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Chronic ketosis and cerebral metabolism.

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Chronic Ketosis and Cerebral Metabolism
Darryl C. DeVivo, MD, Mary P. Leckie, BS, James S. Ferrendelli, MD, and David B. McDougal, Jr, MD
T h e effects of chronic ketosis on cerebral metabolism were determined in adult rats maintained on a high-fat diet for
approximately three weeks and compared to a control group of animals. T h e fat-fed rats had statistically significantly
lower blood glucose concentrations and higher blood p-hydroxybutyrate and acetoacetate concentrations; higher
brain concentrations of bound glucose, glucose 6-phosphate, pyruvate, lactate, P-hydroxybutyrate, citrate,
a-ketoglutarate, alanine, and adenosine triphosphate (ATP); lower brain concentrations of fructose 1,6-diphosphate,
aspartate, adenosine diphosphate (ADP), creatine, cyclic nucleotides, succinyl coenzyme A (CoA), acid-insoluble
CoA, and total CoA; and similar brain concentrations of glucose, malate, calculated oxaloacetate, glutamate,
glutamine, adenosine monophosphate, phosphocreatine, reduced CoA, acetyl CoA, sodium, potassium, chloride, and
water content. T h e metabolite data in the chronically ketotic rats demonstrate an increase in t h e cerebral energy
reserve and energy charge. These data also suggest negative modification of the enzymes phosphofructokinase,
pyruvic dehydrogenase, and a-ketoglutaric dehydrogenase; positive modification of glycogen synthase; and possible augmentation of the hexose transport system. There was no demonstrable difference in brain pH, water content,
or electrolytes i n the two groups of animals. We speculate that the increased brain ATP/ADP ratio is central to most, if
not all, the observed metabolic perturbations and may account for the increased neuronal stability that accompanies
chronic ketosis.
DeVivo DC, Leckie MP, Ferrendelli JS, et al: Chronic ketosis and cerebral metabolism.
Ann Neurol 3:331-337, 1978
T h e anticonvulsant property of a ketogenic diet is well
recognized, but t h e mechanism underlying the effect
remains obscure. Our laboratory has reported previously on an experimental model using rats to study the
high-fat diet El]. Using this animal model w e have
demonstrated an increase in the convulsive threshold
to minimal electroconvulsive shock. We report here
the results of similar experiments designed to characterize various perturbations in cerebral intermediary
metabolism. Some of these findings have been presented previously [7].
Materials and Methods
All reagents used in the substrate assays were of the highest
analytical grade available. Sprague-Dawley rats were maintained on high-fat feedings for 20 days as described previously [ 11. The control animals consumed Wayne Lab-Blox
(Allied Mills, Chicago) ad libitum and were narcotized twice
daily to simulate the gavage feeding conditions. All animals
gained weight during the experiment. The control rats
ranged in weight from 225 to 390 gm when killed. The
high-fat-fed rats grew from 258 & 6 to 274 ? 3 gm during
the feeding period. All rats were housed, 3 per cage, in
air-conditioned animal quarters with alternating twelvehour periods of light and dark. Animals were allowed free
access to water, and the cage bedding was changed daily.
The animals were killed utilizing the freeze-blowing techFrom the Edward Mallinckrodt Department of Pediatrics and the
Departments Of
and Neurosurgery (Neurol"6Y) and
Pharmacology, Washington University School of Medicine, and the
Division of Neurology, St. Louis Children's Hospital, St. Louis,
MO.
