Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin-Treated Rats

Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by ¹³C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2-¹³C₂]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2-¹³C₂]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by ¹³C NMR. In vigabatrin-treated animals [¹³C]glutamine, a common intermediate for [¹³C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2-¹³C₂]glucose or [1,2-¹³C₂]acetate. However, [¹³C]GABA accumulation was sevenfold higher during [1,2-¹³C₂]glucose infusions or twofold higher during [1,2-¹³C₂]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [¹³C]GABA accumulation occurs initially in the neuronal compartment.     

Effect of Stimulation on the Incorporation of 14C from Glial and Neuronal Specific Substrates into Brain Proteins In Vivo and In Vitro

Abstract: The incorporation of amino acids into brain proteins following brachial plexus stimulation (BPS) was studied in anaesthetised Sprague-Dawley rats following injection of radioactive precursors of both neuronal and glial compartments. Following intraperitoneal injection of [¹⁴C]glucose, which is the major neuronal pool precursor, BPS resulted in a significant increase of 379% ( P ± 0.001) in the incorporation of carbon from [¹⁴C]glucose into TCA-insoluble proteins in the contralateral sensorimotor cortex as compared with the ipsilateral area of the same animal. This increase was abolished totally when tetrodotoxin (10 μg ml^-1) was applied topically to the surface of the stimulated area. Following intraperitoneal injection of [¹⁴C]acetate, which is considered to be mainly a glial cell precursor, the same afferent electrical stimuli caused a significant decrease of 21% in the incorporation of amino acids into proteins in the stimulated versus unstimulated sensorimotor cortex. With [4-³H]phenyl-alanine or [l-¹⁴C]leucine as precursors a significant decrease (12%) or no change was recorded, respectively. A similar decrease in protein synthesis in the stimulated sensorimotor cortex was achieved using different routes of injection. No significant changes were observed in the ratio of the specific radioactivities of the total amino acids of the two hemispheres using either precursor. In vitro , synaptosomes showed a large increase in incorporation into proteins after treatment with electrical pulses, both with [¹⁴C]glucose and with [U-¹⁴C]acetate as precursors.     

BIOSYNTHESIS AND BIODEGRADATION OF RAT BRAIN GANGLIOSIDES STUDIED IN VIVO

Abstract— Metabolic relationships between the four major brain gangliosides, GM1, GD1a, GDlb and GT1 were studied in vivo . Labelled acetate and glucosamine were injected intracerebrally into 6–12-day-old rats and the radioactivities of the cerebral gangliosides were analysed. Radioactivity from [³H]acetate was determined in sialic acid, sphingosine and stearic acid and from [1-¹⁴C]glucosamine in hexosamine and sialic acid. The gangliosides were labelled in proportion to their pool size. In 6 day-old rats the labelling was approx. 30 per cent lower in the sialidase-stable sialyl group than in the labile one. When the brain gangliosides were labelled in 12-day-old rats, however, the specific activities of sialidase-labile and stable sialyl groups were the same at 0.5 months after the injection of precursors and disappeared at the same rate. The results indicate that at the age of 6 days a small pool of monosialogangliosides exists, which is converted to di- and trisialogangliosides. The degradation of gangliosides was studied by following the radioactivities in sphingosine and stearic acid from 2 to 6 months after the injection of labelled acetate. The specific activities of sphingosine and stearic acid decreased simultaneously at the same rate in all the four major gangliosides. The specific activity of stearic acid was the same in total brain lipids as in gangliosides. The half-lives for the degradation of the gangliosides were age-dependent and estimated to 60 days in adult rats. They were much shorter in younger rats but no reliable figures could be determined.     

