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The acute effect of valproate on cerebral energy metabolism in mice
Epilepsy Research 47 (2002) 247– 256 www.elsevier.com/locate/epilepsyres
The acute effect of valproate on cerebral energy metabolism in mice Cecilie U. Johannessen a,*, Dirk Petersen b, Frode Fonnum a, Bjørnar Hassel a a
Norwegian Defence Research Establishment, N-2027 Kjeller, Norway b Department of Chemistry, Uni6ersity of Oslo, Oslo, Norway
Received 15 June 2001; received in revised form 3 September 2001; accepted 3 September 2001
Abstract Sodium valproate (VPA) is used in the acute treatment of status epilepticus and mania. We studied the acute effect of VPA on cerebral energy metabolism in awake mice that received VPA 400 mg kg − 1 and [1-13C]glucose or [2-13C]acetate. At 25 min, 13C NMR spectroscopy of brain extracts indicated inhibition of the tricarboxylic acid (TCA) cycle, as could be seen from the accumulation of [4-13C]glutamate and reduction in [13C]aspartate formation. Concomitantly, the level of ATP was reduced by 40%. To identify the enzymatic step at which the TCA cycle was inhibited [U-14C]a-ketoglutarate was injected intracerebrally. Inhibition of a-ketoglutarate dehydrogenase was evident at 25 min, as shown by accumulation of [14C]glutamate. At 45 min the inhibition of a-ketoglutarate dehydrogenase was reversed, shown by both 13C- and 14C-labeling, and the ATP level was normalized. The study shows for the first time that acute administration of VPA causes inhibition of the TCA cycle activity in vivo. The reduction in brain ATP would be expected to reduce neuronal excitability through modulation of sodium channels which may be clinically advantageous in the initial phase of VPA treatment. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Valproate; Cerebral metabolism; Tricarboxylic acid cycle; a-Ketoglutarate dehydrogenase; ATP
1. Introduction Sodium valproate (VPA) is used in high doses in the acute treatment of serial- or prolonged epileptic seizures, status epilepticus, and manic episodes (Price, 1989; Brennan et al., 1994; Gruenze et al., 1999; Naritoku and Mueed, 1999). Abbre6iations: SSA-DH, succinic semialdehyde dehydrogenase; TCA cycle, tricarboxylic acid cycle; VPA, valproate. * Corresponding author. Fax: + 47-6380-7509. E-mail address: [email protected] (C.U. Johannessen).
Studies with positron emission tomography in VPA-treated patients have indicated inhibition of the cerebral metabolism of glucose (Leiderman et al., 1991; Gaillard et al., 1996). These findings raise the question of whether VPA inhibits a specific enzymatic step in the energy metabolism in vivo. In vitro, VPA and metabolites inhibit a-ketoglutarate dehydrogenase (Luder et al., 1990) and enzymes related to GABA metabolism (for review see Lo¨scher, 1999). Such inhibition in vivo could affect cerebral energy production and thereby neuronal activity.
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The use of 13C-labeled energy substrates in conjunction with 13C NMR spectroscopy gives the opportunity to investigate in vivo metabolism in some detail. Glucose has previously been shown to be metabolized predominantly through the neuronal tricarboxylic acid (TCA) cycle, whereas acetate is predominantly metabolized through the glial TCA cycle (Van den Berg et al., 1969; Minchin and Beart, 1974). Analysis of the 13C-labeling of metabolites of [1-13C]glucose or [213 C]acetate gives a detailed picture of neuronal and glial energy metabolism, respectively (Cerdan et al., 1990; Ku¨ nnecke et al., 1993; Shank et al., 1993; Hassel et al., 1997). The aim of the present study was to investigate the acute effect of VPA on cerebral energy metabolism. 13C NMR spectroscopic analysis was carried out on brain extracts of VPA-treated mice that received [1-13C]glucose or [2-13C]acetate as labeled precursors of energy metabolites. The effect of VPA on ATP levels was compared to that of vigabatrin, a specific inhibitor of the GABA shunt. Metabolism of [U-14C]a-ketoglutarate was evaluated after intracerebral administration, to study the effect of VPA on a-ketoglutarate dehydrogenase specifically.
2. Materials and methods
2.1. Materials Female Hsd-Ola ICRF mice, 20 g body weight, (Harlan, UK) were kept in groups of 10 per cage under conditions of constant temperature (21 °C) and humidity (50%), with a 12-h light, 12-h dark cycle and free access to food and water. VPA, [2-13C]acetate and [1-13C]glucose (both 99% 13C enrichment), and reagents for enzymatic measurement of ATP and PCr, were obtained from Sigma (St Louis, MO, USA). [U-14C]a-Ketoglutarate (240 mCi mmol − 1) was obtained from American Radiolabeled Chem. Inc. (St Louis, MO, USA), and vigabatrin was a gift from Marion Merrel Dow (Uxbridge, UK). Before experiments, VPA was dissolved in water to a concentration of 24 mg ml − 1. Vigabatrin was dissolved in water to a concentration of 60
mg ml − 1. [1-13C]Glucose and sodium [213 C]acetate were dissolved in double-distilled water to a concentration of 500 mmol − 1, and pH of all solutions was adjusted to 7.4. The ethanol in which the commercially available [U-14C]a-ketoglutarate was dissolved, was removed by freeze drying for 5 min, and not to dryness, and the [U-14C]a-ketoglutarate was dissolved in NaCl, 150 mmol l − 1, to a final concentration of 1 mCi ml − 1.