nique described by Veech et a1 [36].The brain tissue was
powdered in liquid nitrogen and stored at -80°C until extracted. Acid-methanol-perchlorate extracts were prepared
from the brain powder [ 2 3 ] for direct enzymatic microfluorometric determination of glucose, glucose 6phosphate (G-6-P), fructose 1,G-diphosphate (F-1,6-P2),
pyruvate, lactate, citrate, a-ketoglutarate (a-KG), malate,
adenosine triphosphate (ATP), adenosine diphosphate
(ADP), adenosine monophosphate (AMP), phosphocreatine (PCr), creatine (Cr), and aspartate [19]; for glutamate
and glutamine [ 2 5 ] ; for alanine [13]; and for phydroxybutyrate (P-OHB) [391. The residue from this
perchloric acid extraction was acid hydrolyzed with 1 N
hydrochloric acid to liberate glucose as an indirect measure
of the glycogen content [9]. Cyclic nucleotides were extracted from frozen brain powder and assayed according to
the method of Steiner et a1 [341. Coenzyme A (CoA) and
several of its esters were measured using a modification of
the phosphotransace tylase cycling method of Stadtman,
Novelli, and Lipmann [24, 331 coupled with methods designed to destroy selected CoA esters and free CoA specifically (McDougal, DB Jr, Dargar R: unpublished data). The
entire set of CoA assays requires only 20 to 30 mg of brain.
Acetyl CoA is identified by its specific reaction with
oxaloacetate and citrate synthase. Succinyl CoA is identified
by its reaction with guanosine diphosphate and succinic
thiokinase in the presence of arsenate. Acid-insoluble CoA
includes (long-chain) fatty acyl-CoA esters which are insoluble in perchloric acid.
Accepted for publication Nov 3. 1977.
Address reprint requests Dr DeVjvo, St. Louis
tal, 5oo Kingshighway, St. Louis, MO 6 3 178,
~ ~ 6 4 - 5 ~ 3 4 / ~ 8 / 0 ~ 0 3 - 0 4 0 9@
$ 0I978
~ . 2 5by Darryl C. DeVivo
Hasp,-
331
Blood samples were collected by cardiac puncture immediately after removal of the brain tissue, added to an
equal volume of 3 M perchloric acid, and prepared for assay
of glucose, /3-OHB, and acetoacetate (AcAc) as outlined
previously [8].
Brain sodium, potassium, chloride, and water content also
were determined on a fraction of the frozen brain powder.
The brain powder was placed in a preweighed test tube,
weighed, and placed in an oven at 105°C for 48 hours before
being capped in a desiccator jar for 24 hours and reweighed.
The process was repeated to ensure that a dry weight had
been achieved. The dried tissue was digested with 10 vol
Cwlv) of 0.75 N nitric acid as described previously [ 8 ] for
determination of Na, K, and CI.
Several equations were used to calculate substrate concentrations or metabolic parameters. Oxaloacetate (Oxal)
concentrations were derived from the following equations:
Oxaloacetate = K ~ , l ) i l [Malate] .
Oxaloacetate
=
"AD+](.
[NADH&
[LY-KG][aspartate]
I?;, )T [glu raniate]
&,,,,.
where KMDH
= 278 X lo-' and
= 6.01 at pH 7 . 0 [151.
The cytoplasmic oxidation-reduction potential was calculated from Equation 3
LNAD+](. =
[Pyruvate]
K, 1 ) ~[lactate]
~
[NADH,],.
(3)
where KI.I)H= 111 x IO-" at p H 7.0 [15]. The cerebral
energy charge [ 2 ] was calculated as follows:
Energy charge = 0.5 ( 2 ATP + ADP)
ATP + ADP + AMP
According to this equation che hydrogen ion concentration
is directly proportional to the mass action ratio. A rise in
brain tissue p H therefore is associated with a fall in the mass
action ratio.
Results
Ail animals remained in a n anabolic state t h r o u g h o u t
t h e e x p e r i m e n t as judged by a gain in b o d y weight.
C o n s u m p t i o n of a high-fat d i e t p r o d u c e d a significant
( p < 0.005) decrease in blood glucose and increases in
t h e blood P-OHB and A c A c concentrations (Table 1).
