Mild hypothermia prevents cerebral edema and CSF lactate accumulation in acute liver failure.

Evidence from both clinical and experimental studies demonstrates that mild hypothermia prevents encephalopathy and brain edema in acute liver failure (ALF). As part of a series of studies to elucidate the mechanism(s) involved in this protective effect, groups of rats with ALF resulting from hepatic devascularization were maintained at either 37 degrees C (normothermic) or 35 C (hypothermic), and neurological status was monitored in relation to cerebrospinal fluid (CSF) concentrations of ammonia and lactate. CSF was removed via implanted cisterna magna catheters. Mild hypothermia resulted in a delay in onset of encephalopathy and prevention of brain edema, CSF concentrations of ammonia and lactate were concomitantly decreased. Blood ammonia concentrations, on the other hand, were not affected by hypothermia in ALF rats. These findings suggest that brain edema and encephalopathy in ALF are the consequence of ammonia-induced impairment of brain energy metabolism and open the way for magnetic resonance spectroscopic monitoring of cerebral function in ALF. Mild hypothermia could be beneficial in the prevention of severe encephalopathy and brain edema in patients with ALF awaiting liver transplantation.

Assessment of bioaccumulation and neurotoxicity in rats with portacaval anastomosis and exposed to manganese phosphate: a pilot study.

The use of the additive methylcyclopentadienyl manganese tricarbonyl in unleaded gasoline has resulted in increased attention to the potential toxic effects of manganese (Mn). Hypothetically, people with chronic liver disease may be more sensitive to the adverse neurotoxic effects of Mn. In this work, bioaccumulation of Mn, as well as histopathology and neurobehavioral damage, in end-to-side portacaval anastomosis (PCA) rats exposed to Mn phosphate via inhalation was investigated. During the week before the PCA operation, 4 wk after the PCA operation, and at the end of exposure, the rats were subjected to a locomotor evaluation (day-night activities) using a computerized autotrack system. Then a group of 6 PCA rats (EXP) was exposed to 3050 microg m(-3) (Mn phosphate) for 8 h/day, 5 days/wk for 4 consecutive weeks and compared to a control group (CON), 7 PCA rats exposed to 0.03 microg m(-3). After exposure, the rats were euthanized and Mn content in tissues and organs was determined by neutron activation analysis. The manganese concentrations in blood (0.05 microg/g vs. 0.02 microg/g), lung (1.32 microg/g vs. 0.24 microg/g), cerebellum (0.85 microg/g vs. 0.64 microg/g), frontal cortex (0.87 microg/g vs. 0.61 microg/g), and globus pallidus (3.56 microg/g vs. 1.33 microg/g) were significantly higher in the exposed group compared to the control group (p <.05). No difference was observed in liver, kidney, testes, and caudate putamen between the two groups. Neuronal cell loss was assessed by neuronal cell counts. The loss of cells in globus pallidus and caudate putamen as well as in frontal cortex was significantly higher (p <.05) for the EXP group. Assessment of the locomotor activities did not reveal any significant difference. This study constitutes a first step toward our understanding of the potential adverse effects of Mn in sensitive populations.

L-ornithine-L-aspartate in experimental portal-systemic encephalopathy: therapeutic efficacy and mechanism of action.

Strategies aimed at the lowering of blood ammonia remain the treatment of choice in portal-systemic encephalopathy (PSE). L-ornithine-L-aspartate (OA) has recently been shown to be effective in the prevention of ammonia-precipitated coma in humans with PSE. These findings prompted the study of mechanisms of the protective effect of OA in portacaval-shunted rats in which reversible coma was precipitated by ammonium acetate administration (3.85 mmol/kg i.p.). OA infusions (300 mg/kg/h, i.v) offered complete protection in 12/12 animals compared to 0/12 saline-infused controls. This protective effect was accompanied by significant reductions of blood ammonia, concomitant increases of urea production and significant increases in blood and cerebrospinal fluid (CSF) glutamate and glutamine. Increased CSF concentrations of leucine and alanine also accompanied the protective effect of OA. These findings demonstrate the therapeutic efficacy of OA in the prevention of ammonia-precipitated coma in portacaval-shunted rats and suggest that this protective effect is both peripherally-mediated (increased urea and glutamine synthesis) and centrally-mediated (increased glutamine synthesis).