2.2. Animal experiments and brain extract preparation The mice were given a dose of 400 mg kg − 1 VPA which is a commonly used dose in mouse experiments. This apparently high dosage was used because of a low plasma/brain distribution ratio, and since the metabolism of VPA is very rapid in mice with a half-life of 0.8 h, compared to 9–18 h in man (Lo¨ scher, 1985, 1999). Maximal serum concentration is achieved 25 min after VPA administration (Lo¨ scher, 1999). Mice that were fasted overnight, received an intraperitoneal (i.p.) injection of VPA (400 mg kg − 1). The animals were tested on a rotarod made in our laboratory. The rod (10 cm length, 2.5 cm diameter) rotated at 20 rev./min. At 10 or 30 min after administration of VPA the animals received a bolus injection of 0.3 ml [1-13C]glucose or sodium [2-13C]acetate in a tail vein, and after another 15 min the animals were sacrificed by cervical dislocation and decapitated. The animals were handled in accordance with institutional and national guidelines for animal research. The brains were homogenized in 3 ml perchloric acid, 7% (vol/vol), protein was removed by centrifugation, and the supernatant was neutralized with KOH. The precipitate, KClO4, was removed by centrifugation, and the supernatant was lyophilized to dryness and redissolved in D2O, 600 ml, with dioxane, 0.01%, as internal standard. The samples were analyzed by 13C NMR spectroscopy as described below. To obtain brain extracts for ATP and PCr measurements, mice received an i.p. injection of VPA 400 mg kg − 1 (survival time 25 or 45 min), vigabatrin 1 g kg − 1 (survival time 75 min) or saline (survival time 45 min). The animals were
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killed by cervical dislocation and immediately immersed in liquid nitrogen for 30 s after cervical dislocation. The brains were removed in the frozen state and were crushed in a mortar cooled with liquid nitrogen together with 2.5 ml perchloric acid, 7% (vol/vol). The extract was neutralized with KOH, 9 mol l − 1, to pH 4– 5. One group of animals received [U-14C]a-ketoglutarate intrastriatally. At 15 or 35 min after injection of VPA, 400 mg kg − 1, or saline, the mice were anesthetized with an i.p. injection of (per kg b.w.): fentanyl 0.2 mg, fluanisone 10 mg, midazolam 5 mg. A hole was drilled in the skull 0.5 mm anterior to the bregma, and 1.5 mm lateral to the midline. A syringe was inserted 2 mm into the brain and 1 mCi [U-14C]a-ketoglutarate (1 ml) was injected into striatum. After 5 min the animals were decapitated. The striatum was dissected out on ice and homogenized in 1 ml perchloric acid, 3.5%. Following removal of protein and perchlorate, as described above, amino acids were separated by HPLC. The eluate was collected in 1-min fractions that were mixed with 10 ml scintillation fluid and analyzed by scintillation counting as described by Hassel et al. (1992). The levels of amino acids was determined by HPLC after derivatization with o-phthaldialdehyde using aaminoadipate as internal standard (Hassel et al., 1997).
2.3.
13
C NMR spectroscopy
For 13C NMR spectroscopy, broad band decoupled 125.76 MHz 13C NMR spectra were obtained on a Bruker Avance DRX 500 MHz spectrophotometer using a 30° pulse angle and 39 kHz spectral width with 65 K data points. The acquisition time was 0.826 s, and the relaxation delay was 3 s. For all analyses 10 K (10.240) scans with a line broadening of 1.0 Hz were used. All spectra were recorded at ambient temperature. Some spectra were also inverse-gated decoupled only during acquisition to avoid nuclear Overhauser effects. From the two sets of spectra, correction factors were calculated. Correction for saturation was not necessary for the resonances used for the calculation, since the relaxation time
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of dioxane was similar to that of the analyzed amino acids. A Silicon Graphics computer was used for data processing. The percent enrichment of the various carbon positions of the amino acids was determined after subtracting the naturally abundant 13C (1.1% of total carbon) from the total amount of the amino acids as determined by HPLC (see above) (Badar-Goffer et al., 1990).
2.4. Spectrophotometric methods ATP and PCr were measured by the method of Lowry and Passonneau (1972). Glucose, lactate, and ammonia were determined by reflectance spectroscopy using a DT60 Echtachem (Kodak). For the ammonia analysis, serum samples were drawn after 10 min of VPA treatment and immediately measured. The content of a-ketoglutarate in brain was measured spectrophotometrically as described by Lowry and Passonneau (1972) after extraction of a-ketoglutarate with 2.5% trichloroacetic acid. The samples were lyophilized to dryness, and after the lyophilisates were redissolved in water, 100 ml substrate was added to 100 ml imidazole buffer. Each experiment was carried out in triplicates in independent trials with 3–4 animals in each group. Succinate was measured spectrophotometrically using a kit (number 176 281) from Boehringer Mannheim (Mannheim, Germany). Protein was quantified by the method of Lowry et al. (1951). The total amount of amino acids was analyzed by HPLC as described above. The brain concentration of VPA was measured with an enzyme immunoassay (EMIT®, SyvaDade-Behring) on a spectrophotometric analyzer (Cobas Mira S, Roche). Differences between the groups obtained were analyzed statistically by Sigma Stat using one-way ANOVA and Dunnett’s method for multiple comparisons. The data obtained with 2-[13C]acetate were analyzed with a two-tailed Student’s t-test. 3. Results
3.1. Beha6ioural changes The VPA-treated mice appeared slightly sedated during the first 10–15 min although one
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or two mice intermittently had a more active behaviour than controls. At 25 min after injection of VPA, there were no obvious differences between controls and VPA-treated mice regarding their spontaneous behaviour. There were no differences between the groups with respect to their performance on the rotarod. The animals that received vigabatrin, 1 g kg − 1, showed signs of sedation 15 min after injection and tended to lie down more than controls, as previously described (Hassel et al., 1998).
3.2. Effects of VPA on cerebral metabolism: NMR spectroscopy and ATP le6els
13
C
At 25 min after injection of VPA, there was an accumulation of glutamate and glutamine labeled in the C-4 position from [1-13C]glucose, and the 13 C enrichment of the amino acids increased by 32 and 84%, respectively (Table 1). The synthesis of [13C]aspartate labeled in the C-2 or C-3 positions, decreased by approximately 70% in the VPAtreated animals both at 25 and 45 min (Table 1). The accumulation of [13C]glutamate and the decrease in [13C]aspartate labeling indicated inhibition of the TCA cycle between a-ketoglutarate dehydrogenase and malate dehydrogenase. The C-4 position in glutamate is the first carbon in glutamate and glutamine to become labeled from [1-13C]glucose, and the C-3 position is
labeled after a full turn of the label through the TCA cycle (Shank et al., 1993; Hassel et al., 1995). The glutamate C-3/C-4 labeling ratio has therefore been used as an indication of the TCA cycle activity in neurons. At 25 min after injection of VPA, the glutamate C-3/C-4 ratio had decreased from 0.279 0.02 nmol mg protein − 1 in controls to 0.1590.02 nmol mg protein − 1 (mean9 S.E.M.) (PB 0.001), indicating a reduction in the turnover of the TCA cycle. An accumulation of [2-13C]GABA of 53% was seen, while [2-13C]alanine decreased by 39% (Table 1). The reduction in the levels of ATP and PCr by 40% at 25 min could be due to the observed inhibition of the TCA cycle, but this may be a causal effect, since no experiments were performed to check directly this possibility. This effect was reversed at 45 min. The control values of brain ATP and PCr were in agreement with previous results (Siesjo¨ , 1978) (Fig. 1). The levels of ATP and PCr were not altered by administration of the irreversible GABA transaminase inhibitor, vigabatrin, 1 g kg − 1 (Fig. 1). At 45 min, the TCA cycle was no longer inhibited, as could be seen from the 13C labeling of glutamate, glutamine, and GABA being similar to control values (Table 1). The glutamate C-3/C-4 labeling ratio was reversed (0.2290.02 nmol mg protein − 1).