AXONAL TRANSPORT OF LIPIDS IN THE RABBIT OPTIC SYSTEM

Abstract— Axonal transport of lipids was demonstrated in the rabbit optic system using [2-³H]glycerol and [3-¹⁴C]serine. Following intraocular injection of these precursors, radioactive lipids were detected in the optic tract, superior colliculus and lateral geniculate body over a 31 day period. The bulk of lipid appeared to migrate at a rate equivalent to that of rapidly transported protein which, when combined with a prolonged period of release into the axon, led to a peak of transported radioactivity at 6-10 days for the 3 tissues. The suggestion of a second peak at 17 days indicated the possibility of a smaller slow component, although another interpretation is suggested. Analysis of individual transported lipids revealed [2-³H]glycerol to label phosphoglycerides preferentially and [3-¹⁴C]serine to be an effective precursor for sphingolipids and certain of the phosphoglycerides. [3-¹⁴C]Serine labeled axonally transported proteins to an even greater extent than lipids, revealing the same fast and slow components previously shown with other amino acids.     

Trifluoperazine inhibits the incorporation of labelled precursors into lipids, proteins and DNA of Mycobacterium tuberculosis H37Rv

Abstract We have recently demonstrated that the calmodulin antagonist trifluoperazine has antitubercular activity in vitro against Mycobacterium tuberculosis H₃₇R_v susceptible and resistant to isoniazid. It is shown that trifluoperazine at a concentration of 50 μ g ml⁻¹ when added to the cells along with the labelled precursors inhibited the incorporation of [¹⁴C]acetate into lipids (63%) and uptake of [¹⁴C]glycine (74%) and [^3H]thymidine (52%) bu whole cells of M. tuberculosis H₃₇R_v by 6 h of exposure. After 48 h, the inhibition was 87%, 97% and 74%, respectively. However, when the drug was added to cells taking up and metabolizing the labelled precursors at a later point (3 h for [¹⁴C]acetate and [³H]thymidine and 12 h for [¹⁴C]glycine) it inhibited completely the uptake of all the precursors, at least up to 24 h. The onset of inhibitory action was very rapid, i.e. 3 h. It is suggested that trifluoperazine has multiple sites of action and acts probably by affecting the synthesis of lipids, proteins and DNA.     

Pathway of propionate degradation in Desulfobulbus propionicus

Abstract The degradation of [1-¹⁴C]- and [2-¹⁴C] propionate to acetate and bicarbonate by the sulfate- reducing bacterium Desulfobulbus propionicus was studied. When [1-¹⁴C]propionate was used, more than 95% of the label was recovered in the HCO₃⁻ fraction. [2-¹⁴C]Propionate was quantitatively converted into labeled acetate of which the methyl and carboxyl group were equally labeled. These results are in accordance with a randomizing route such as the methylmalonyl-CoA pathway for propionate degradation and support earlier evidence for the functioning of this pathway on the basis of enzyme assays.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

Confirmation of operative acetate, H2/CO2 and methanol methanogenic pathways in the solid-state refuse fermentation

By use of the radiolabelled substrates sodium [1–¹⁴C] acetate, sodium [2–¹⁴C] acetate, NaH¹⁴CO₃ and ¹⁴CH₃OH, three of the possible methanogenic pathways in fermenting refuse were confirmed. Due to the absence of a methanol pool, however, the relative contribution of each could not be determined. Circumstantial evidence for an operative trimethylamine pathway was gained but not confirmed whilst preliminary attempts to stimulate methanogenesis in refuse by supplementation with mono-and dimethylamine proved unsuccessful.     