T h e brain electrolytes ( N a , K, C1) a n d water c o n t e n t
w e r e the s a m e in t h e control and high-fat-fed g r o u p s
(Table 2). Several significant differences in cerebral
metabolites w e r e noted b e t w e e n t h e two groups, as
s h o w n i n T a b l e 3. T h e b o u n d glucose (reflecting
glycogen content), G-6-P, pyruvate, lactate, /3-OHB,
citrate, a-KG, alanine, and ATP concentrations w e r e
significantly increased in t h e high-fat-fed g r o u p ;
F-1 ,6-P2, ADP, aspartate, succinyl CoA, acidinsoluble CoA, total CoA, a n d t h e cyclic nucleotides
w e r e significantly decreased in t h e high-fat-fed g r o u p ;
and f r e e glucose, malate, glutamate, glutamine, calculated oxaloacetate, A M P , P C r , r e d u c e d C o A , and
acetyl CoA w e r e similar in t h e two g r o u p s ( s e e T a b l e
3).
The cerebral lactate/pyruvate ratio a n d t h e cytoplasmic oxidation-reduction potential w e r e similar in
t h e t w o g r o u p s of animals (Table 4). T h e cerebral
energy charge a n d t h e ATPlADP ratio, cerebral
Tuble 2. Efferts of Diet on Bruin
Electrolytes and Water Content
The cerebral energy reserve [ 2 0 ]was calculated from Equarion 5 :
Energy reserve = (PCr + ADP) +
2 (ATP glucose)
+
+ 2.9 (glycogen)
(5)
Brain pH was estimated from the mass action ratio of the
creatine phosphokinase reaction [3 11 as derived from Equation 6:
KVIVK
=
[Crl[ATPl
[PCr] [ADP]
1
[H']
Determination
Control
(N = 22)
High-Fat
Diet
( N = 13)
Sodium
Potassium
Chloride
Water (%)
44.7
97.6
32.0
79.3
44.7 ? 0 . 5 N S
98.7 +- 0.8 N S
31.7 ? 0.3 N S
78.9 ? 0.2 NS
2
2
2
2
0.4
0.8
0.3
0.2
Significance
Values represent mean 2 SEM for the dumber of animals indicated.
Electrolytes are expressed as milliequivalents per kilogram of wet
[issue.
NS = not significant.
Table I . Effpcts of Diet on Blood Gfucooseand Ketone Body Concentration.(
Metabolite
Control
( N = 22)
High-Fat
Diet
( N = 13)
Significance
Glucose
/3-H ydroxy butyrate
Acetoacetate
7,080 2 142
117 2 8
73
4
6,290 2 218
578 2 39
111
10
p < 0.005
p < 0.005
p < 0.00s
*
Values are in micromoles per liter and represent mean
332
Annals of Neurology
Vol 3 No 4
%
*
SEM for the number of animals indicated.
April 1978
Tablr
3 . Effects of Diet on Vurious Rrain
Metabolites
Metabolite
Control (N= 2 2 )
High-Fat Diet
(N = 1 3 )
Glucose [free)
Glucose (bound)
G-6-P
F- 1,6-P2
Pyruvate
Lactate
2,130
3,400
156
25.3
107
1,560
13.5
298
189
279
4.62
6.71
680
2,640
11,270
4,870
2,440
681
68
3,995
5,620
2.92
0.068
15.36
2.85
4.01
43.6
69.9
2,290
4,130
175
18.5
132
1,990
34.8
314
209
272
4.55
6.34
730
2,270
11,270
5,140
2,600
640
62
3,990
5,380
2.58
0.054
14.80
2.80
2.98
38.0
62.9
P-OHB
Citrate
a-KG
Malate
(OxalhDH
!Oxal),,,
Alanine
Aspartate
Glutamate
Glutamine
ATP
ADP
AMP
PCr
Cr
Cyclic AMP
Cyclic G M P
Reduced CoA
Aceryl CoA
Succinyl CoA
Acid-insoluble CoA
Total CoA
t 63
2
50
t
t
2
1.2
3
2 39
t
3.0
2
2
t
2
t
?
f
2
t
2
2
t
2
*
f
f
?
t
t
f
f
t
5
2
4
0.17
0.12
20
60
130
110
22
14
3
35
70
0.11
0.008
0.41
0.10
0.28
1.2
1.8
Cerebral metabolites expressed as micromoles per kilogram of wet tissue
?
Significance
f 84
*
90
4
1.2
f 4
5 60
f
2.4
?
