Effect of ammonium ions on synaptic transmission in the mammalian central nervous system.

It is not surprising that a compound with such unique properties as NH3/NH4+, should have a large variety of biochemical and neurological effects and to find itself implicated in many pathological conditions. Its undissociated (NH3) or dissociated (NH4+) forms, having different physicochemical properties, enter neurons and other cells through differing pathways. These two forms then change internal pH in opposite directions, and initiate a variety of regulatory processes that attempt to overcome these pH changes. In addition, ammonia has a central role in normal intermediary metabolism, and when present in excess, it can disturb reversible reactions in which it participates. The challenge in interpreting these various observations lies in the difficulty in assigning to them a role in the generation of symptoms seen in experimental and clinical hyperammonemias. In this review we have attempted to summarize information available on the effects of ammonium ions on synaptic transmission, a central process in nervous system function. Evidence has been presented to show that ammonium ions, in pathologically relevant concentrations, interfere with glutamatergic excitatory transmission, not by decreasing the release of glutamate, but by preventing its action on post-synaptic AMPA receptors. Furthermore, NH4+ depolarizes neurons to a variable degree, without consistently changing membrane resistance, probably by reducing [K+]i. A decrease in EK+ may also be responsible for decreasing the effectiveness of the outward chloride pump, thus explaining the well known inhibitory effect of NH4+ on the hyperpolarizing IPSP. There is a consensus of opinion that chronic hyperammonemia increases 5HT turnover and this may be responsible for altered sleep patterns seen in hepatic encephalopathy. There does not seem to be a consistent effect on catecholaminergic transmission in hyperammonemias. However, chronic hyperammonemia causes pathological changes in perineuronal astrocytes, which may lead to a reduced uptake of released glutamate and a decreased detoxification of ammonia by the brain. Chronic moderate increase in extracellular glutamate results in a down-regulation of NMDA receptors, while the decreased detoxification of ammonia makes the central nervous system more vulnerable to a sudden hyperammonemia, due, for instance, to an increased dietary intake of proteins or to gastrointestinal bleeding in patients with liver disease. Clearly, data summarized in this review represent only the beginning in the elucidation of the mechanism of ammonia neurotoxicity. It should help, we hope, to direct future investigations towards some of the questions that need to be answered.

Increased densities of binding sites for the "peripheral-type" benzodiazepine receptor ligand [3H]PK11195 in vulnerable regions of the rat brain in thiamine deficiency encephalopathy.

Quantitative receptor autoradiography was used to evaluate the density of high-affinity binding sites for the "peripheral-type" benzodiazepine receptor (PTBR) ligand [3H]PK11195 in brain regions of the rat at different stages of pyrithiamine-induced thiamine deficiency encephalopathy, an experimental model of the Wernicke-Korsakoff syndrome (WKS). Assessment of the density of [3H]PK11195 binding sites in thiamine-deficient animals showing no neurologic signs of thiamine deficiency encephalopathy, and revealed no significant alterations compared with pair-fed control animals in any brain region studied. Densities of [3H]PK11195 binding sites were, however, significantly increased in brain regions of the rat at the symptomatic stage, where increased densities were seen in the inferior colliculus (233% increase, p < 0.001), inferior olivary nucleus (154% increase, p < 0.001) and thalamus (up to 107% increase, p < 0.001). Histologic studies of these same brain regions revealed evidence of neuronal cell loss and concomitant gliosis. Densities of [3H]PK11195 binding sites in nonvulnerable brain regions that showed no histologic evidence of neuronal loss, such as the cerebral cortex, hippocampus, and caudate-putamen, were not significantly different from those in control animals. Increased densities of binding sites for the PTBR ligand probably reflect glial proliferation and are consistent with an excitotoxic mechanism in the pathogenesis of neuronal cell loss in thiamine deficiency encephalopathy. Positron emission tomography (PET) using [11C]PK11195 could offer a potentially useful diagnostic tool in WKS in humans.

Peripheral benzodiazepine binding sites in Alzheimer's disease frontal and temporal cortex.

