Protective effect of long term ammonium ingestion against acute ammonium intoxication

Rats were fed for 15 days a diet containing ammonium acetate (20% w/w) and then injected i. p. with ammonium acetate (7 mmol/Kg). Only 1 out of 18 control rats but 9 of 18 rats fed ammonium survived, indicating a protective effect of ammonium ingestion against an acute ammonia challenge. Blood ammonia returned to normal levels sooner in hyperammonemic rats, suggesting more rapid detoxication. In controls, blood urea levels rose immediately reaching a maximum at 15 min, however in hyperammonemic rats urea levels did not change during the first hour, then rose slowly up to 3 hours. These results suggest that in the ammonium fed rats ammonia is initially sequestered and finally eliminated as urea.     

Morin a flavonoid exerts antioxidant potential in chronic hyperammonemic rats: a biochemical and histopathological study

Plant flavonoids are emerging as potent therapeutic drugs effective against a wide range of free radical-mediated diseases. Morin (3,5,7,2′,4′-pentahydroxyflavone), a member of flavonols, is an important bioactive compound by interacting with nucleic acids, enzymes and protein. In this study, we found that morin (30 mg/kg body weight) by oral administration offers protection against hyperammonemia by means of reducing blood ammonia, oxidative stress and enhancing antioxidant status in ammonium chloride-induced (100 mg/kg body weight; i.p) hyperammonemic rats. Enhanced blood ammonia, plasma urea, lipid peroxidation in circulation and tissues (liver and brain) of ammonium chloride-treated rats was accompanied by a significant decrease in the tissues levels of superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione peroxidase (GPx). Morin administered rats showed a significant reduction in ammonia, urea, lipid peroxidation with a simultaneous elevation in antioxidant levels. Cotreatment with morin prevented the elevation of liver marker enzymes induced by ammonium chloride. The body weight of the animals decreased significantly on ammonium chloride administration when compared with control group. However, cotreatment with morin significantly prevented the decrease of the body weight caused by ammonium chloride. Hyperammonemic rats show liver fibrosis, steatosis, sinusoidal dilatation, etc., along with necrosis, microcystic degeneration in brain. All these changes were reduced in hyperammonemic rats treated with Morin, which too correlated with the biochemical observations. In conclusion, these findings indicate that morin exert antioxidant potential and offer protection against ammonium chloride-induced hyperammonemia. But the exact underlying mechanism needs to be elucidated.     

Paradoxical protection of both protein-free and high protein diets against acute ammonium intoxication

Rats were fed standard (20% protein), protein-free or high protein (80%) diets for 15 days and then injected intraperitoneally with ammonium acetate (7 mmol/Kg). Survival was 6%, 75% and 100%, respectively, for rats fed standard, protein-free and high protein diets. After injection of 6 mmol/Kg of ammonium acetate, blood ammonia reached a peak (at ca. 2 mM) after 7, 25 and 30 min for rats fed high protein, protein-free and standard diets, respectively. The results presented indicate that protection in the high protein group is due to faster detoxication of ammonia via a more active urea cycle while the tolerance of the protein-free group to higher levels of ammonia remains to be clarified.     

Ammonium ingestion prevents depletion of hepatic energy metabolites induced by acute ammonium intoxication

Ingestion of an ammonium containing diet produces hyperammonemia and protects rats against acute ammonium intoxication. Acute ammonium toxicity has been attributed to the depletion of energy metabolite intermediates. We show here that hyperammonemia affords considerable protection against depletion of hepatic energy metabolites evoked by ammonium acetate injection. In control rats there were marked decreases in the content of acetoacetate, beta-hydroxybutyrate, ATP, 2-oxoglutarate, lactate, and pyruvate while phosphoenolpyruvate increased markedly. In hyperammonemic rats beta-hydroxybutyrate, ATP, 2-oxoglutarate, and lactate were not significantly affected while pyruvate increased markedly and phosphoenolpyruvate slightly. These results suggest that in controls the activity of pyruvate kinase is inhibited after ammonium injection while in hyperammonemic rats it is not inhibited. The content of alanine (an inhibitor of pyruvate kinase) reached 2.8 mumol/g in controls and 1.6 mumol/g in hyperammonemic rats, 15 min after ammonium injection. This could explain the different effects of ammonium injection on control and hyperammonemic rats.     