Table 1 Effect of VPA on the labeling of cerebral amino acids from [1-13C]glucose or sodium [2-13C]acetate No. animals
Glutamate
Glutamine
GABA
Aspartate
Alanine
C-4
C-4
C-2
C-2
C-3
C-3
0.34 9 0.08 0.219 0.08* 0.29 9 0.05
[1 -13C]glucose Control VPA, 25 min VPA, 45 min
18 9 8
6.7990.52 8.88 90.68* 7.329 0.60
1.12 9 0.19 2.07 9 0.40* 0.94 9 0.25
1.02 9 0.19 1.56 90.19* 1.35 9 0.20
1.23 9 0.15 0.38 9 0.12* 0.37 9 0.14*
1.23 9 0.14 0.44 9 0.12* 0.49 9 0.14*
[2 -13C]acetate Control VPA, 45 min
8 8
5.64 90.82 3.549 0.26*
3.33 9 0.64 2.85 9 1.03*
0.79 90.25 0.56 9 0.17
0.79 9 0.15 0.42 9 0.07*
0.71 9 0.14 0.53 9 0.15*
Fasted mice received VPA, 400 mg kg −1, or saline i.p. At 10 or 30 min, 0.3 ml 500 mmol l−1 [1-13C]glucose or sodium [2-13C]acetate was injected i.v. At 25 or 45 min, the animals were killed. Values are means (nmol 13C mg protein−1) 9S.E.M. * Statistically significant changes compared to controls (PB0.05).
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atally and measured the radiolabeling of amino acids associated with the TCA cycle. VPA treatment caused the 14C labeling of glutamate to increase by 82% and that of aspartate to decrease by 81% at 25 min after injection of VPA (Table 2). The radiolabeling of glutamine and GABA was also increased (Table 2). At 45 min, the radiolabeling of amino acids was similar to control values (Table 2). Fig. 1. Brain levels of ATP and PCr. Mice were given an i.p. injection of VPA, 400 mg kg − 1, or vigabatrin, 1 g kg − 1, or saline. After 25 or 45 min with VPA, respectively, or 75 min with vigabatrin, the animals were decapitated and immediately frozen in liquid nitrogen for 30 s. The brains were prepared as described. There were 4 –7 animals in each group presented as means9 S.E.M. The ATP (white) and PCr (black) levels decreased significantly (*PB 0.05) after administration of VPA at 25 min. The vigabatrin group is presented as a control substance which will inhibit the GABA shunt only in the TCA cycle.
Interestingly, in animals that received the glial substrate [2-13C]acetate, VPA treatment led to a decrease in the 13C labeling of glutamate and glutamine by 38 and 15%, respectively (Table 1). These results were in marked contrast to the labeling obtained with [1-13C]glucose.
3.3. Inhibition of h-ketoglutarate dehydrogenase by VPA in 6i6o. Metabolism of [U-14C]h-ketoglutarate To establish more precisely the enzymatic step of the TCA cycle where VPA might cause inhibition we injected [U-14C]a-ketoglutarate intrastri-
3.4. Cerebral le6els of VPA, amino acids, and related metabolites, and serum le6els of ammonia and glucose The brain level of VPA was 0.6390.05 mmol l − 1 at 25 min, and at 45 min it was remarkably similar, 0.5490.06 mmol l − 1. At 25 min after VPA administration, the brain level of GABA increased by 40%, and the level of aspartate decreased by 45%. The level of glutamate was constant during VPA treatment. The level of lactate increased by 44% at 25 min after administration of VPA (Table 3) reflecting energy impairment. At 45 min after VPA administration the level of GABA was increased by 27%, whereas aspartate was decreased by 35% (Table 3). At this time point the level of glutamine had increased by 34% (Table 3). The levels of a-ketoglutarate and succinate remained constant and were similar to those reported previously for brain (Siesjo¨ , 1978). The serum level of ammonia increased from 50.69 9.8 to 1019 15 mmol l − 1 at 25 min after administration of VPA (PB 0.02). VPA treatment did not cause significant changes in the serum level of glucose which was 3.99 0.5 mmol l − 1
Table 2 Effect of VPA on incorporation of label from [U-14C]a-ketoglutarate into amino acids in the striatum
Control VPA 25 min VPA 45 min
No. animals
Glutamate
Glutamine
GABA
Aspartate
4 5 4
8.79 0.9 15.9 9 2.5** 9.993.6
139.6 922 205.5 950* 146.5 929
5.0 91.5 7.5 90.5* 6.0 91.5
44.1 9 7.3 8.4 91.3** 31.2 9 7.8
Mice were injected i.p. with VPA, 400 mg kg−1, or saline. At 15 or 35 min, the animals were anesthetized, a hole was drilled in the skull, and 1 ml [U-14C]a-ketoglutarate (240 mCi mmol−1) was injected into striatum. At 25 or 45 min, the animals were killed, and the subcortical injection area was dissected out. Values are means (dpm mg protein−1) 9S.E.M. * Statistically significant changes compared to controls (PB0.05). ** Statistically significant changes compared to controls (PB0.01).
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Control VPA 25 min VPA 45 min
No. animals
Glutamate
Glutamine
18 9 16
115.3 94.3 34.2 9 1.3 111.3 95.8 39.1 9 4.5 118.9 95.6 45.8 9 3.4**
GABA
Aspartate
Alanine
a-Ketoglutarate Succinate
Lactate
Glucose
VPA
14.5 9 0.6 20.3 9 0.9** 18.4 9 0.9**
22.7 9 1.9 12.5 9 0.9** 14.7 9 1.3**
7.8 9 0.5 6.3 91.0 7.5 91.2
2.1 9 0.3 2.4 9 0.2 2.59 0.2
31.392.5 45.19 4.5* 33.094.4
24.892.5 23.5 9 2.5 25.99 2.3
0 9.0 90.9 7.7 9 0.9
4.89 0.5 4.69 0.4 4.79 0.6
Fasted mice were injected i.p. with VPA, 400 mg kg−1, or saline. At 25 or 45 min, the animals were killed. Values are means (nmol mg protein−1) 9S.E.M. * Statistically significant changes compared to controls (PB0.05). ** Statistically significant changes compared to controls (PB0.01).