Much evidence has accumulated to suggest that the peripheral type benzodiazepine (PBZ) binding site has a predominantly glial localization. Elevated PBZ binding densities have been reported in various models of brain damage, apparently reflecting glial proliferation in response to neurodegeneration. In the present study, PBZ receptor densities were examined in frontal and temporal cortex of Alzheimer's disease (AD) patients using the ligand [3H]PK 11195. There was a highly significant (p less than 0.01) increase in PBZ binding sites in the temporal cortex from AD patients. In the frontal cortex, a moderate increase was observed that approached statistical significance (p = 0.07). Decreased choline acetyltransferase activity was observed in both regions. These findings offer support for the potential use of the PBZ binding site as a marker for gliosis associated with neuronal cell death.

Uptake and efflux of 14-C-dopamine in platelets: evidence for a generalized defect in Parkinson's disease.

Thirty-five parkinsonian patients (five untreated, six with levodopa only, seven with levodopa plus Ro 4-4602, nine with anticholinergic and/or antihistaminic medication, and eight with the anticholinergic/antihistaminic medication plus amantadine) and 35 age-matched control subjects were studied. Platelets isolated from each individual plasma were incubated with 14C-dopamine. Uptake was found to be decreased to a significant degree in all treated or untreated parkinsonian patients when compared with control subjects. Anticholinergic and/or antihistaminic medication, with or without amantadine, further decreased the dopamine uptake into platelets, while levodopa alone or with Ro 4-4602 returned uptake values to near normal. Dopamine efflux paralleled exactly the uptake values. The fact that parkinsonian platelets exhibit impaired dopamine uptake, while age-matched control platelets do not, constitutes the first direct evidence in favor of a generalized dopamine defect in Parkinson's disease.

Brain extracellular quinolinic acid in chronic experimental hepatic encephalopathy as assessed by in vivo microdialysis: acute effects of L-tryptophan.

Increased brain quinolinic acid (QUIN) levels have been suggested to play a role in hepatic encephalopathy (HE). Previous brain tissue studies have been unable to confirm this hypothesis. Because QUIn is a potent NMDA-receptor agonist, it also is relevant to determine brain extracellular QUIN levels in HE. For this purpose, we assessed frontal neocortical extracellular QUIN levels by in vivo microdialysis in rats subjected to a portacaval shunt (PCS). We also evaluated the acute effects of altered L-tryptophan (L-TRP) availability on brain extracellular QUIN levels. The basal extracellular L-TRP levels were significantly (p < .001) higher in the PCS rats than in the sham-operated controls. However, the QUIN level (p < .05) and the QUIN to L-TRP ratio (p < .01) were significantly lower in the PCS rats. Elevated L-TRP availability increased the QUIN levels to a similar degree in both sham and PCS rats. This study, in conjunction with our previous results, does thereby not support a major involvement of QUIN in the pathogenesis of HE.

Neurochemical and electrophysiological studies on the inhibitory effect of ammonium ions on synaptic transmission in slices of rat hippocampus: evidence for a postsynaptic action.

To elucidate the mechanisms involved in the inhibition of synaptic transmission by ammonium ions, the effects of NH4Cl on glutamate release and on synaptic transmission from Schaffer collaterals to CA1 pyramidal cells were measured in fully submerged slices of rat hippocampus. The large, Ca(2+)-dependent release of glutamate evoked by electrical-field stimulation or by 56 mM K+ was not reduced by 5 mM NH4Cl. In contrast, 5 mM NH4Cl decreased the smaller, field stimulation-induced release of glutamate observed in the presence of low concentrations of Ca2+ (0.1 mM), as well as the spontaneous release of glutamate both in normal and low Ca2+. Unlike the Ca(2+)-dependent release of glutamate, synaptic transmission was reversibly depressed even by 1 mM NH4 Cl. Firing of CA1 pyramidal cells evoked by iontophoretically applied glutamate was significantly inhibited by 2 or 5 mM NH4Cl. This depression was increased in the presence of 25 microM bicuculline. Results suggest that ammonium ions do not depress the Ca(2+)-dependent release of glutamate originating from synaptic vesicles, which is involved in synaptic transmission. Rather, ammonium ions inhibit synaptic transmission by a postsynaptic action, a conclusion strengthened by the inhibitory effect of NH4Cl on glutamate-induced firing. However, NH4Cl may inhibit the formation of cytoplasmic glutamate, the source of spontaneous and Ca(2+)-independent release.

Effect of sodium benzoate on cerebral and hepatic energy metabolites in spf mice with congenital hyperammonemia.