3
t 4
_f
*
NS
< 0.005
< 0.005
< 0.005
< 0.005
< 0.005
< 0.005
< 0.005
< 0.005
p
p
p
p
p
p
p
p
f
NS
NS
*
p < 0.05
p < 0.05
p < 0.005
7
t
0.16
f 0.17
f 30
50
2 90
f 150
t 14
f 14
2
3
f 65
t 30
t
0.08
f
0.003
t 0.72
t
0.08
f
0.18
t
1.0
f
1.3
NS
NS
p < 0.005
p < 0.05
NS
NS
p < 0.025
p < 0.025
p < 0.005
NS
NS
p < 0.005
p < 0.05
p < 0.005
SEM
G-6-P = glucose 6-phosphate; F- 1,6-P2= fructose 1,B-diphosphate;P-OHB = P-hydroxybutyrate; u - K G = cr-ketoglutaratc; ( ~ x a l ) ~ , =
,,,
oxaloacetate calculated from equation 1; (oxal),,,,,, = oxaloacetate calculated from equation 2; ATP = adenosine triphosphate; ADP =
adenosine diphosphate; A M P = adenosine monophosphate; PCr = phosphocreatine; Cr = creatine; G M P = guanine monophosphate; CoA =
coenzyme A; NS = not significant.
Table 4. Effects of Diet
011
Cerebral Metabolism
Determination
(N
Control
= 22)
tN
Lactatelpyruvate
~NAD+l,./[NADH,I,
Energy charge
ATPl ADP
Energy reserve
Braidblood glucose
[Crl [ATPI
[PCrl [ADPI
14.8 t 0.3
617
2 14
0.872 t 0.003
3.63 2 0.10
t 0.2
23.6
0.303 2 0.011
601
0.884
4.08
26.4
0.367
5.20 t 0.17
High-Fat Diet
= 13)
15.1
t 0.3
f 13
f 0.002
f 0.10
t 0.6
5
0.014
5.52 t 0.17
Significance
NS
NS
p < 0.005
p < 0.005
p < 0.005
p < 0.005
NS
“AD+], = nicotinamide-adenine dinucleotide; [NADH,],. = nicotinarnide-adenine dinucleotide, reduced state; ATP = adenosine triphosphate; ADP = adenosine diphosphate; C r = creatine; PCr = phosphocreatine; NS = not significant.
DeVivo et al: Ketosis and Cerebral Metabolism
333
energy reserve, and brain glucose/blood glucose ratio
were significantly higher (p < 0.005) in the high-fatfed group. The creatine phosphokinase mass action
ratio was similar in the two groups of animals.
Discussion
Our observations in this study lend themselves t o
several interpretations. First, we noted a significant
elevation in the brain concentrations of glycogen
(bound glucose) and G-6-P and a significant increase
in the brain glucose/blood glucose ratio in the highfat-fed animals (see Table 3). G-6-P is the product of
the reaction catalyzed by hexokinase (Figure). This
enzyme reaction is subject to product inhibition, and
as such it controls the flux of glucose into the brain
pool of phosphorylated intermediates [4]. G-6-P also
influences glycogen synthesis by acting as a positive
modifier of the dependent (or active) form ofglycogen
synthase, the rate-limiting reaction involving formation of glycogen [ 181. The increased concentrations of
G-6-P and ATP therefore may partly explain the 22%
increase in brain glycogen (see Table 3 ) that we observed in the high-fat-fed group. The increased brain
glucose/blood glucose ratio in the high-fat-fed group
is the consequence of a significantly lower blood glucose concentration (see Table 1) and a higher (although not statistically significant) brain glucose concentration (see Table 3). This altered ratio could be
interpreted as evidence either for slower phosphorylation of brain glucose (for the reasons stated above)
or for enhanced transport of glucose from the blood
into the brain. Glucose transport across the bloodbrain barrier is known to be carrier mediated (facilitation), with a Michaelis-Menten constant (K,) of approximately 5 mM [26]. At the moment there is no
evidence that the hexose transport system across the
blood-brain barrier can be induced by nutritional
states. However, augmentation of another transport
system (for monocarboxylic acids) during fasting has
recently been demonstrated [51. Movement of ketone
bodies into the brain is dependent upon the monocarboxylic transport system. The design of our present
study does not permit us to discriminate between
these two possibilities as explanations for the elevated
brain glucose/blood glucose ratio in the high-fat-fed
group.