Protective effect of L-carnitine in ammonia-precipitated encephalopathy in the portacaval shunted rat.

L-carnitine administration prevents the neurological symptoms of acute ammonia toxicity. To further evaluate its efficacy in the prevention of hepatic encephalopathy in hyperammonemic conditions, L-carnitine (16 mmol/kg, intraperitoneally [i.p.] was administered 1 hour before ammonium acetate (NH4OAc) (8.5 mmol/kg, subcutaneously) to portacaval shunted (PCS) rats. Cerebrospinal fluid (CSF) ammonia, lactate, and amino acid levels were measured in relation to deteriorating neurological status in these animals. None of 35 L-carnitine-treated animals showed neurological deterioration after NH4OAC administration compared with saline-treated controls; the latter manifested severe encephalopathy progressing through loss of righting reflex to coma. Survival rate was 100% in the L-carnitine-treated group compared with 5% in saline-treated controls. Following NH4OAC administration to PCS rats, CSF ammonia increased to 0.93 +/- 0.15 mmol/L and 1.24 +/- 0.15 mmol/L at precoma and coma stages of encephalopathy (P < .01) respectively. Treatment with L-carnitine reduced CSF ammonia at both precoma and coma stages; the time-course of this protective effect paralleled blood and CSF L-carnitine accumulation. CSF alanine and lactate increases following NH4OAC administration to PCS rats were significantly attenuated following L-carnitine treatment. However, L-carnitine treatment did not lead to significant reductions in plasma ammonia nor CSF or brain glutamine in these animals. These findings show the therapeutic efficacy of L-carnitine in ammonia-precipitated coma in PCS rats and suggest that this protective effect is centrally mediated involving improved mitochondrial respiration. L-carnitine could be of therapeutic benefit in the prevention of hepatic encephalopathy precipitated by ammoniagenic conditions in humans with chronic liver disease.

Increased blood manganese in cirrhotic patients: relationship to pallidal magnetic resonance signal hyperintensity and neurological symptoms.

Increasing evidence suggests that manganese deposition is responsible for the T1-weighted magnetic resonance imaging (MRI) signal hyperintensity consistently observed in pallidum of cirrhotic patients. However, the relationship between blood manganese and the etiology or severity of liver disease, as well as the neurological symptomatology in these patients, has not been well established. In the present study, blood manganese concentrations were measured by atomic absorption spectrometry together with MRI and neurological evaluation in 57 cirrhotic patients with various etiologies and severity of liver disease. Blood manganese concentrations were elevated in 67% of cirrhotic patients and were significantly higher in patients with previous portacaval anastomoses or transjugular intrahepatic portosystemic shunt (TIPS). Pallidal signal hyperintensity was observed in 88% of patients, and significant correlations were demonstrated between blood manganese and pallidal index (PI) (a measure of pallidal signal hyperintensity), as well as Child-Pugh score. Assessment of extrapyramidal symptoms using the Columbia rating scale revealed a significant incidence of tremor, rigidity, or akinesia in up to 89% of cirrhotic patients. However, there was no significant correlation between blood manganese and extrapyramidal symptoms, although severity of akinesia was significantly greater in Child-Pugh C patients. Extrapyramidal symptoms could result from a toxic effect of manganese on basal ganglia dopaminergic function. These findings further support a role for manganese in the etiology of pallidal MRI signal hyperintensity in patients with chronic liver disease.

Portacaval anastomosis results in altered neuron--astrocytic metabolic trafficking of amino acids: evidence from 13C-NMR studies.

13C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-13C]glucose, whereas [2-13C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-13C]glucose as precursor. Brain [1-13C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The 13C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-13C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats.

Choline acetyltransferase and acetylcholinesterase activities are unchanged in brain in human and experimental portal-systemic encephalopathy.