A single transient episode of hyperammonemia induces long-lasting alterations in protein kinase A

Hepatic encephalopathy in patients with liver disease is associated with poor prognosis. This could be due to the induction by the transient episode of hepatic encephalopathy of long-lasting alterations making patients more susceptible. We show that a single transient episode of hyperammonemia induces long-lasting alterations in signal transduction. The content of the regulatory subunit of the protein kinase dependent on cAMP (PKA-RI) is increased in erythrocytes from cirrhotic patients. This increase is reproduced in rats with portacaval anastomosis and in rats with hyperammonemia without liver failure, suggesting that hyperammonemia is responsible for increased PKA-RI in patients. We analyzed whether there is a correlation between ammonia levels and PKA-RI content in patients. All cirrhotic patients had increased content of PKA-RI. Some of them showed normal ammonia levels but had suffered previous hyperammonemia episodes. This suggested that a single transient episode of hyperammonemia could induce the long-lasting increase in PKA-RI. To assess this, we injected normal rats with ammonia and blood was taken at different times. Ammonia returned to basal levels at 2 h. However, PKA-RI was significantly increased in blood cells from rats injected with ammonia 3 wk after injection. In conclusion, it is shown that a single transient episode of hyperammonemia induces long-lasting alterations in signal transduction both in blood and brain. These alterations may contribute to the poor prognosis of patients suffering hepatic encephalopathy.     

The effect of adrenal denervation on the metabolic effects of hyperammonemia in sheep

The metabolic effect of intravenous infusion of ammonium chloride (60 mumol/(kg body weight.min] was compared in five sheep before and after adrenal denervation. Adrenal denervation completely abolished the hyperglycemic effect of ammonium chloride, diminished the rise of pyruvate and lactate concentration, and failed to influence the lipolytic effect of NH4Cl. It is suggested that the metabolic effects of ammonia are in a different degree related to the action of ammonia on the central nervous system and (i) the hyperammonemic effect of ammonia completely depends on the neurogenic increase of adrenal medullary hormones; (ii) the rise of blood lactate and pyruvate level observed during hyperammonemia is only partially mediated by adrenaline; and (iii) the lipolytic effect of ammonia ion does not depend on the nerve-controlled secretion of adrenal medullary hormones.     

The effect of protein content of the diet on the rate of urea formation in sheep liver

1. In the livers of six sheep given a high-protein diet, the concentrations of certain urea-cycle enzymes [ornithine transcarbamoylase, arginine synthetase (combined activity of argininosuccinate synthetase and argininosuccinase) and arginase] were significantly greater than when the sheep were given a low-protein diet. Alkaline phosphatase activity/mg. of liver protein was not significantly affected by diet. 2. Three sheep previously given the high-protein diet showed no significant rise in the concentration of ammonia in the blood after the administration of urea (0·5g./kg. body wt.). The concentration of ammonia in the blood of the three sheep given the low-protein diet rose exponentially with time after dosing with urea and all sheep died. 3. It is suggested that tolerance to ammonia toxicity in the sheep is at least partly a function of the activity of the urea-cycle enzymes in the liver.     

Glutamine synthetase in brain: effect of ammonia

Glutamine synthetase (GS) in brain is located mainly in astrocytes. One of the primary roles of astrocytes is to protect neurons against excitotoxicity by taking up excess ammonia and glutamate and converting it into glutamine via the enzyme GS. Changes in GS expression may reflect changes in astroglial function, which can affect neuronal functions.Hyperammonemia is an important factor responsible of hepatic encephalopathy (HE) and causes astroglial swelling. Hyperammonemia can be experimentally induced and an adaptive astroglial response to high levels of ammonia and glutamate seems to occur in long-term studies. In hyperammonemic states, astroglial cells can experience morphological changes that may alter different astrocyte functions, such as protein synthesis or neurotransmitters uptake. One of the observed changes is the increase in the GS expression in astrocytes located in glutamatergic areas. The induction of GS expression in these specific areas would balance the increased ammonia and glutamate uptake and protect against neuronal degeneration, whereas, decrease of GS expression in non-glutamatergic areas could disrupt the neuron-glial metabolic interactions as a consequence of hyperammonemia.Induction of GS has been described in astrocytes in response to the action of glutamate on active glutamate receptors. The over-stimulation of glutamate receptors may also favour nitric oxide (NO) formation by activation of NO synthase (NOS), and NO has been implicated in the pathogenesis of several CNS diseases. Hyperammonemia could induce the formation of inducible NOS in astroglial cells, with the consequent NO formation, deactivation of GS and dawn-regulation of glutamate uptake. However, in glutamatergic areas, the distribution of both glial glutamate receptors and glial glutamate transporters parallels the GS location, suggesting a functional coupling between glutamate uptake and degradation by glutamate transporters and GS to attenuate brain injury in these areas.In hyperammonemia, the astroglial cells located in proximity to blood-vessels in glutamatergic areas show increased GS protein content in their perivascular processes. Since ammonia freely crosses the blood-brain barrier (BBB) and astrocytes are responsible for maintaining the BBB, the presence of GS in the perivascular processes could produce a rapid glutamine synthesis to be released into blood. It could, therefore, prevent the entry of high amounts of ammonia from circulation to attenuate neurotoxicity. The changes in the distribution of this critical enzyme suggests that the glutamate-glutamine cycle may be differentially impaired in hyperammonemic states.     