The second set of observations focuses on the brain
concentrations of G-6-P, F-l ,6-P2, pyruvate, lactate,
alanine, acetyl CoA, citrate, a-KG, and succinyl CoA.
These metabolites represent reactants of important
regulatory enzymes controlling glycolysis and oxidative metabolism. G-6-P and fructose 6-phosphate are
at equilibrium; as such, either metabolite reflects the
activity of phosphofructokinase, the key regulatory
enzyme in glycolysis. F-l,6-P2 is the product of this
reaction (see the Figure). An increase in the G-6-P
334 Annals of Neurology Vol 3 No 4 April 1978
concentration and decrease in the F-1 ,6-Pz concentration as seen in the high-fat-fed group (Table 3) suggest
negative modification of phosphofructokinase. The
increased ATP and citrate concentrations we observed may be important in this regard since both
intermediates are negative modifiers of this reaction
[20, 301. The similarity between the creatine
phosphokinase mass action ratios (see Table 4 )
suggests that a decrease in brain p H is not a plausible
explanation for the observed inhibition of phosphofructokinase in the high-fat-fed group of animals.
By similar reasoning, increased concentrations of
pyruvate and a - K G suggest decreased activities of
pyruvate dehydrogenase and a-ketoglutaric dehydrogenase, respectively (see the Figure). These two
multienzyme reaction sequences are tightly regulated
and share some biochemical similarities, as has been
discussed recently [28, 3 2 , 371. The pyruvic dehydrogenase ( P D H ) complex is regulated by a phosphorylation-dephosphorylation mechanism [37]. The
dephosphorylated (active) form of the complex is inactivated by a phosphorylation reaction catalyzed by
the enzyme P D H kinase; conversely, dephosphoryla-
The metabolic relationships betuwen glycolysis, the tricarboxylicacid cycle, and ketone body oxidation. Thepercentages in parentheses indicate significant increases (+) or decreases ( -) of the
respectivebrain substrates in the high-fat-fed rats compared
udth the control rats. Enzymes referred t o i n the text are indicated by the letters: (a) = hexokinase; (b) = phosphofructokinase; (c) = pyrucic dehydrogenase: (d) = glutamir-pyruiiic
transaminase; ( e )= citvate synthase; ( f ) = a-ketoglutaric dehydrogenase; ( g )= 3-oxoacid-CoA transferase.
GLYCOGEN ( + 2 2 % )
G LUCOS E
-0
0
-
G 6 - P ( + 12 %)
F - 1 t, 6 - P 2 ( - 2 7 % )
(-14%) ASP
CoA
M ALATE
CIT R ATE (+5% )
J
a-KG (+I1%)
t
SUCCINATE
t
s UCC I N Y L - Co A (- 2 6 %)
(+ I 57 % P O H B
T
tion of the inactive form is catalyzed by the enzyme
P D H phosphatase. P D H kinase is inhibited by A D P
and therefore is dependent upon the ATP/ADP ratio
[37]. The higher ATP/ADP ratio which we observed
in the high-fat-fed group may explain the apparent
inhibition of the P D H complex. T h e acetyl CoA/CoA
ratio also controls activity of the PDH complex. However, we observed no significant difference in this
ratio between the two groups of animals. T h e acetyl
CoA concentrations, in fact, were very similar. This
observation probably reflects the alternate synthesis
of acetyl CoA fromP-OHB and AcAc (see the Figure)
to compensate for the presumed decreased synthesis
of acetyl CoA from pyruvate. The higher alanine concentrations in the high-fat-fed group apparently represent a shift in the mass action ratio of the reaction
catalyzed by glutamic-pyruvic transaminase associated
with increased pyruvate concentrations (see the Figure). Similarly, lactate concentrations increased in
parallel with pyruvate concentrations in the high-fatfed group, thereby maintaining a relatively constant
lactate/pyruvate ratio. This observation also suggests
that the cytoplasmic oxidation-reduction potential is
not perturbed by chronic anabolically induced ketosis
as opposed to acute starvation-induced ketosis [ 8 ] .