Activities of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase were studied in the frontal cortex, temporal cortex, cerebellum and caudate nucleus obtained at autopsy from eight alcoholic cirrhotic patients who died in hepatic coma and from an equal number of age-matched subjects free from hepatic, neurological or psychiatric disorders. Activities of these enzymes were unaltered in the brains of cirrhotics compared to controls. Choline acetyltransferase and cholinesterase activities were also studied in the cerebral cortex, cerebellum, brain stem and striatum of rats four weeks following portacaval anastomosis and their sham-operated controls. Portacaval-shunting did not cause any statistically significant differences in the activities of choline acetyltransferase, acetyl or butyrylcholinesterases. These results argue against a presynaptic cholinergic lesion in human and experimental portal-systemic encephalopathy.

Effects of ornithine aminotransferase inactivation by 5-fluoromethylornithine in rats following portacaval anastomosis.

5-Fluoromethylornithine (5FMOrn) is a selective inactivator of ornithine aminotransferase. Administration of this compound to rodents causes a prominent increase of tissue ornithine concentrations, and prevents the neurological consequences of acute ammonia intoxication. However, long-term treatment with 5FMOrn of rats with portacaval shunts did not result in decreased circulating ammonia concentrations, nor did it prevent other pathologic manifestations of shunting. The sensitivity to ammonia intoxication of rats with portacaval shunts was also unaffected by pretreatment with 5FMOrn, although liver ornithine concentrations were significantly elevated; specific activities of urea cycle enzymes were slightly higher in portacaval shunted compared to sham-operated controls following 5-FMOrn treatment. Administration of 5FMOrn dramatically elevated urinary excretion of several amino acids in rats with portacaval shunts, but not in sham-operated animals, suggesting that the reabsorption of amino acids from the glomerular filtrate may be impaired in shunted rats. These results suggest that, in contrast to acute hyperammonemic syndromes, 5-FMOrn may be of limited therapeutic value in chronic hyperammonemia syndromes in which there is significant portal-systemic shunting.

Densities of binding sites for the "peripheral-type" benzodiazepine receptor ligand 3H-PK11195 are increased in brain 24 hours following portacaval anastomosis.

Quantitative receptor autoradiography was used to measure the densities of binding sites for the "peripheral-type" benzodiazepine receptor ligand 3H-PK11195 in regions of the rat brain 1, 3, 7 and 28 days following portacaval anastomosis (PCA) and in sham-operated control animals. The results demonstrate that densities of 3H-PK11195 binding sites were significantly increased in the cerebral cortex (by 40%, p < 0.05) as early as 24 hours following PCA. In the thalamus significant increases in densities of 3H-PK11195 binding sites were seen 3 days after PCA, whereas in brain regions such as the striatum and cerebellum, significant increases in 3H-PK11195 binding sites were not evident until 7 days following PCA. By 28 days following PCA increased densities of 3H-PK11195 binding sites were well established and widespread throughout the brain. Previous studies demonstrate early increases of brain ammonia following PCA. PTBRs or their endogenous ligands could play an important role in the early astrocytic response (mitochondrial proliferation, swelling) to ammonia following PCA.

Ammonia-induced brain edema and intracranial hypertension in rats after portacaval anastomosis.

Brain edema, leading to intracranial hypertension and brain herniation, is a major cause of death in fulminant liver failure. Astrocyte swelling is a prominent neuropathological feature in experimental fulminant liver failure. It has been postulated that the osmotic effects of glutamine, generated in astrocytes from ammonia and glutamate in a reaction catalyzed by glutamine synthetase, could mediate brain swelling. Normal rats and rats that received a portacaval anastomosis were infused with ammonium acetate or a sodium acetate control; brain water in cerebral cortex was measured with the gravimetry method, intracranial pressure by means of a cisterna magna catheter and cortical amino acids using high-performance liquid chromatography. Although brain edema was detected in both groups receiving ammonia, it was of a greater magnitude in portacaval anastomosis rats (80.94% + 0.17% vs. 80.24% + 0.09%, p < 0.01), resulting in the development of intracranial hypertension. When portacaval anastomosis rats were infused with ammonium acetate and pretreated with 150 mg/kg methionine-sulfoximine, an inhibitor of glutamine synthetase activity, brain edema was ameliorated and intracranial pressure did not rise. A dose-dependent reduction in brain glutamine levels was seen with increasing doses of methionine-sulfoximine; however, brain edema did not decrease beyond the 150 mg/kg dose, suggesting that the increase in brain water was not solely a result of glutamine accumulation. We conclude that brain edema of a magnitude that results in intracranial hypertension is more likely to develop in rats after portacaval anastomosis receiving a continuous ammonia infusion. The osmotic effects of glutamine appear to mediate, but only in part, the increase in brain water seen in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-specific alterations of binding sites for peripheral-type benzodiazepine receptor ligand [3H]PK11195 in rats following portacaval anastomosis.