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Table 3 Levels of amino acids, related metabolites, and VPA in the brains of mice after administration of VPA
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compared to 3.590.6 and 3.390.6 mmol l − 1, in controls and with VPA for 25 and 45 min, in fasted animals. 4. Discussion
4.1. VPA inhibits h-ketoglutarate dehydrogenase in 6i6o The present study shows that acute administration of VPA leads to inhibition of the a-ketoglutarate dehydrogenase step of the TCA cycle in vivo. This conclusion was ultimately based on the accumulation of [14C]glutamate and the reduced formation of [14C]aspartate in striatum from [U14 C]a-ketoglutarate. These findings agree with an in vitro study by Luder et al. (1990), who showed that VPA and its metabolites potently inhibit a-ketoglutarate dehydrogenase. a-Ketoglutarate dehydrogenase is a rate-limiting step of the TCA cycle (Lai et al., 1977), and any impairment of the activity of this enzyme would be expected to cause a reduction in energy production. This assumption was confirmed in our study by the low brain levels of ATP and PCr in VPA-treated mice at 25 min, accompanied by an accumulation of lactate. Although the inhibition of a-ketoglutarate dehydrogenase was confirmed with radiolabeling techniques, inhibition of the TCA cycle was strongly suggested by the labeling pattern obtained with 13C NMR spectroscopy. We may exclude that VPA treatment caused inhibition of the TCA cycle at an enzymatic step distal to a-ketoglutarate dehydrogenase, e.g. succinate dehydrogenase or fumarase, because previous studies have shown that such inhibition causes accumulation of succinate and no accumulation of [4-13C]glutamate (Hassel and Sonnewald, 1995). In the present study there was no accumulation of succinate, confirming that succinate dehydrogenase or fumarase were not inhibited by VPA. In vitro studies on rat brain and liver mitochondria have concluded that the site of inhibition of the TCA cycle by VPA is proximal to succinyl-CoA, also excluding succinate dehydrogenase, fumarase, and malate dehydrogenase as a target of VPA (Cunningham et al., 1980; Haas et al., 1981).
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Interestingly, we found that the inhibition of a-ketoglutarate dehydrogenase was reversed at 45 min after administration of VPA, when the brain level of VPA was still high. An explanation for the apparent reactivation of the enzyme could be that the a-ketoglutarate dehydrogenase activity became stimulated, for instance by a decrease in intramitochondrial calcium and pH that are known to modulate the enzyme activity (Lai and Cooper, 1986), and these changes would be dependent on the level of ATP. Furthermore, the enzyme could be reversed due to a local increase in the substrate for the enzyme, a-ketoglutarate. Alternatively, redistribution of brain VPA from the water phase to the lipid phase could explain the discrepancy between the high brain level of VPA and the lack of enzyme inhibition. VPA has been shown to interact with phospholipids in cell membranes (Chang et al., 2001). Active metabolites of VPA, such as E-D2-VPA or the CoAderivatives of VPA and E-D2-VPA, were not measured. Metabolites of VPA have been shown to inhibit a-ketoglutarate dehydrogenase more potently than VPA itself (Luder et al., 1990). It cannot be excluded that the inhibition and apparent reactivation of a-ketoglutarate dehydrogenase in the present study were due to variations in the levels of VPA metabolites rather than to changes in the level of VPA itself. Ammonia is an inhibitor of a-ketoglutarate dehydrogenase (Lai and Cooper, 1986), but any inhibitory effect of ammonia on a-ketoglutarate dehydrogenase activity was probably blunted by the increase in the level of glutamine after VPA administration. Amidation of glutamate to glutamine is the main pathway for removal of excess of ammonium in the brain (Berl et al., 1962; Collins et al., 1994). The accumulation of [2-13C]GABA from [113 C]glucose indicates inhibition of GABA metabolism (for reviews see Lo¨ scher, 1999; Johannessen, 2000). Accumulation of GABA has been considered one of the main antiepileptic mechanisms of VPA (Godin et al., 1969; Fariello et al., 1995). Probably, the reduction in ATP levels was caused by inhibition of a-ketoglutarate dehydrogenase and not by inhibition of GABA metabolism, since treatment with vigabatrin, an
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inhibitor of GABA transaminase, did not affect ATP levels (Fig. 1). Judging from the accumulation of GABA, (Hassel et al., 1998) vigabatrin, 1 g kg − 1, causes a greater inhibition of GABA metabolism than does VPA, 400 mg kg − 1, as used in the present study. In GABAergic neurons, both the TCA cycle and the a-ketoglutarate dehydrogenase was probably inhibited, based on previous in vitro studies (Van der Laan et al., 1979; Luder et al., 1990). A brain concentration similar to that of the present study may be expected to be reached in humans in the acute treatment of status epilepticus or mania (Price, 1989; Brennan et al., 1994). Therefore, the pharmacological effects observed in this study are likely to occur in man during acute treatment.
4.2. Theoretical consequences of a reduced cerebral ATP le6el As a theoretical consideration, a reduction in cerebral ATP levels could have a wide range of effects on brain function. Metabolic inhibition leads to a reduction in neuronal excitability through modulation of sodium channels, and such reduction in excitability is caused by a fall in ATP levels (Jiang and Haddad, 1992; Cummins et al., 1993). Generally, VPA is believed to act at the voltage-dependent sodium channel, inhibiting high-frequency firing of neurons (Slater and Johnston, 1978; McLean and Macdonald, 1986; Thomas et al., 1996; Vreugdenhil et al., 1998). The reduction in the level of ATP induced by VPA may have contributed to the apparent blocking effect of VPA on the voltage-dependent sodium channel in some of these studies where the ATP levels were not monitored. A decrease in neuronal excitability would also be expected during a reduction in ATP levels since ATP is an excitatory neurotransmitter (Burnstock, 1977; Tschopl et al., 1992; Chen et al., 1994). The reduction in ATP levels may have contributed to the sedation observed in the present study during the initial 15 min of VPA treatment. Therefore the reduction in ATP levels may have a clinical effect in the acute treatment of VPA.
4.3. Uptake of transmitter glutamate and of acetate during VPA treatment Glutamatergic neurotransmission was apparently maintained during VPA treatment, since the labeling of glutamine from [1-13C]glucose was maintained. The 13C labeling of glutamine from [1-13C]glucose is thought mainly to reflect glial uptake of 13C-labeled transmitter glutamate (Mason et al., 1992; Hassel et al., 1997; Sibson et al., 1998, 2001). A 40% reduction in the level of ATP would be expected to impair most cellular functions. The transmembrane sodium pump, upon which glial uptake of glutamate is dependent, must have been sufficiently active to allow such uptake of glutamate, although the uptake of glutamate indirectly depends on the sodium/ potassium-gradient. In VPA-treated animals, there was a reduction in the labeling of glutamine and glutamate from [2-13C]acetate, in contrast to the results obtained with [1-13C]glucose. This finding suggests that VPA inhibits the uptake of acetate and possibly other monocarboxylates into the brain or into the glial cells in vivo. Uptake of acetate is mediated by monocarboxylate carriers (Waniewski and Martin, 1998), and VPA has previously been shown to inhibit monocarboxylate transport in vitro (Benavides et al., 1982; Kang et al., 1990; Terasaki et al., 1991; Adkinson and Shen, 1996).
Acknowledgements The study was supported by the Norwegian Research Council. The authors wish to thank Dr Svein I. Johannessen for helpful discussions and his laboratory at The National Center for Epilepsy, Sandvika, for assistance with the EMIT analysis of VPA. Frode Fonnum is a VISTA professor.