High Ammonia Levels in Brain Induce Tubulin in Cerebrum but Not in Cerebellum

Ingestion of large amounts of ammonium increases markedly the content of tubulin in brain. The effect on tubulin induction of ammonium ingestion for up to 100 days was investigated. Brain tubulin content showed a rapid initial increase (28%) at 2 days and reached 50% after 100 days on the diet. To discern if ammonia, the increase in urea synthesis, or both was responsible for tubulin induction, rats were maintained at several levels of uremia (by administering diets containing 0 to 80% protein) or in hyperammonemia (by urease treatment). Only ammonium administration in the diet and urease injection induced tubulin in brain. Tubulin was quantified in three different brain regions. There was a regional selectivity of tubulin induction by ammonia in rat brain. Whereas the cerebellum remained unaltered, the paleencephalon showed the highest increase, and the cerebral cortex exhibited only a modest increase.     

The potentiation of ammonia toxicity by sodium benzoate is prevented by L-carnitine

Sodium benzoate has been recommended and even been used for the treatment of hyperammonemia in humans. More recently, a note of caution was raised since it has been shown that in experimental animals, sodium benzoate potentiates ammonia toxicity and inhibits urea synthesis in vitro. This has been further confirmed in the work presented here and the mechanism by which benzoate increases mortality and the levels of blood ammonia in mice given ammonium acetate have also been studied. In hyperammonemia, urea production and N-acetylglutamate levels were decreased by sodium benzoate. Pretreatment of mice with L-carnitine suppressed mortality following ammonium acetate plus sodium benzoate administration. Under these conditions L-carnitine lowered blood ammonia and increased urea production and N-acetylglutamate levels.     

Effects of Acute Hyperammonemia on Cerebral Amino Acid Metabolism and pHi In Vivo, Measured by 1H and 31P Nuclear Magnetic Resonance

The effects of an acute intravenous infusion of ammonium acetate on rat cerebral glutamate and glutamine concentrations, energy metabolism, and intracellular pH were measured in vivo with 1H and 31P nuclear magnetic resonance (NMR). The level of blood ammonia maintained by the infusion protocol used in this study (approximately 500 microM, arterial blood) did not cause significant changes in arterial PCO2, PO2, or pH. Cerebral glutamate levels fell to at least 80% of the preinfusion value, whereas glutamine concentrations increased 170% relative to the preinfusion controls. The fall in brain glutamate concentrations followed a time course similar to that of the rise of brain glutamine. There were no detectable changes in the content of phosphocreatine (PCr) or nucleoside triphosphates (NTP), within the brain regions contributing to the sensitive volume of the surface coil, during the ammonia infusion. Intracellular pH, estimated from the chemical shift of the inorganic phosphate resonance relative to the resonance of PCr in the 31P spectrum, was also unchanged during the period of hyperammonemia. 1H spectra, specifically edited to allow quantitation of the brain lactate content, indicated that lactate rose steadily during the ammonia infusion. Detectable increases in brain lactate levels were observed approximately 10 min after the start of the ammonia infusion and by 50 min of infusion had more than doubled. Spectra acquired from rats that received a control infusion of sodium acetate were not different from the spectra acquired prior to the infusion of either ammonium or sodium acetate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperammonemia induces polymerization of brain tubulin

Rats were made hyperammonemic by feeding them a diet containing ammonium acetate. The tubulin content in their brain increased 30% after 20 days on the diet. All the increase was found in polymerized tubulin; no increase in free tubulin was noted. When rats on the ammonium diet were then fed the standard diet, the tubulin increased slightly on the first day but decreased markedly on the second day, reaching control values on the third day. It should be noted that brain tubulin synthesis, was not reduced on the first day of feeding the standard diet but was markedly inhibited (to 40% of control) on the second day, returning to control values on the third day. On the first day of refeeding there is a remarkable disassembly of microtubules with a large, proportional increase (50%) of free tubulin. Both free and polymerized tubulin levels returned to control values on the third day. These results indicate that in hyperammonemia changes in the degree of polymerization of tubulin preceded those in tubulin synthesis.     

