny J. The biochemistry of ketogenesis and its role in
weight management, neurological disease and oxidative stress. J Physiol
Biochem. 2012;68(1):141–51. doi:10.1007/s13105-011-0112-4.
56 Moore J, Eric C, Westman M. Cholesterol Clarity: What the HDL is Wrong
With my Numbers. Las Vegas: Victory Belt Publishing Inc; 2013.
57 Dekaban A. Plasma lipids in epileptic children treated with the high fat diet.
Arch Neurol. 1966;15(2):177–84.
58 Chesney D, Brouhard B, Wyllie E, Powaski K. Biochemical abnormalities of
the ketogenic diet in children. Clin Pediatr. 1999;38(2):107–9. doi:10.1177/
000992289903800207.
59. Schwartz R, Boyes S, Aynsley-Green A. Metabolic effects of three ketogenic diets
in the treatment of severe epilepsy. Dev Med Child Neurol. 1989;31(2):152–60.
60. Katyal N, Koehler A, McGhee B, Foley C, Crumrine P. The ketogenic diet in
refractory epilepsy: the experience of Children’s Hospital of Pittsburgh. Clin
Pediatr. 2000;39(3):153–9. doi:10.1177/000992280003900303.
61. Ellenbroek J, van Dijck L, Töns H, Rabelink T, Carlotti F, Ballieux B, et al.
Long-term ketogenic diet causes glucose intolerance and reduced beta and
alpha cell mass but no weight loss in mice. Am J Physiol Endocrinol Metab.
2014;306(5):E552–8. doi:10.1152/ajpendo.00453.2013.
62. Bergqvist A. Long-term monitoring of the ketogenic diet: Do’s and Don’ts.
Epilepsy Res. 2012;100(3):261–6. doi:10.1016/j.eplepsyres.2011.05.020.
63. Groesbeck D, Bluml R, Kossoff E. Long-term use of the ketogenic diet in the
treatment of epilepsy. Dev Med Child Neurol. 2006;48(12):978–81. doi:10.
1017/s0012162206002143.
64. Patel A, Pyzik P, Turner Z, Rubenstein J, Kossoff E. Long-term outcomes of
children treated with the ketogenic diet in the past. Epilepsia. 2010;51(7):
1277–82. doi:10.1111/j.1528-1167.2009.02488.x.
65. Brehm BJ, Seeley RJ, Daniels SR, D’Alessio DA. A randomized trial comparing
a very low carbohydrate diet and a calorie-restricted low fat diet on body
weight and cardiovascular risk factors in healthy women. J Clin Endocrinol
Metab. 2003;88(4):1617–23. doi:10.1210/jc.2002-021480.
66. Shai I, Schwarzfuchs D, Henkin Y, Shahar DR, Witkow S, Greenberg I, et al.
Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet. N Engl
J Med. 2008;359(3):229–41. doi:10.1056/NEJMoa0708681.
67. Volek JS, Phinney SD, Forsythe CE, Quann EE, Wood RJ, Puglisi MJ, et al.
Carbohydrate restriction has a more favorable impact on the metabolic
syndrome than a low fat diet. Lipids. 2009;44(4):297–309. doi:10.1007/
s11745-008-3274-2.
68. Feinman RD, Volek JS. Low carbohydrate diets improve atherogenic
dyslipidemia even in the absence of weight loss. Nutr Metab. 2006;3:24. doi:
10.1186/1743-7075-3-24.
69. Sharman MJ, Gomez AL, Kraemer WJ, Volek JS. Very low-carbohydrate and
low-fat diets affect fasting lipids and postprandial lipemia differently in
overweight men. J Nutr. 2004;134(4):880–5.
70. Sharman MJ, Kraemer WJ, Love DM, Avery NG, Gomez AL, Scheett TP, et al.
A ketogenic diet favorably affects serum biomarkers for cardiovascular
disease in normal-weight men. J Nutr. 2002;132(7):1879–85.
71. Westman EC, Mavropoulos J, Yancy WS, Volek JS. A review of low-
carbohydrate ketogenic diets. Curr Atheroscler Rep. 2003;5(6):476–83.