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The acute effect of valproate on cerebral energy metabolism in mice Cecilie U. Johannessen a,*, Dirk Petersen b, Frode Fonnum a, Bjørnar Hassel a a
Norwegian Defence Research Establishment, N-2027 Kjeller, Norway b Department of Chemistry, Uni6ersity of Oslo, Oslo, Norway
Received 15 June 2001; received in revised form 3 September 2001; accepted 3 September 2001
Abstract Sodium valproate (VPA) is used in the acute treatment of status epilepticus and mania. We studied the acute effect of VPA on cerebral energy metabolism in awake mice that received VPA 400 mg kg − 1 and [1-13C]glucose or [2-13C]acetate. At 25 min, 13C NMR spectroscopy of brain extracts indicated inhibition of the tricarboxylic acid (TCA) cycle, as could be seen from the accumulation of [4-13C]glutamate and reduction in [13C]aspartate formation. Concomitantly, the level of ATP was reduced by 40%. To identify the enzymatic step at which the TCA cycle was inhibited [U-14C]a-ketoglutarate was injected intracerebrally. Inhibition of a-ketoglutarate dehydrogenase was evident at 25 min, as shown by accumulation of [14C]glutamate. At 45 min the inhibition of a-ketoglutarate dehydrogenase was reversed, shown by both 13C- and 14C-labeling, and the ATP level was normalized. The study shows for the first time that acute administration of VPA causes inhibition of the TCA cycle activity in vivo. The reduction in brain ATP would be expected to reduce neuronal excitability through modulation of sodium channels which may be clinically advantageous in the initial phase of VPA treatment. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Valproate; Cerebral metabolism; Tricarboxylic acid cycle; a-Ketoglutarate dehydrogenase; ATP
1. Introduction Sodium valproate (VPA) is used in high doses in the acute treatment of serial- or prolonged epileptic seizures, status epilepticus, and manic episodes (Price, 1989; Brennan et al., 1994; Gruenze et al., 1999; Naritoku and Mueed, 1999). Abbre6iations: SSA-DH, succinic semialdehyde dehydrogenase; TCA cycle, tricarboxylic acid cycle; VPA, valproate. * Corresponding author. Fax: + 47-6380-7509. E-mail address: [email protected] (C.U. Johannessen).
Studies with positron emission tomography in VPA-treated patients have indicated inhibition of the cerebral metabolism of glucose (Leiderman et al., 1991; Gaillard et al., 1996). These findings raise the question of whether VPA inhibits a specific enzymatic step in the energy metabolism in vivo. In vitro, VPA and metabolites inhibit a-ketoglutarate dehydrogenase (Luder et al., 1990) and enzymes related to GABA metabolism (for review see Lo¨scher, 1999). Such inhibition in vivo could affect cerebral energy production and thereby neuronal activity.
0920-1211/02/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 0 - 1 2 1 1 ( 0 1 ) 0 0 3 0 8 - 4
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The use of 13C-labeled energy substrates in conjunction with 13C NMR spectroscopy gives the opportunity to investigate in vivo metabolism in some detail. Glucose has previously been shown to be metabolized predominantly through the neuronal tricarboxylic acid (TCA) cycle, whereas acetate is predominantly metabolized through the glial TCA cycle (Van den Berg et al., 1969; Minchin and Beart, 1974). Analysis of the 13C-labeling of metabolites of [1-13C]glucose or [213 C]acetate gives a detailed picture of neuronal and glial energy metabolism, respectively (Cerdan et al., 1990; Ku¨ nnecke et al., 1993; Shank et al., 1993; Hassel et al., 1997). The aim of the present study was to investigate the acute effect of VPA on cerebral energy metabolism. 13C NMR spectroscopic analysis was carried out on brain extracts of VPA-treated mice that received [1-13C]glucose or [2-13C]acetate as labeled precursors of energy metabolites. The effect of VPA on ATP levels was compared to that of vigabatrin, a specific inhibitor of the GABA shunt. Metabolism of [U-14C]a-ketoglutarate was evaluated after intracerebral administration, to study the effect of VPA on a-ketoglutarate dehydrogenase specifically.
2. Materials and methods
2.1. Materials Female Hsd-Ola ICRF mice, 20 g body weight, (Harlan, UK) were kept in groups of 10 per cage under conditions of constant temperature (21 °C) and humidity (50%), with a 12-h light, 12-h dark cycle and free access to food and water. VPA, [2-13C]acetate and [1-13C]glucose (both 99% 13C enrichment), and reagents for enzymatic measurement of ATP and PCr, were obtained from Sigma (St Louis, MO, USA). [U-14C]a-Ketoglutarate (240 mCi mmol − 1) was obtained from American Radiolabeled Chem. Inc. (St Louis, MO, USA), and vigabatrin was a gift from Marion Merrel Dow (Uxbridge, UK). Before experiments, VPA was dissolved in water to a concentration of 24 mg ml − 1. Vigabatrin was dissolved in water to a concentration of 60
mg ml − 1. [1-13C]Glucose and sodium [213 C]acetate were dissolved in double-distilled water to a concentration of 500 mmol − 1, and pH of all solutions was adjusted to 7.4. The ethanol in which the commercially available [U-14C]a-ketoglutarate was dissolved, was removed by freeze drying for 5 min, and not to dryness, and the [U-14C]a-ketoglutarate was dissolved in NaCl, 150 mmol l − 1, to a final concentration of 1 mCi ml − 1.