Long-term ingestion of ammonium increases acetylglutamate and urea levels without affecting the amount of carbamoyl-phosphate synthase

Rats were fed the following diets: standard (20% protein), high-protein (80%), protein-free, standard plus ammonium and protein-free plus ammonium for six weeks. The standard plus ammonium diet was prepared to contain ammonia equivalent to that supplied by the high-protein diet. Addition of ammonium acetate (20% by mass) to the 20% protein or protein-free diets results in 2.3- and 10-fold increases of urea excretion respectively, without increase of carbamoyl-phosphate synthase. Supplementation of the standard diet with ammonium increases the mitochondrial content of acetylglutamate from 830 to 1590 pmol/mg protein, and of the protein-free diet from 130 to 1040 pmol/mg. However, ingestion of ammonium did not increase the activity of acetylglutamate synthase. Therefore the efflux of acetylglutamate from mitochondria was determined. After 30 min at 37 degrees C liver mitochondria from rats on standard diet released 61% of the initial acetylglutamate while mitochondria from animals on standard plus ammonium diet released only 20%. These results indicate that ingestion of ammonium increases the content of acetylglutamate in rat liver by decreasing its efflux from mitochondria. This effect is similar to that produced in mice by a high protein diet [Morita et al. (1982) J. Biochem. (Tokyo) 91, 563-569]. However, while the high-protein diet increases carbamoylphosphate synthase content, the ammonium diet does not.     

Cell-selective effects of ammonia on glutamate transporter and receptor function in the mammalian brain

Increased brain ammonia concentrations are a hallmark feature of several neurological disorders including congenital urea cycle disorders, Reye's syndrome and hepatic encephalopathy (HE) associated with liver failure. Over the last decade, increasing evidence suggests that hyperammonemia leads to alterations in the glutamatergic neurotransmitter system. Studies utilizing in vivo and in vitro models of hyperammonemia reveal significant changes in brain glutamate levels, glutamate uptake and glutamate receptor function. Extracellular brain glutamate levels are consistently increased in rat models of acute liver failure. Furthermore, glutamate transport studies in both cultured neurons and astrocytes demonstrate a significant suppression in the high affinity uptake of glutamate following exposure to ammonia. Reductions in NMDA and non-NMDA glutamate receptor sites in animal models of acute liver failure suggest a compensatory decrease in receptor levels in the wake of rising extracellular levels of glutamate. Ammonia exposure also has significant effects on metabotropic glutamate receptor activation with implications, although less clear, that may relate to the brain edema and seizures associated with clinical hyperammonemic pathologies. Therapeutic measures aimed at these targets could result in effective measures for the prevention of CNS consequences in hyperammonemic syndromes.     

Selective regional distribution of tubulin induced in cerebrum by hyperammonemia

Ingestion of ammonium induces hyperammonemia which increases tubulin content in cerebrum but not in cerebellum. We have dissected 11 discrete areas of cerebrum and quantified the tubulin content in control and hyperammonemic rats. An heterogeneity in the induction of tubulin is shown. The areas more affected are ventral hippocampus, dorsal hippocampus, hypothalamus, septum, reticular formation and frontal cortex, in which tubulin content increased by 63%, 27%, 32%, 48%, 45%, and 25%, respectively, after two months of feeding the ammonium diet.     

Protective effect of ethanol on acute ammonia intoxication in mice

A single i.p injection of 12 mmoles ammonium acetate/kg produced 100% mortality in mice. Ethanol in doses of 11 to 75 mmoles/kg administered along with the ammonium acetate decreased dramatically the mortality, the maximum protective effect being at 75 mmoles/kg. Blood and brain ammonia levels were also significantly reduced, while blood ethanol was higher in animals injected with ammonia and ethanol. Methanol and butanol also had some protective effect.