The elevated a-KG and decreased succinyl CoA
concentrations imply a block at the enzymatic step
catalyzed by cu-ketoglutaric dehydrogenase (see the
Figure). Recent evidence suggests that the succinyl
CoA/CoA ratio is the major regulating mechanism for
this reaction, with little o r no influence being exerted
by the guanine or adenine nucleotides [32]. In our
study the succinyl CoA/CoA ratio was lower in the
high-fat-fed group (0.203 & 0.02 1 vs 0.248 2 0.017;p
< 0.10) and cannot be invoked as the mechanism of
enzyme inhibition under these experimental conditions. The pyridine nucleotide ratio also exerts a regulatory effect upon this reaction. Unfortunately, the
cerebral intramitochondrial oxidation-reduction potential is difficult to estimate for reasons that have
been reviewed recently [2 13. The undetectable brain
concentrations of AcAc preclude estimation of
the intramitochondrial redox potential using the
P-hydroxybutyric dehydrogenase reaction. One might
anticipate that the mitochondria1 redox potential
could shift to a more oxidized state if pyruvate is
partially replaced by /3-OHB or AcAc as a fuel source
for oxidative metabolism [8]. Such a redox shift, however, would favor rather than inhibit a-ketoglutaric
dehydrogenase activity. O u r inability to suggest a
mechanism that might inhibit the a-ketoglutaric dehydrogenase complex might imply that the statistically
significant elevation of a-KG (see Table 3) is biologically unimportant. The identical concentrations of
brain glutamate and similar concentrations of brain
y-aminobutyric acid [ 11 observed in the two groups of
rats are consistent with this formulation. The relatively greater decrease in the mean succinyl CoA concentration might reflect greater conversion of this
metabolite to succinate via the reaction catalyzed
by the enzyme 3-oxoacid-CoA transferase during
chronic ketosis. As shown in the Figure, succinyl CoA
is essential for the metabolism of AcAc.
An increased availability of NAD+ might facilitate
the pyridine nucleotide-dependent reactions in the
citric acid cycle involved in the oxidation of various
intermediates. The potential advantage of this speculation in relation to malic dehydrogenase is to further
counter the unfavorable kinetics of malate oxidation
to oxaloacetate, thereby augmenting intramitochondrial oxaloacetate formation and the malate-aspartate
shuttle. Availability of oxaloacetate as a cosubstrate
probably is the most important mechanism controlling
the velocity of citrate synthase [17, 381. The K,,, for
oxaloacetate varies from approximately 1 to 1 6 p M in
most biological systems. The velocity of the reaction
therefore is probably linearly related to the concentration of oxaloacetate, and in view of the similarities in
the calculated concentrations of oxaloacetate, this rate
might be expected not to differ greatly in the two
groups of animals. Clearly, appropriate experiments
need to be designed to assess the credibility of these
speculations.
The third set of observations focuses on the energy
state of the tissue. We noted no change in the PCr
concentrations, a significant increase in ATP, and a
significant decrease in A D P (see Table 3). Expressed
as energy charge or as ATP/ADP ratio, these observations can be translated into a favorable shift in the
cellular energy state as shown in Table 4. Perhaps
more meaningful (in the sense that the calculations
also include glycogen and PCr) is the observed significant increase in cerebral energy reserve (Table 4).
This observation suggests that the brain may be
metabolically more stable from moment to moment
and less easily compromised by subtle fluctuations in
glucose availability or body temperature. For this reason we suggest that the anticonvulsant property of a
ketogenic diet derives from the observed increase in
cerebral energy reserve, which in turn reflects the
higher ATP/ADP ratio in the high-fat-fed group.