72. Wood RJ, Volek JS, Davis SR, Dell’Ova C, Fernandez ML. Effects of a
carbohydrate-restricted diet on emerging plasma markers for cardiovascular
disease. Nutr Metab. 2006;3:19. doi:10.1186/1743-7075-3-19.
73. Volek JS, Sharman MJ, Forsythe CE. Modification of lipoproteins by very
low-carbohydrate diets. J Nutr. 2005;135(6):1339–42.
74. Volek JS, Westman EC. Very-low-carbohydrate weight-loss diets revisited.
Cleve Clin J Med. 2002;69(11):849. 53, 56–8 passim.
75. Volek JS, Sharman MJ, Gomez AL, DiPasquale C, Roti M, Pumerantz A, et al.
Comparison of a very low-carbohydrate and low-fat diet on fasting lipids,
LDL subclasses, insulin resistance, and postprandial lipemic responses in
overweight women. J Am Coll Nutr. 2004;23(2):177–84.
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 14 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

76. Volek JS, Sharman MJ. Cardiovascular and hormonal aspects of very-low-
carbohydrate ketogenic diets. Obes Res. 2004;12 Suppl 2:115s–23. doi:10.
1038/oby.2004.276.
77. Volek JS, Sharman MJ, Gomez AL, Scheett TP, Kraemer WJ. An isoenergetic
very low carbohydrate diet improves serum HDL cholesterol and
triacylglycerol concentrations, the total cholesterol to HDL cholesterol ratio
and postprandial pipemic responses compared with a low fat diet in
normal weight, normolipidemic women. J Nutr. 2003;133(9):2756–61.
78. Schoeler NE, Wood S, Aldridge V, Sander JW, Cross JH, Sisodiya SM.
Ketogenic dietary therapies for adults with epilepsy: feasibility and
classification of response. Epilepsy & behavior : E&B. 2014;37:77–81. doi:10.
1016/j.yebeh.2014.06.007.
79. Sengupta P. The laboratory Rat: relating its Age with Human’s. Int J Prev
Med. 2013;4(6):624–30.
80. Tsuchiya N, Harada Y, Taki M, Minematsu S, Maemura S, Amagaya S. Age-related
changes and sex differences on the serum chemistry values in Sprague–Dawley
rats–I. 6–30 weeks of age. Exp Anim. 1995;43(5):671–8.
81. Saito K, Ishikawa M, Murayama M, Urata M, Senoo Y, Toyoshima K, et al.
Effects of sex, age, and fasting conditions on plasma lipidomic profiles of
fasted Sprague–Dawley rats. PLoS ONE. 2013;9(11):e112266. doi:10.1371/
journal.pone.0112266.
82. Ellington A, Kullo I. Atherogenic lipoprotein subprofiling. Adv Clin Chem.
2008;46:295–317.
83. Mudd J, Borlaug B, Johnston P, Kral B, Rouf R, Blumenthal R, et al. Beyond
low-density lipoprotein cholesterol: defining the role of low-density
lipoprotein heterogeneity in coronary artery disease. J Am Coll Cardiol.
2007;50(18):1735–41. doi:10.1016/j.jacc.2007.07.045.
84. Sacks F, Campos H. Clinical review 163: cardiovascular endocrinology :
Low-density lipoprotein size and cardiovascular disease: a reappraisal. J
Clin Endocrinol Metab. 2003;88(10):4525–32. doi:10.1210/jc.2003-030636.
85. Wierzbicki A. Quality as well as quantity? Beyond low-density lipoprotein-
cholesterol - the role of particle size. Int J Clin Pract. 2007;61(11):1780–2. doi:
10.1111/j.1742-1241.2007.01571.x.
86. Tantibhedhyangkul P, Hashim S, Van Itallie T. Effects of ingestion of long-chain
triglycerides on glucose tolerance in man. Diabetes. 1967;16(11):796–9. doi:10.
2337/diab.16.11.796.
87. Eckel R, Hanson A, Chen A, Berman J, Yost T, Brass E. Dietary substitution of
medium-chain triglycerides improves insulin-mediated glucose metabolism
in NIDDM subjects. Diabetes. 1992;41(5):641–7. doi:10.2337/diab.41.5.641.