Kinetics of binding of [3H]PK11195, an antagonist ligand with high selectivity for the peripheral-type (mitochondrial) benzodiazepine receptor (PTBR), was studied in homogenates of cerebral cortex, kidney, heart, and testis of portacaval shunted rats and sham-operated controls. Portacaval anastomosis resulted in a significant two- to threefold increase in the number of [3H]PK11195 binding sites in cerebral cortex and kidney. A reduction in the number of [3H]PK11195 binding sites was observed in testis preparations, while the number of binding sites in the heart remained unaltered. These differences in the response of PTBRs to portacaval anastomosis, in different organs suggest that the physiological function of these receptors and the factors regulating them are modulated by distinct mechanisms. The finding of increased densities of [3H]PK11195 binding sites in brain and kidney following portacaval anastomosis parallels the cellular hypertrophy in these tissues and, together with previous observations of similar increases of these binding sites in brain and kidney in congenital hyperammonemia, suggest a pathophysiologic role for ammonia in these changes. In contrast, the significant loss of [3H]PK11195 binding sites in testicular preparations following portacaval anastomosis together with the known effects of steroid hormones on these sites suggests a role for PTBRs in the pathogenesis of testicular atrophy in chronic liver disease.

Determination of orotic acid in urine.

Orotic acid was separated from other urinary constituents by ion-pair formation with tetrabutylammonium, and isocratic elution from a reversed-phase column. Absorbance at 280 nm was recorded for quantitation. Owing to the better column characteristics the separations are somewhat faster, and the sensitivity of the method is higher than those of analogous methods using anion-exchange columns. The method was used for the determination of orotic acid in human urine, in urine of rats with portacaval shunts and in small (30 microliters) urine samples from sparse fur mice. Shunted rats excreted ca. 100% more orotic acid per 24 h than sham-operated controls, in spite of their considerably lower body weight. Excessive orotic acid in urine indicates a conditional deficiency of ornithine. Sparse fur mice are congenitally hyperammonemic because of a defective hepatic ornithine carbamoyltransferase. Determination of orotic acid in the urine is a suitable method to identify those animals among litter mates which have the hereditary enzyme defect.

Increased cerebrospinal fluid lactate reflects deterioration of neurological status in experimental portal-systemic encephalopathy.

Increased brain and CSF lactate have been described in human and experimental portal-systemic encephalopathy (PSE). Using a recently described cisterna magna catheter technique, CSF lactate was measured in relation to deterioration of neurological status in portacaval shunted rats administered ammonium acetate to precipitate severe PSE. Loss of righting reflex (precoma stage of PSE) was accompanied by 2-3 fold increased CSF lactate and onset of coma by 4-fold increases of lactate (p less than 0.001 compared to either sodium acetate treated portacaval shunted rats or sham-operated controls administered ammonium acetate). The most likely explanation for increased CSF lactate is ammonia-induced inhibition of malate-aspartate shuttle and/or inhibition of tricarboxylic acid cycle flux in brain. Similar mechanisms could be involved in the pathogenesis of PSE in patients with chronic liver disease.

Cerebrospinal fluid amino acids in relation to neurological status in experimental portal-systemic encephalopathy.

Using an indwelling cisterna magna catheter technique, serial CSF samples were analyzed for amino acid content in rats at various stages of portal-systemic encephalopathy resulting from ammonium acetate administration following portacaval anastomosis. Anastomosis alone resulted in increased CSF concentrations of glutamine, tyrosine, phenylalanine, glutamate and alanine. GABA levels, on the other hand were not significantly changed. Onset of severe neurological symptoms following ammonium acetate administration resulted in selectively increased CSF alanine. Other amino acids were not further increased at severe stages of encephalopathy. Increased CSF alanine probably results from increased glutamine transamination in the brains of portacaval shunted rats.