The anticonvulsant effect of a ketogenic diet
evolves slowly over several days but can be obliterated
in three hours [35]o r one to two days [ 11with resumption of an antiketogenic regimen. The metabolic adaptation after a ketogenic diet is begun also parallels the
augmentation of the monocarboxylic acid transport
system [51, further supporting our contention that
ketone body utilization is fundamental to the anticonvulsant effect of a ketogenic diet. W e presume that the
temporal profile describing the rise in convulsive
threshold after the onset of ketosis reflects cerebral
DeVivo et al: Ketosis and Cerebral Metabolism 335
metabolic adaptation to ketone body utilization. The
biochemical mechanism or mechanisms underlying
the increased ATP/ADP ratio, however, remain to be
defined.
It should be noted that similar metabolic perturbations have been defined in other tissues during
ketosis, including heart [27] and diaphragm [3], and
that this intracellular change may underlie the carbohydrate intolerance that develops during starvation o r
high-fat feeding [ l o , 161. Relevant studies have also
been carried out using cerebral slices from animals
[12, 291. Rolleston and Newsholme [29] demonstrated that j3-OHB decreased the rate of glucose
oxidation without influencing the rate of glucose utilization by guinea pig cerebral cortex slices. The increased lactate production by the tissue under these
conditions was equivalent to a glucose-sparing effect.
Similar effects of P-OHB on glucose metabolism by
rat cerebral slices were observed by Ide et a1 [121.
Our findings can be compared with the observed
biochemical effects of various anticonvulsants including phenobarbital [ 141 and trimethadione, ethosuximide, and chlordiazepoxide [22]. In general, these
anticonvulsant medications have similar apparent
effects on cerebral glucose transport, glycogen
synthesis, and glycolysis. However, these agents appear to behave like sedatives by depressing some citric
acid cycle intermediates. This latter effect is the opposite of our observations in the high-fat-fed group, in
which we noted increased cerebral concentrations of
citrate and a-KG and similar levels of malate and
calculated oxaloacetate. The animals maintained on
the high-fat diet did not have a significant alteration in
brain p H as determined by the reactants involved in
the reaction catalyzed by creatine phosphokinase (see
Table 4); nor did we see any important changes in the
brain tissue content of water, sodium, potassium, o r
chloride (see Table 2). It would appear therefore that
presumed alterations of brain tissue p H , electrolytes,
or water content cannot be invoked to explain the
anticonvulsant effect of a high-fat diet.
Whether ketosis affects the rate of cerebral metabolism remains unanswered. The decreased concentrations of cyclic nucleotides (see Table 3 ) and the
increased ATP/ADP ratio could be interpreted as evidence for depression of cerebral metabolism. However, a recent study of rat brain metabolism during
fasting demonstrated no major differences in cerebral
blood flow or cerebral oxygen consumption [6].Experimental studies designed to evaluate the profile of
citric acid cycle intermediates under various metabolic
conditions have revealed some distinctive changes
[ 111. Hyperthermia was associated with elevated brain
concentrations of pyruvate, a-KG, and malate; ether
anesthesia was associated with decreased levels of
pyruvate, a-KG, and malate; and anoxic ischemia was
336 Annals of Neurology Vol 3 No 4 April 1978
associated with elevated concentrations of pyruvate
and malate and a reduced concentration of a-KG. The
cerebral metabolic profile we observed in the chronically ketotic rats (elevated pyruvate and a-KG and
unchanged malate concentrations) differs from each
of these three metabolic conditions, allowing us to
suggest that chronic ketosis neither increases nor decreases cerebral metabolic oxygen consumption.
Further studies are in progress to clarify the effect of
ketosis on brain metabolism in the rat model used in
the present study. In any event, it appears that a rise in
the ATP/ADP ratio is central to most, if not all, of the
metabolic perturbations described in our study and
might provide a reasonable explanation for the increased neuronal stability that develops during
chronic ketosis.
Supported in part by Public Health Service Grants NS09808,
NS06800, and NS13104.
References
1. Appleton DB, DeVivo DC: An animal model for the
ketogenic diet. Epilepsia 15:2 11-227, 1974
2. Atkinson DE, Walton GM: Adenosine triphosphate conservation in metabolic regulation: rat liver citrate cleavage enzyme. J
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