88. Yost T, Erskine J, Gregg T, Podlecki D, Brass E, Eckel R. Dietary substitution of
medium chain triglycer ides in subjects with non-insulin-dependent
diabetes mellitus in an ambulatory setting: impact on glycemic control
and insulin-mediated glucose metabolism. J Am Coll Nutr. 1994;13(6):
615–22. doi:10.1080/07315724.1994.10718457.
89. Kashiwaya Y, Pawlosky R, Markis W, King MT, Bergman C, Srivastava S, et al.
A ketone ester diet increases brain malonyl-CoA and Uncoupling proteins 4
and 5 while decreasing food intake in the normal Wistar Rat. J Biol Chem.
2010;285(34):25950–6. doi:10.1074/jbc.M110.138198.
90. Senior B, Loridan L. Direct regulatory effect of ketones on lipolysis and on
glucose concentrations in man. Nature. 1968;219(5149):83–4.
91. Miles JM, Haymond MW, Gerich JE. Suppression of glucose production and
stimulation of insulin secretion by physiological concentrations of ketone bodies
in man. J Clin Endocrinol Metab. 1980;52(1):34–7. doi:10.1210/jcem-52-1-34.
92. Kristian HM, Thomas S, Niels HS, Thomas G, Gerrit van H. Systemic, cerebral
and skeletal muscle ketone body and energy metabolism during acute
hyper-D-β-hydroxybutyrataemia in post-absorptive healthy males. J Clin
Endocrin Metabol. 2014. doi:10.1210/jc.2014-2608
93. Owen OE, Morgan AP, Kemp HG, Sullivan JM, Herrera MG, Cahill GF. Brain
metabolism during fasting. J Clin Invest. 1967;46(10):1589–95. doi:10.1172/
JCI105650.
94. Papamandjaris AA, MacDougall DE, Jones PJ. Medium chain fatty acid
metabolism and energy expenditure: obesity treatment implications. Life
Sci. 1997;62(14):1203–15.
95. Linde R, Hasselbalch S, Topp S, Paulson O, Madsen P. Global cerebral blood
flow and metabolism during acute hyperketonemia in the awake and
anesthetized rat. J Cereb Blood Flow Metab. 2006;26(2):170–80. doi:10.1038/
sj.jcbfm.9600177.
96. Azzam R, Azar N. Marked seizure reduction after MCT supplementation.
Case reports in neurological medicine. 2013;2013:809151. doi:10.1155/2013/
809151.
97. Corwin RL. Binge-type eating induced by limited access in rats does not
require energy restriction on the previous day. Appetite. 2004;42(2):139–42.
doi:10.1016/j.appet.2003.08.010.
98. Keenan KP, Ballam GC, Dixit R, Soper KA, Laroque P, Mattson BA, et al. The
effects of diet, overfeeding and moderate dietary restriction on Sprague–
Dawley rat survival, disease and toxicology. J Nutr. 1997;127(5 Suppl):851S–6.
99. Keenan KP, Smith PF, Hertzog P, Soper K, Ballam GC, Clark RL. The effects of
overfeeding and dietary restriction on Sprague–Dawley Rat survival and
early pathology biomarkers of aging. Toxicol Pathol. 1994. doi:10.1177/
019262339402200308.
100. Kashiwaya Y, Bergman C, Lee J-H, Wan R, King M, Mughal M, et al. A ketone
ester diet exhibits anxiolytic and cognition-sparing properties, and lessens
amyloid and tau pathologies in a mouse model of Alzheimer’s disease.
Neurobiol Aging. 2013;34(6):1530–9. doi:10.1016/j.neurobiolaging.2012.11.023.
101. Birkhahn R, McCombs C, Clemens R, Hubbs J. Potential of the
monoglyceride and triglyceride of DL-3-hydroxybutyrate for parenteral
nutrition: synthesis and preliminary biological testing in the rat. Nutrition.
1997;13(3):213–9. doi:10.1016/s0899-9007(96)00404-2.
102. Puchowicz M, Smith C, Bomont C, Koshy J, David F, Brunengraber H.
Dog model of therapeutic ketosis indu ced by oral administration of R,
S-1,3-butanediol diacetoacetate. J Nutr Biochem. 2000;11(5):281–7.
103. Brunengraber H. Potential of ketone body esters for parenteral and oral
nutrition. Nutrition. 1997;13(3):233–5.
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