The sparse-fur (spf) mutant mouse has an X-linked deficiency of ornithine transcarbamylase and develops congenital hyperammonemia similar to that seen in human patients. We studied the effect of sodium benzoate (2.5, 5 and 10 mmol/kg body wt) on ammonia, glutamine and glutamate, as well as various intermediates of energy metabolism in brain and liver of normal CD-1/Y and hyperammonemic spf/Y mice. The ammonia concentration of brain was decreased with 2.5 mmol sodium benzoate in spf/Y mice, whereas higher doses resulted in a significant increase in both liver and brain. Cerebral glutamine content decreased generally in a dose-dependent manner, both in normal and affected mice, following treatment with various doses of sodium benzoate. Cerebral glutamate concentrations were increased only in spf mice treated with sodium benzoate, whereas ATP and acetyl CoA were decreased (P < 0.001), in both normal and affected mice, indicating that glutamine synthesis may be affected by ATP availability. Free CoA levels were decreased (P < 0.05) only in liver in both groups of treated mice, whereas pyruvate concentrations were elevated (P < 0.05) in affected mice following sodium benzoate administration. The results demonstrate that a dose of 2.5 mmol sodium benzoate/kg body wt has a beneficial effect in reducing cerebral ammonia with a concomitant decrease in glutamine. However, the results suggest that many of the metabolite changes observed following higher doses of benzoate could be due to depletion of ATP, free CoA and acetyl CoA levels, possibly secondary to benzoyl CoA accumulation. The response of the spf/Y mouse to sodium benzoate was different from that of the control CD-1/Y mouse, which could be due to its urea cycle dysfunction and a chronic hyperammonemic state. Hence, the spf/Y mouse may be the ideal animal model for studying the pharmacology of sodium benzoate in hyperammonemic disorders at both the cerebral and hepatic levels.

Trace amines in hepatic encephalopathy.

In 1971 Fischer and Baldessarini proposed the hypothesis that hepatic encephalopathy (HE), a neuropsychiatric syndrome associated with hepatic dysfunction, could result from the direct decarboxylation of amino acids leading to trace amines such as tyramine and octopamine which could then act as false neurotransmitters. This was supported by the observation that the clinical symptoms of HE appeared to improve following treatment with L-Dopa, which cannot be metabolized to either of these trace amines. In addition to serum and urine levels of octopamine correlating roughly with the grade of clinical HE, levels of octopamine were also significantly increased in rat brain following coma induced by hepatic devascularization and in portacaval-shunted rats fed high aromatic amino acid content diets. This hypothesis was questioned, however, given the lack of observable adverse behavioural effects following treatments with octopamine. Finally, the equivocal results of a limited number of clinical trials (using L-Dopa) argued against a direct intervention by catecholamine-like trace amines in HE. An alternative hypothesis was advanced by Sourkes in 1978 implicating increased tryptophan metabolism as a factor in the etiology of HE. Hepatic dysfunction in humans alters CNS concentrations of tryptophan which correlate well with levels of the tryptamine metabolite indoleacetic acid (IAA). Furthermore, regional densities of [3H]tryptamine receptors in HE patient brain tissue are significantly decreased. These data support a pathophysiologic role for tryptophan and its neuroactive trace amine metabolite tryptamine in HE.

A high-affinity [3H]tryptamine binding site in human brain.

In vitro filtration binding revealed high-affinity specific [3H]tryptamine binding sites in human brain. These binding sites are heterogeneously distributed throughout brain, ranging from 280 fmol/mg protein in hippocampus and thalamus to approximately 90 fmol/mg protein in medulla oblongata and cerebellum. Preliminary autoradiographic studies indicate a heterogeneous distribution within layers of the frontal cortex. The observed stereoselectivity of the site, the interaction of the site with a G protein and the observed region-selective downregulation of the site in a human pathological condition, i.e. hepatic encephalopathy (Mousseau et al., 1994), suggests that this binding site is a functional [3H]tryptamine receptor. A similarity in kinetics and distribution of the [3H]tryptamine receptor in human and rat brain indicates that these two entities represent homologous structures, although the difference in pharmacological profiles suggests species variants. One cannot exclude the possibility that the rat and human [3H]tryptamine receptors do represent distinct subtypes. Finally, the suggested role for tryptamine in neuropsychiatric disorders as originally suggested by Dewhurst (1968) is supported by the present series of experiments.