RAPID TRANSPORT OF FUCOSYL GLYCOPROTEINS TO NERVE ENDINGS IN MOUSE BRAIN

Abstract— Mice were injected intracerebrally with mixtures of [³H]fucose and [¹⁴C]gluco-samine, and incorporation into macromolecules in various subcellular fractions of brain was studied at 1, 2, 3 and 4 h after administration of the precursors. There was a lag of several hours between the incorporation of [³H]fucose into the glycoproteins of the whole brain fractions and of that into the soluble and particulate glycoproteins of the nerve ending fractions. In contrast, no lag was observed between the incorporation of [¹⁴C]glucosamine into the macromolecules of the whole brain fractions and of that into the soluble macro-molecules of the nerve ending fraction. We conclude that fucosyl glycoproteins of the nerve ending fraction were synthesized in the nerve cell bodies and transported to nerve endings by rapid axoplasmic transport, whereas a substantial proportion of the glucosamine in the soluble macromolecules of the nerve ending fraction was incorporated by the nerve endings themselves. In addition, our evidence indicates that cyclobeximide inhibited fucose incorporation into brain glycoproteins by inhibiting the synthesis of acceptor proteins rather than fucosyl transferase.     

Propionate metabolism in a methanogenic enrichment culture. Direct reductive carboxylation and acetogenesis pathways

Abstract Serial dilutions of methanogenic sludges in propionate medium gave a methanogenic non-acetoclastic enrichment degrading 1 mol of propionate to 1.6 mol of acetate and 0.17 mol of methane, with a transient accumulation of butyrate. NMR recordings showed the conversion of [2-¹³C]- and [3-¹³C]-propionate to [3-¹³C]- and [4-¹³C]-butyrate, respectively, thus demonstrating a reductive carboxylation of propionate to butyrate. The labelling found in the accumulated acetate and fermentation balances also suggested that reductive carboxylation was the major pathway involved in propionate conversion to acetate.     

Metabolism of [5-3H]Kynurenine in the Rat Brain In Vivo: Evidence for the Existence of a Functional Kynurenine Pathway

Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN-lesioned rat striata after a focal injection of [5-³H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5-³H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µ M [5-³H]KYN. Labeled QUIN, KYNA, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µ M [5-³H]KYN, a lesion-induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [³H]KYNA (5.0 vs. 1.8%), [³H]3-HK (20.9 vs. 4.5%), [³H]XA (1.5 vs. 0.4%), and [³H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion-induced increases of the activities of kynurenine-3-hydroxylase (3.6-fold), kynureninase (7.6-fold), kynurenine aminotransferase (1.8-fold), and 3-hydroxyanthranilic acid oxygenase (4.2-fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.     

Membrane-bound glucosamine acetyltransferase in coleoptile segments of Avena sativa

The in vivo metabolism of D-[U-¹⁴C]glucosamine and the in vitro properties of glucosamine acetyltransferase (EC 2.3.1.3), the first committed enzyme in the metabolism of exogenously supplied D-glucosamine, were studied in coleoptile segments of Avena sativa L. cv. Sole II. D-[U-¹⁴C]glucosamine was taken up by oat coleoptile segments and sequentially metabolised to radioactive N-acetylglucosamine, N-acetylglucosamine 6-P, N-acetylglucosamine 1-P, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, N-acetylglucosamine residues were incorporated into glycoproteins and glycolipids of the cells. All glucosamine acetyltransferase activity was found to be membrane-bound. The enzyme was solubilized by either digitonin or CHAPS. The specificities and the kinetics of the membrane-bound and soluble glucosamine acetyltransferase were determined. The effects of ions, nucleotides, nucleoside diphosphate amino sugars, coenzymes and group-specific chemical probes on the rate of membrane-bound and CHAPS-solubilized enzyme were investigated. Our data indicate that UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine do not exert a feed-back control on the glucosamine acetyltransferase either in vivo or in vitro. Further, some nucleotides and the metal ions Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺ and Co²⁺ affect the activity of the enzyme in vitro.     