2.2. Animal experiments and brain extract preparation The mice were given a dose of 400 mg kg − 1 VPA which is a commonly used dose in mouse experiments. This apparently high dosage was used because of a low plasma/brain distribution ratio, and since the metabolism of VPA is very rapid in mice with a half-life of 0.8 h, compared to 9–18 h in man (Lo¨ scher, 1985, 1999). Maximal serum concentration is achieved 25 min after VPA administration (Lo¨ scher, 1999). Mice that were fasted overnight, received an intraperitoneal (i.p.) injection of VPA (400 mg kg − 1). The animals were tested on a rotarod made in our laboratory. The rod (10 cm length, 2.5 cm diameter) rotated at 20 rev./min. At 10 or 30 min after administration of VPA the animals received a bolus injection of 0.3 ml [1-13C]glucose or sodium [2-13C]acetate in a tail vein, and after another 15 min the animals were sacrificed by cervical dislocation and decapitated. The animals were handled in accordance with institutional and national guidelines for animal research. The brains were homogenized in 3 ml perchloric acid, 7% (vol/vol), protein was removed by centrifugation, and the supernatant was neutralized with KOH. The precipitate, KClO4, was removed by centrifugation, and the supernatant was lyophilized to dryness and redissolved in D2O, 600 ml, with dioxane, 0.01%, as internal standard. The samples were analyzed by 13C NMR spectroscopy as described below. To obtain brain extracts for ATP and PCr measurements, mice received an i.p. injection of VPA 400 mg kg − 1 (survival time 25 or 45 min), vigabatrin 1 g kg − 1 (survival time 75 min) or saline (survival time 45 min). The animals were
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killed by cervical dislocation and immediately immersed in liquid nitrogen for 30 s after cervical dislocation. The brains were removed in the frozen state and were crushed in a mortar cooled with liquid nitrogen together with 2.5 ml perchloric acid, 7% (vol/vol). The extract was neutralized with KOH, 9 mol l − 1, to pH 4– 5. One group of animals received [U-14C]a-ketoglutarate intrastriatally. At 15 or 35 min after injection of VPA, 400 mg kg − 1, or saline, the mice were anesthetized with an i.p. injection of (per kg b.w.): fentanyl 0.2 mg, fluanisone 10 mg, midazolam 5 mg. A hole was drilled in the skull 0.5 mm anterior to the bregma, and 1.5 mm lateral to the midline. A syringe was inserted 2 mm into the brain and 1 mCi [U-14C]a-ketoglutarate (1 ml) was injected into striatum. After 5 min the animals were decapitated. The striatum was dissected out on ice and homogenized in 1 ml perchloric acid, 3.5%. Following removal of protein and perchlorate, as described above, amino acids were separated by HPLC. The eluate was collected in 1-min fractions that were mixed with 10 ml scintillation fluid and analyzed by scintillation counting as described by Hassel et al. (1992). The levels of amino acids was determined by HPLC after derivatization with o-phthaldialdehyde using aaminoadipate as internal standard (Hassel et al., 1997).
2.3.
13
C NMR spectroscopy
For 13C NMR spectroscopy, broad band decoupled 125.76 MHz 13C NMR spectra were obtained on a Bruker Avance DRX 500 MHz spectrophotometer using a 30° pulse angle and 39 kHz spectral width with 65 K data points. The acquisition time was 0.826 s, and the relaxation delay was 3 s. For all analyses 10 K (10.240) scans with a line broadening of 1.0 Hz were used. All spectra were recorded at ambient temperature. Some spectra were also inverse-gated decoupled only during acquisition to avoid nuclear Overhauser effects. From the two sets of spectra, correction factors were calculated. Correction for saturation was not necessary for the resonances used for the calculation, since the relaxation time
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of dioxane was similar to that of the analyzed amino acids. A Silicon Graphics computer was used for data processing. The percent enrichment of the various carbon positions of the amino acids was determined after subtracting the naturally abundant 13C (1.1% of total carbon) from the total amount of the amino acids as determined by HPLC (see above) (Badar-Goffer et al., 1990).
2.4. Spectrophotometric methods ATP and PCr were measured by the method of Lowry and Passonneau (1972). Glucose, lactate, and ammonia were determined by reflectance spectroscopy using a DT60 Echtachem (Kodak). For the ammonia analysis, serum samples were drawn after 10 min of VPA treatment and immediately measured. The content of a-ketoglutarate in brain was measured spectrophotometrically as described by Lowry and Passonneau (1972) after extraction of a-ketoglutarate with 2.5% trichloroacetic acid. The samples were lyophilized to dryness, and after the lyophilisates were redissolved in water, 100 ml substrate was added to 100 ml imidazole buffer. Each experiment was carried out in triplicates in independent trials with 3–4 animals in each group. Succinate was measured spectrophotometrically using a kit (number 176 281) from Boehringer Mannheim (Mannheim, Germany). Protein was quantified by the method of Lowry et al. (1951). The total amount of amino acids was analyzed by HPLC as described above. The brain concentration of VPA was measured with an enzyme immunoassay (EMIT®, SyvaDade-Behring) on a spectrophotometric analyzer (Cobas Mira S, Roche). Differences between the groups obtained were analyzed statistically by Sigma Stat using one-way ANOVA and Dunnett’s method for multiple comparisons. The data obtained with 2-[13C]acetate were analyzed with a two-tailed Student’s t-test. 3. Results
3.1. Beha6ioural changes The VPA-treated mice appeared slightly sedated during the first 10–15 min although one
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or two mice intermittently had a more active behaviour than controls. At 25 min after injection of VPA, there were no obvious differences between controls and VPA-treated mice regarding their spontaneous behaviour. There were no differences between the groups with respect to their performance on the rotarod. The animals that received vigabatrin, 1 g kg − 1, showed signs of sedation 15 min after injection and tended to lie down more than controls, as previously described (Hassel et al., 1998).
3.2. Effects of VPA on cerebral metabolism: NMR spectroscopy and ATP le6els
13
C
At 25 min after injection of VPA, there was an accumulation of glutamate and glutamine labeled in the C-4 position from [1-13C]glucose, and the 13 C enrichment of the amino acids increased by 32 and 84%, respectively (Table 1). The synthesis of [13C]aspartate labeled in the C-2 or C-3 positions, decreased by approximately 70% in the VPAtreated animals both at 25 and 45 min (Table 1). The accumulation of [13C]glutamate and the decrease in [13C]aspartate labeling indicated inhibition of the TCA cycle between a-ketoglutarate dehydrogenase and malate dehydrogenase. The C-4 position in glutamate is the first carbon in glutamate and glutamine to become labeled from [1-13C]glucose, and the C-3 position is
labeled after a full turn of the label through the TCA cycle (Shank et al., 1993; Hassel et al., 1995). The glutamate C-3/C-4 labeling ratio has therefore been used as an indication of the TCA cycle activity in neurons. At 25 min after injection of VPA, the glutamate C-3/C-4 ratio had decreased from 0.279 0.02 nmol mg protein − 1 in controls to 0.1590.02 nmol mg protein − 1 (mean9 S.E.M.) (PB 0.001), indicating a reduction in the turnover of the TCA cycle. An accumulation of [2-13C]GABA of 53% was seen, while [2-13C]alanine decreased by 39% (Table 1). The reduction in the levels of ATP and PCr by 40% at 25 min could be due to the observed inhibition of the TCA cycle, but this may be a causal effect, since no experiments were performed to check directly this possibility. This effect was reversed at 45 min. The control values of brain ATP and PCr were in agreement with previous results (Siesjo¨ , 1978) (Fig. 1). The levels of ATP and PCr were not altered by administration of the irreversible GABA transaminase inhibitor, vigabatrin, 1 g kg − 1 (Fig. 1). At 45 min, the TCA cycle was no longer inhibited, as could be seen from the 13C labeling of glutamate, glutamine, and GABA being similar to control values (Table 1). The glutamate C-3/C-4 labeling ratio was reversed (0.2290.02 nmol mg protein − 1).