The astrocytic ("peripheral-type") benzodiazepine receptor: role in the pathogenesis of portal-systemic encephalopathy.

An increasing body of evidence supports the notion that activation of astrocytic (peripheral-type) benzodiazepine receptors contributes to the pathogenesis of the central nervous system symptoms which are characteristic of portal-systemic encephalopathy (PSE). Binding site densities for the PTBR ligand [3H-PK11195] are increased in autopsied brain tissue from PSE patients as well as in the brains of animals with experimental chronic liver failure. In the case of the animal studies, increased PTBR sites resulted from increased PTBR gene expression. Exposure of cultured astrocytes to ammonia or manganese (two neurotoxic agents which under normal circumstances are removed by the hepatobiliary system and which are found to accumulate in brain in PSE) results in increased densities of [3H-PK11195] binding sites. Activation of PTBR is known to result in increased cholesterol uptake and increased synthesis in brain of neurosteroids some of which have potent positive allosteric modulator properties on the GABA-A receptor system. Accumulation of such substances in the brain in chronic liver failure could explain the neural inhibition characteristics of PSE.

Neurotransmitter function in thiamine-deficiency encephalopathy.

The encephalopathy caused by severe thiamine depletion of the mammalian CNS is accompanied by regionally selective changes in neurotransmitter function. Thiamine deficiency induced by administration of the central thiamine antagonist, pyrithiamine, causes more widespread lesions and accompanying changes in neurotransmitter function than does the deficiency state induced by chronic deprivation of the vitamin. There is convincing evidence for a central muscarinic cholinergic lesion in pyrithiamine-treated rats and neuropharmacological studies show that this lesion is partially responsible for the neurological deficit resulting from this treatment. There is also good evidence to suggest that thiamine deprivation selectively affects cerebellar afferent and efferent systems. Included in these are a loss of serotoninergic mossy fibres and of the functional integrity of glutamatergic granule cells. In addition, abnormalities of both nerve terminals and glial cells are found in lateral vestibular nucleus and it has been proposed that a loss of Purkinje cell terminals and concomitant decreases of pontine GABA may reflect these changes. The selective vulnerability of brain structures to thiamine deprivation is reflected in (i) the turnover rate of total thiamine in these areas and (ii) the selective decreases in activity of the thiamine pyrophosphate dependent enzyme pyruvate dehydrogenase.

Effects of maternal thiamine deficiency on the development of thiamine-dependent enzymes in regions of the rat brain.

Previous studies suggest that developing rat brain is susceptible to reduced thiamine intake. In order to assess the metabolic basis for this susceptibility, activities of three thiamine-dependent enzymes (pyruvate dehydrogenase complex, ?-ketoglutarate dehydrogenase and transketolase) were measured in homogenates of brain tissue from the offspring of thiamine-deficient mothers. Control groups of animals were pair-fed to equal food consumption with the thiamine-deficient animals. The study revealed region-selective delays in the establishment of adult activities of thiamine-dependent enzymes as a result of maternal thiamine deficiency. Pyruvate dehydrogenase complex activities in cerebral cortex were significantly reduced (by 20% P < 0.05); ?-ketoglutarate dehydrogenase activities were also reduced in cerebral cortex (by 30% P < 0.05). In the case of transketolase, enzyme activities were significantly reduced in cerebral cortex, cerebellum and brainstem. Following thiamine replenishment, defective enzyme activities were restored to normal in all cases. However, since thiamine-dependent enzymes are important for the establishment of adult patterns of cerebral energy metabolism and also in myelin synthesis, maternal thiamine deficiency resulting in reductions of thiamine-dependent enzymes at a vulnerable period in brain development could have serious metabolic consequences leading to permanent neurological sequellae in the offspring.

Increased expression of glyceraldehyde-3-phosphate dehydrogenase in cultured astrocytes following exposure to manganese.

Manganism is a disorder characterized by hyperintensities in basal ganglia structures on magnetic resonance imaging which may be the consequence of manganese deposition in these areas. Since manganese is taken up avidly into astrocytes and is known to interfere with cerebral energy metabolism, we studied the effect of this metal on the expression and activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in primary cultures of astrocytes. Treatment with 100 microM manganese for 7 days increased both the Vmax and Km values for GAPDH which was not reproducible with other divalent metals. Using RT-PCR, increased GAPDH expression was detected in cells exposed to manganese compared with controls. No changes in cytochrome oxidase activity or ATP levels were observed, and lactate production was unaffected, in manganese-treated cells. These findings provide evidence of a possible role for GAPDH in the mediation of the effects of manganese on central nervous system function.