Interrelationships of Ubiquinone and Sterol Syntheses in Cultured Cells of Neural Origin

Abstract: Ubiquinone synthesis has been studied in cultured C-6 glial and neuroblastoma cells by utilizing an inhibitor, 3-β-(2-diethylaminoethoxy) androst-5-en-17-one hydrochloride (U18666A), of cholesterol biosynthesis. Exposure of C-6 glial cells to nanomolar quantities of U18666A caused a marked inhibition of total sterol synthesis from [¹⁴C]acetate or [³H]mevalonate within minutes. A 95% inhibition was apparent after a 3-h exposure to 200 ng/ml of U18666A. These observations, together with studies of the incorporation of radioactivity from the two precursors into cholesterol, desmosterol, lanosterol, and squalene, indicated that although the most sensitive site to inhibition by U18666A is desmosterol reduction to cholesterol, a major site of inhibition is demonstrable at a more proximal site, perhaps squalene synthetase. As a consequence of the latter inhibition, exposure of C-6 glial cells to U18666A caused a marked stimulation of incorporation of [¹⁴C]acetate or [³H]mevalonate into ubiquinone. Over a wide range of U18666A concentrations, the increase in ubiquinone synthesis was accompanied by an approximately similar decrease in total sterol synthesis. Whereas in the absence of U18666A only approximately 7% of the radioactivity incorporated from [³H]mevalonate into isoprenoid compounds was found in ubiquinone, in the presence of the drug approximately 90% of incorporated radioactivity was found in ubiquinone. The reciprocal effects of U18666A on ubiquinone and sterol syntheses were apparent also in the neuronal cells. The data thus demonstrate a tight relationship between ubiquinone and sterol biosyntheses in cultured cells of neural origin. In such cells ubiquinone synthesis is exquisitely sensitive to the availability of isoprenoid precursors derived from the cholesterol biosynthetic pathway.     

THE INCORPORATION OF DOUBLE-LABELLED ACETATE INTO GLUTAMATE AND RELATED AMINO ACIDS FROM ADULT MOUSE BRAIN: COMPARTMENTATION OF AMINO ACID METABOLISM IN BRAIN

Abstract– We have determined the incorporation of [³H]-, [1-¹⁴C]- and [2-¹⁴C]acetate into glutamate, glutamine and aspartate of the adult mouse brain. All these three acetates were incorporated more extensively into glutamine than into glutamate. This has been reported by several authors for each of these labelled acetates in separate experiments. It was shown that [³H, 2-¹⁴C]acetate can be used to obtain an acetate labelling ratio analogous to the previously used [2-¹⁴C]acetate/[1-¹⁴C]acetate labelling ratio. From these acetate labelling ratios of glutamine and glutamate conclusions can be deduced about the dynamic relationship of these amino acids with each other and with the tricarboxylic acid cycle.
A fairly large isotope effect between acetate and glutamate was observed. As this isotope effect is very likely caused by the citrate synthase reaction, it can be argued that citrate synthase involved in the conversion of labelled acetate into glutamate is far out of equilibrium in vivo. Comparing our data with literature data, the possibility can be suggested that citrate synthase in the acetate metabolizing compartment is in situ kinetically distinct from citrate synthase in other compartments of the brain.     

Effects of Ketone Bodies on Astrocyte Amino Acid Metabolism

Abstract: The effects of acetoacetate and 3-hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with ¹⁵N as a metabolic probe and gas chromatography-mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3-hydroxybutyrate (5 m M ) to the incubation medium did not alter the initial rate of appearance of [¹⁵N]glutamate in the glia, but it did inhibit transamination of glutamate to [¹⁵N]aspartate. Addition of acetoacetate also inhibited formation of [2-¹⁵N]glutamine, but 3-hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 m M ) was also associated with diminished production of [¹⁵N]aspartate and [2-¹⁵N]glutamine with [¹⁵N]glutamate as precursor. Studies with [2-¹⁵N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [¹⁵N]GABA to [2-¹⁵N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3-hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.     