Table 1 Effect of VPA on the labeling of cerebral amino acids from [1-13C]glucose or sodium [2-13C]acetate No. animals
Glutamate
Glutamine
GABA
Aspartate
Alanine
C-4
C-4
C-2
C-2
C-3
C-3
0.34 9 0.08 0.219 0.08* 0.29 9 0.05
[1 -13C]glucose Control VPA, 25 min VPA, 45 min
18 9 8
6.7990.52 8.88 90.68* 7.329 0.60
1.12 9 0.19 2.07 9 0.40* 0.94 9 0.25
1.02 9 0.19 1.56 90.19* 1.35 9 0.20
1.23 9 0.15 0.38 9 0.12* 0.37 9 0.14*
1.23 9 0.14 0.44 9 0.12* 0.49 9 0.14*
[2 -13C]acetate Control VPA, 45 min
8 8
5.64 90.82 3.549 0.26*
3.33 9 0.64 2.85 9 1.03*
0.79 90.25 0.56 9 0.17
0.79 9 0.15 0.42 9 0.07*
0.71 9 0.14 0.53 9 0.15*
Fasted mice received VPA, 400 mg kg −1, or saline i.p. At 10 or 30 min, 0.3 ml 500 mmol l−1 [1-13C]glucose or sodium [2-13C]acetate was injected i.v. At 25 or 45 min, the animals were killed. Values are means (nmol 13C mg protein−1) 9S.E.M. * Statistically significant changes compared to controls (PB0.05).
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atally and measured the radiolabeling of amino acids associated with the TCA cycle. VPA treatment caused the 14C labeling of glutamate to increase by 82% and that of aspartate to decrease by 81% at 25 min after injection of VPA (Table 2). The radiolabeling of glutamine and GABA was also increased (Table 2). At 45 min, the radiolabeling of amino acids was similar to control values (Table 2). Fig. 1. Brain levels of ATP and PCr. Mice were given an i.p. injection of VPA, 400 mg kg − 1, or vigabatrin, 1 g kg − 1, or saline. After 25 or 45 min with VPA, respectively, or 75 min with vigabatrin, the animals were decapitated and immediately frozen in liquid nitrogen for 30 s. The brains were prepared as described. There were 4 –7 animals in each group presented as means9 S.E.M. The ATP (white) and PCr (black) levels decreased significantly (*PB 0.05) after administration of VPA at 25 min. The vigabatrin group is presented as a control substance which will inhibit the GABA shunt only in the TCA cycle.
Interestingly, in animals that received the glial substrate [2-13C]acetate, VPA treatment led to a decrease in the 13C labeling of glutamate and glutamine by 38 and 15%, respectively (Table 1). These results were in marked contrast to the labeling obtained with [1-13C]glucose.
3.3. Inhibition of h-ketoglutarate dehydrogenase by VPA in 6i6o. Metabolism of [U-14C]h-ketoglutarate To establish more precisely the enzymatic step of the TCA cycle where VPA might cause inhibition we injected [U-14C]a-ketoglutarate intrastri-
3.4. Cerebral le6els of VPA, amino acids, and related metabolites, and serum le6els of ammonia and glucose The brain level of VPA was 0.6390.05 mmol l − 1 at 25 min, and at 45 min it was remarkably similar, 0.5490.06 mmol l − 1. At 25 min after VPA administration, the brain level of GABA increased by 40%, and the level of aspartate decreased by 45%. The level of glutamate was constant during VPA treatment. The level of lactate increased by 44% at 25 min after administration of VPA (Table 3) reflecting energy impairment. At 45 min after VPA administration the level of GABA was increased by 27%, whereas aspartate was decreased by 35% (Table 3). At this time point the level of glutamine had increased by 34% (Table 3). The levels of a-ketoglutarate and succinate remained constant and were similar to those reported previously for brain (Siesjo¨ , 1978). The serum level of ammonia increased from 50.69 9.8 to 1019 15 mmol l − 1 at 25 min after administration of VPA (PB 0.02). VPA treatment did not cause significant changes in the serum level of glucose which was 3.99 0.5 mmol l − 1
Table 2 Effect of VPA on incorporation of label from [U-14C]a-ketoglutarate into amino acids in the striatum
Control VPA 25 min VPA 45 min
No. animals
Glutamate
Glutamine
GABA
Aspartate
4 5 4
8.79 0.9 15.9 9 2.5** 9.993.6
139.6 922 205.5 950* 146.5 929
5.0 91.5 7.5 90.5* 6.0 91.5
44.1 9 7.3 8.4 91.3** 31.2 9 7.8
Mice were injected i.p. with VPA, 400 mg kg−1, or saline. At 15 or 35 min, the animals were anesthetized, a hole was drilled in the skull, and 1 ml [U-14C]a-ketoglutarate (240 mCi mmol−1) was injected into striatum. At 25 or 45 min, the animals were killed, and the subcortical injection area was dissected out. Values are means (dpm mg protein−1) 9S.E.M. * Statistically significant changes compared to controls (PB0.05). ** Statistically significant changes compared to controls (PB0.01).
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Control VPA 25 min VPA 45 min
No. animals
Glutamate
Glutamine
18 9 16
115.3 94.3 34.2 9 1.3 111.3 95.8 39.1 9 4.5 118.9 95.6 45.8 9 3.4**
GABA
Aspartate
Alanine
a-Ketoglutarate Succinate
Lactate
Glucose
VPA
14.5 9 0.6 20.3 9 0.9** 18.4 9 0.9**
22.7 9 1.9 12.5 9 0.9** 14.7 9 1.3**
7.8 9 0.5 6.3 91.0 7.5 91.2
2.1 9 0.3 2.4 9 0.2 2.59 0.2
31.392.5 45.19 4.5* 33.094.4
24.892.5 23.5 9 2.5 25.99 2.3
0 9.0 90.9 7.7 9 0.9
4.89 0.5 4.69 0.4 4.79 0.6
Fasted mice were injected i.p. with VPA, 400 mg kg−1, or saline. At 25 or 45 min, the animals were killed. Values are means (nmol mg protein−1) 9S.E.M. * Statistically significant changes compared to controls (PB0.05). ** Statistically significant changes compared to controls (PB0.01).