Evidence for a serotonin transporter deficit in experimental acute liver failure.

It has been suggested that alterations of serotonin transport may be implicated in the pathogenesis of the neuropsychiatric symptoms encountered in acute liver failure. In order to address this issue, microdialysate concentrations of serotonin, its precursor L-tryptophan and metabolite 5-hydroxyindoleacetic acid (5-HIAA) as well as brain regional distribution of serotonin transporter ([3H]-citalopram) sites were measured in rats with acute liver failure resulting from hepatic devascularization. A significant loss of [3H]-citalopram sites was observed in dorsal Raphe nucleus, in frontal and frontoparietal cortices as well as in substantia nigra of rats with severe encephalopathy resulting from acute liver failure. In frontal cortex, this loss of transporter binding sites was accompanied by significant increases of L-tryptophan, serotonin and 5-HIAA concentrations in extracellular fluid. Pharmacological manipulation of the brain serotonin system could afford a novel therapeutic approach to the prevention of the neuropsychiatric symptoms characteristic of acute liver failure in humans.

Loss of noradrenaline transporter sites in frontal cortex of rats with acute (ischemic) liver failure.

There is increasing evidence that central noradrenaline (NA) transport mechanisms are implicated in the central nervous system complications of acute liver failure. In order to assess this possibility, binding sites for the high affinity NA transporter ligand [3H]-nisoxetine were measured by quantitative receptor autoradiography in the brains of rats with acute liver failure resulting from hepatic devascularization and in appropriate controls. In vivo microdialysis was used to measure extracellular brain concentrations of NA. Severe encephalopathy resulted in a significant loss of [3H]-nisoxetine sites in frontal cortex and a concomitant increase in extracellular brain concentrations of NA in rats with acute liver failure. A loss of transporter sites was also observed in thalamus of rats with acute liver failure. This loss of NA transporter sites could result from depletion of central NA stores due to a reserpine-like effect of ammonia which is known to accumulate to millimolar concentrations in brain in ischemic liver failure. Impaired NA transport and the consequent increase in synaptic concentrations and increased stimulation of neuronal and astrocytic noradrenergic receptors could be implicated in the pathogenesis of the encephalopathy and brain edema characteristic of acute liver failure.

Effects of ammonia on glutamate transporter (GLAST) protein and mRNA in cultured rat cortical astrocytes.

Ammonia is a neurotoxic substance which accumulates in brain in liver failure and it has been suggested that ammonia plays a key role in contributing to the astrocytic dysfunction characteristic of hepatic encephalopathy. In particular, the effects of ammonia may be responsible for the reduced astrocytic uptake of neuronally-released glutamate and high extracellular glutamate levels consistently seen in experimental models of hepatic encephalopathy. To further address this issue, [(3)H]-D-aspartate uptake was examined in primary rat cortical astrocyte cultures exposed to 5 mM ammonium chloride for a period of 7 days. In addition, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot studies were performed to examine the mRNA and protein expression respectively of the glutamate transporter GLAST in ammonia-treated cells. Studies revealed a 57% (p<0.05) decrease in [(3)H]-D-aspartate uptake and a concomitant significant decrease in GLAST transporter protein (43%, p<0.05) and mRNA (32%, p<0.05) expression. The reduced capacity of astrocytes to reuptake glutamate following ammonia exposure may result in compromised neuron-astrocyte trafficking of glutamate and could thus contribute to the pathogenesis of the cerebral dysfunction characteristic of hyperammonemic syndromes such as hepatic encephalopathy.

Portacaval anastomosis causes selective alterations of peripheral-type benzodiazepine receptor expression in rat brain and peripheral tissues.