Synthesis of Acetylcholine from Acetate in a Sympathetic Ganglion

Abstract: The present experiments tested whether acetate plays a role in the provision of acetyl-CoA for acetylcholine synthesis in the cat's superior cervical ganglion. Labeled acetylcholine was identified in extracts of ganglia that had been perfused for 20 min with Krebs solution containing choline (10⁻⁵ M ) and [³H], [1-⁴C], or [2-¹⁴C]acetate (10³ M ); perfusion for 60 min or with [³H]acetate (10⁻² M ) increased the labeling. The acetylcholine synthesized from acetate was available for release by a Ca²⁺-dependent mechanism during subsequent periods of preganglionic nerve stimulation. When ganglia were stimulated via their preganglionic nerves or by exposure to 46 m M K⁺, the labeling of acetylcholine from [³H]acetate was reduced when compared with resting ganglia. The reduced synthesis of acetylcholine from acetate during stimulation was not due to acetate recapture, shunting of acetate into lipid synthesis, or the transmitter release process itself. In ganglia perfused with [2-¹⁴C]glucose, the amount of labeled acetylcholine formed was clearly enhanced during stimulation. An increase in acetylcholine labeling from [³H]acetate was shown during a 15-min resting period following a 60-min period of preganglionic nerve stimulation (20 Hz). It is concluded that acetate is not the main physiological acetyl precursor for acetylcholine synthesis in this sympathetic ganglion, and that during preganglionic nerve stimulation there is enhanced delivery of acetyl-CoA to choline acetyltransferase from a source other than acetate.     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

GLUTAMATE METABOLISM IN OCTOPUS BRAIN IN VIVO;ABSENCE OF A WAELSCH EFFECT

Abstract— The metabolism of [U-¹⁴C]glutamate was followed in vivo in the octopus Eledone cirrhosa following intracranial injection, and compared with that in the mammalian brain.
By contrast with the rat brain, the specific activity of glutamine recovered from Eledone optic and vertical lobes was lower than that of glutamate at short time intervals after injection. Thus the Waelsch effect was not apparent in this species. Again, in contrast with the rat brain, radioactivity could be found in alanine but not in GABA following [U-¹⁴C]glutamate injection. This was compatible with observations made previously in vitro.
The significance of these intraspecies differences in metabolism and compartmentation is discussed.     

TRANSPORT OF MICROTUBULAR PROTEIN IN AXONS OF RETINAL GANGLION CELLS

Abstract— Following intraocular injection of [³H]leucine, microtubular protein was isolated by two different methods from the optic pathway of the rabbit. It was found that most of this protein was transported at a slow rate in the axons of retinal ganglion cells. After arrival at the nerve terminals in the lateral geniculate body, the microtubular protein decayed with a half-life of 9 days.     

METABOLISM OF γ-HYDROXY-[1-14C] BUTYRATE BY RAT BRAIN: RELATIONSHIP TO THE KREBS CYCLE AND METABOLIC COMPARTMENTATION OF AMINO ACIDS

Abstract— Ninhydrin decarboxylation experiments were carried out on the labelled amino acids produced following intraventricular injection of either γ-hydroxy-[1-¹⁴C]butyric acid (GHB) or [1-¹⁴C] succinate. The loss of isotope (as ¹⁴CO₂) was similar for both substances. The [1-¹⁴C]GHB metabolites lost 75% of the label and the [1-¹⁴C] succinate metabolites lost 68%. This observation gives support to the hypothesis that the rat brain has the enzymatic capacity to metabolize [1-¹⁴C]GHB to succinate and to amino acids that have the isotope in the carboxylic acid group adjacent to the a-amino group. These results also indicate that the label from [1-¹⁴C]GHB does not enter the Krebs cycle as acetate. The specific activity ratio of radiolabelled glutamine to glutamic acid was determined in order to evaluate which of the two major metabolic compartments preferentially metabolize GHB. It was found that for [1-¹⁴C]GHB this ratio was 4.20 ± 0.18 (S.E. for n = 7) and for [l-¹⁴C]succinate this ratio was 7.71 (average of two trials, 7.74 and 7.69). These results suggest that the compartment thought to be associated with glial cells and synaptosomal structures is largely responsible for the metabolism of GHB. Metabolism as it might relate to the neuropharmacological action of GHB is discussed.