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Table 3 Levels of amino acids, related metabolites, and VPA in the brains of mice after administration of VPA
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compared to 3.590.6 and 3.390.6 mmol l − 1, in controls and with VPA for 25 and 45 min, in fasted animals. 4. Discussion
4.1. VPA inhibits h-ketoglutarate dehydrogenase in 6i6o The present study shows that acute administration of VPA leads to inhibition of the a-ketoglutarate dehydrogenase step of the TCA cycle in vivo. This conclusion was ultimately based on the accumulation of [14C]glutamate and the reduced formation of [14C]aspartate in striatum from [U14 C]a-ketoglutarate. These findings agree with an in vitro study by Luder et al. (1990), who showed that VPA and its metabolites potently inhibit a-ketoglutarate dehydrogenase. a-Ketoglutarate dehydrogenase is a rate-limiting step of the TCA cycle (Lai et al., 1977), and any impairment of the activity of this enzyme would be expected to cause a reduction in energy production. This assumption was confirmed in our study by the low brain levels of ATP and PCr in VPA-treated mice at 25 min, accompanied by an accumulation of lactate. Although the inhibition of a-ketoglutarate dehydrogenase was confirmed with radiolabeling techniques, inhibition of the TCA cycle was strongly suggested by the labeling pattern obtained with 13C NMR spectroscopy. We may exclude that VPA treatment caused inhibition of the TCA cycle at an enzymatic step distal to a-ketoglutarate dehydrogenase, e.g. succinate dehydrogenase or fumarase, because previous studies have shown that such inhibition causes accumulation of succinate and no accumulation of [4-13C]glutamate (Hassel and Sonnewald, 1995). In the present study there was no accumulation of succinate, confirming that succinate dehydrogenase or fumarase were not inhibited by VPA. In vitro studies on rat brain and liver mitochondria have concluded that the site of inhibition of the TCA cycle by VPA is proximal to succinyl-CoA, also excluding succinate dehydrogenase, fumarase, and malate dehydrogenase as a target of VPA (Cunningham et al., 1980; Haas et al., 1981).
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Interestingly, we found that the inhibition of a-ketoglutarate dehydrogenase was reversed at 45 min after administration of VPA, when the brain level of VPA was still high. An explanation for the apparent reactivation of the enzyme could be that the a-ketoglutarate dehydrogenase activity became stimulated, for instance by a decrease in intramitochondrial calcium and pH that are known to modulate the enzyme activity (Lai and Cooper, 1986), and these changes would be dependent on the level of ATP. Furthermore, the enzyme could be reversed due to a local increase in the substrate for the enzyme, a-ketoglutarate. Alternatively, redistribution of brain VPA from the water phase to the lipid phase could explain the discrepancy between the high brain level of VPA and the lack of enzyme inhibition. VPA has been shown to interact with phospholipids in cell membranes (Chang et al., 2001). Active metabolites of VPA, such as E-D2-VPA or the CoAderivatives of VPA and E-D2-VPA, were not measured. Metabolites of VPA have been shown to inhibit a-ketoglutarate dehydrogenase more potently than VPA itself (Luder et al., 1990). It cannot be excluded that the inhibition and apparent reactivation of a-ketoglutarate dehydrogenase in the present study were due to variations in the levels of VPA metabolites rather than to changes in the level of VPA itself. Ammonia is an inhibitor of a-ketoglutarate dehydrogenase (Lai and Cooper, 1986), but any inhibitory effect of ammonia on a-ketoglutarate dehydrogenase activity was probably blunted by the increase in the level of glutamine after VPA administration. Amidation of glutamate to glutamine is the main pathway for removal of excess of ammonium in the brain (Berl et al., 1962; Collins et al., 1994). The accumulation of [2-13C]GABA from [113 C]glucose indicates inhibition of GABA metabolism (for reviews see Lo¨ scher, 1999; Johannessen, 2000). Accumulation of GABA has been considered one of the main antiepileptic mechanisms of VPA (Godin et al., 1969; Fariello et al., 1995). Probably, the reduction in ATP levels was caused by inhibition of a-ketoglutarate dehydrogenase and not by inhibition of GABA metabolism, since treatment with vigabatrin, an
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inhibitor of GABA transaminase, did not affect ATP levels (Fig. 1). Judging from the accumulation of GABA, (Hassel et al., 1998) vigabatrin, 1 g kg − 1, causes a greater inhibition of GABA metabolism than does VPA, 400 mg kg − 1, as used in the present study. In GABAergic neurons, both the TCA cycle and the a-ketoglutarate dehydrogenase was probably inhibited, based on previous in vitro studies (Van der Laan et al., 1979; Luder et al., 1990). A brain concentration similar to that of the present study may be expected to be reached in humans in the acute treatment of status epilepticus or mania (Price, 1989; Brennan et al., 1994). Therefore, the pharmacological effects observed in this study are likely to occur in man during acute treatment.
4.2. Theoretical consequences of a reduced cerebral ATP le6el As a theoretical consideration, a reduction in cerebral ATP levels could have a wide range of effects on brain function. Metabolic inhibition leads to a reduction in neuronal excitability through modulation of sodium channels, and such reduction in excitability is caused by a fall in ATP levels (Jiang and Haddad, 1992; Cummins et al., 1993). Generally, VPA is believed to act at the voltage-dependent sodium channel, inhibiting high-frequency firing of neurons (Slater and Johnston, 1978; McLean and Macdonald, 1986; Thomas et al., 1996; Vreugdenhil et al., 1998). The reduction in the level of ATP induced by VPA may have contributed to the apparent blocking effect of VPA on the voltage-dependent sodium channel in some of these studies where the ATP levels were not monitored. A decrease in neuronal excitability would also be expected during a reduction in ATP levels since ATP is an excitatory neurotransmitter (Burnstock, 1977; Tschopl et al., 1992; Chen et al., 1994). The reduction in ATP levels may have contributed to the sedation observed in the present study during the initial 15 min of VPA treatment. Therefore the reduction in ATP levels may have a clinical effect in the acute treatment of VPA.
4.3. Uptake of transmitter glutamate and of acetate during VPA treatment Glutamatergic neurotransmission was apparently maintained during VPA treatment, since the labeling of glutamine from [1-13C]glucose was maintained. The 13C labeling of glutamine from [1-13C]glucose is thought mainly to reflect glial uptake of 13C-labeled transmitter glutamate (Mason et al., 1992; Hassel et al., 1997; Sibson et al., 1998, 2001). A 40% reduction in the level of ATP would be expected to impair most cellular functions. The transmembrane sodium pump, upon which glial uptake of glutamate is dependent, must have been sufficiently active to allow such uptake of glutamate, although the uptake of glutamate indirectly depends on the sodium/ potassium-gradient. In VPA-treated animals, there was a reduction in the labeling of glutamine and glutamate from [2-13C]acetate, in contrast to the results obtained with [1-13C]glucose. This finding suggests that VPA inhibits the uptake of acetate and possibly other monocarboxylates into the brain or into the glial cells in vivo. Uptake of acetate is mediated by monocarboxylate carriers (Waniewski and Martin, 1998), and VPA has previously been shown to inhibit monocarboxylate transport in vitro (Benavides et al., 1982; Kang et al., 1990; Terasaki et al., 1991; Adkinson and Shen, 1996).
Acknowledgements The study was supported by the Norwegian Research Council. The authors wish to thank Dr Svein I. Johannessen for helpful discussions and his laboratory at The National Center for Epilepsy, Sandvika, for assistance with the EMIT analysis of VPA. Frode Fonnum is a VISTA professor.
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