There is a growing body of evidence to suggest that peripheral-type benzodiazepine receptors (PTBRs) and their endogenous ligands are implicated in the pathogenesis of end-organ failure in chronic liver disease. Portal-systemic encephalopathy, a major neuropsychiatric complication associated with chronic liver disease, results in activation of brain PTBR and probably in peripheral organs. In order to address these issues, PTBR mRNA was measured using semi-quantitative RT-PCR in extracts of cerebral cortex, kidney and testis of rats four weeks after end-to-side portacaval anastomosis and sham-operation (controls). Densities of PTBR sites were measured concomitantly by in vitro receptor binding using the selective PTBR ligand [3H]PK11195. Portacaval shunting resulted in a 2 to 3-fold increase in expression of PTBR in brain and kidney and a 37% reduction in expression in testis. Densities of [3H]PK11195 sites changed in parallel with the alterations of gene expression. These findings suggest that selective alterations of PTBR expression are implicated in the pathogenesis of peripheral tissue hypertrophy (kidney) and/or atrophy (testis) which accompanies portal-systemic shunting in chronic liver failure. In brain, activation of PTBR could result in an increase in the production of neurosteroids with potent inhibitory action in the CNS, which could contribute to the pathogenesis of portal-systemic encephalopathy.

Increased "peripheral-type" benzodiazepine receptor sites and mRNA in thalamus of thiamine-deficient rats.

"Peripheral-type" benzodiazepine receptors (PTBRs) are highly expressed on the outer mitochondrial membrane of several types of glial cells. In order to further elucidate the nature of the early glial cell changes in thiamine deficiency, PTBR sites and PTBR mRNA were measured in thalamus, a brain structure which is particularly vulnerable to thiamine deficiency, of thiamine-deficient rats at presymptomatic and symptomatic stages of deficiency. PTBR sites were measured using an in vitro binding technique and the selective radio ligand [3H]-PK11195. PTBR gene expression was measured by RT-PCR using oligonucleotide primers based upon the published sequence of the cloned rat PTBR. Microglial and astrocytic changes in thalamus due to thiamine deficiency were assessed using immunohistochemistry and antibodies to specific microglial (ED-1) and astrocytic (GFAP) proteins respectively. Significant increases of [3H]-PK11195 binding sites and concomitantly increased PTBR mRNA were observed in thalamus at the symptomatic stage of thiamine deficiency, coincident with severe neuronal cell loss and increased GFAP-immunolabelling (indicative of reactive gliosis). Positron Emission Tomography using 11C-PK11195 could provide a novel approach to the diagnosis and assessment of the extent of thalamic damage due to thiamine deficiency in humans with Wernicke's Encephalopathy.

Mechanisms of selective neuronal cell death due to thiamine deficiency.

Multiple mechanisms contribute to the selective brain lesions observed in WKS and experimental thiamine deficiency. Recent evidence of early microglial activation and increased free radical production suggest that oxidative stress processes play an important early role in the brain damage associated with thiamine deficiency.

Effect of L-carnitine on cerebral and hepatic energy metabolites in congenitally hyperammonemic sparse-fur mice and its role during benzoate therapy.

Sparse-fur (spf) mutant mice with X-linked ornithine transcarbamylase deficiency were used to study the effect of L-carnitine on energy metabolites in congenital hyperammonemia. L-Carnitine was used at doses of 2, 4, 8, or 16 mmol/kg body weight (BW), and levels of ammonia, glutamine, glutamate, and some intermediates of energy metabolism were measured in brain and liver of spf/Y mice. Cerebral and hepatic levels of ammonia were decreased with 4 mmol L-carnitine (P < .001), whereas other doses did not seem to have any effect on this metabolite. Cerebral levels of glutamine were decreased following administration of L-carnitine at doses of up to 4 mmol/kg BW, whereas hepatic glutamine levels remained unaltered at all doses of L-carnitine. Both cerebral and hepatic levels of pyruvate, lactate, and alpha-ketoglutarate were decreased at doses of up to 8 mmol L-carnitine/kg BW. L-Carnitine treatment elevated adenosine triphosphate (ATP), free coenzyme A (CoA), and acetyl CoA levels in both brain and liver of spf/Y mice. Cytosolic and mitochondrial redox ratios of spf/Y mice, which were altered by congenital chronic hyperammonemia, were partially corrected by L-carnitine administration. L-Carnitine supplementation to spf/Y mice during sodium benzoate therapy also restored the availability of free CoA and ATP, thus counteracting the adverse effects of higher doses of sodium benzoate. These changes in free CoA and acetyl CoA levels could be due to the deinhibition of pantothenate kinase and stimulation of fatty acid oxidation by L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)