The effect of sodium benzoate on ammonia toxicity in rats

At 9.5 mmoles/kg body weight, sodium benzoate sharply increased mortality in rats subsequently challenged with ammonia. Fasted animals were less sensitive to potentiation of ammonia toxicity by benzoate than were fed animals. At 2.5 mmoles/kg body weight, benzoate was observed to protect fasted animals against ammonia toxicity. Measurements of ammonia disappearance, urea formation, and hippurate synthesis in suspensions of isolated hepatocytes indicate that benzoate potentiates ammonia toxicity by inhibiting the urea cycle.     

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.     

Effects of ammonia and allopurinol on rat hippocampal NMDA receptors

Ammonia is considered to be the main agent responsible for hepatic encephalopathy which progressively leads to altered mental status. N‐methyl‐D‐aspartate (NMDA) is an ionotropic glutamate receptor, which is involved in synaptogenesis, memory and neurotoxicity. The aim of this study was to investigate the effects of ammonia intoxication and allopurinol, a xanthine oxidase (XO) inhibitor, on NMDA receptor subunits, NR2A and NR2B, in the hippocampus of rats. Thirty‐six male rats were divided into three groups (n = 12/group) as follows: (1)control group (phosphate buffered saline (PBS) solution); (2)ammonia group (ammonium acetate, 2.5 mmol/kg), (3)ammonia + allopurinol group (ammonium acetate, 2.5 mmol/kg, allopurinol, 50 mg/kg). Each rat received intraperitoneal injection for 28 days. Western Blotting technique was used for detecting NR2A and NR2B expressions. Both NR2A and NR2B subunit expressions decreased 27 and 11%, respectively, in ammonia group with respect to the control group. Ammonium acetate decreased significantly in NR2A subunit expressions in the hippocampus (p < 0.01). Administration of ammonia + allopurinol caused statistically significant increases in NR2A subunit expressions compared to the ammonia group (p < 0.001). The down‐regulation of NMDA receptors caused by ammonium acetate suggest that these receptors may play role in the process of hepatic encephalopathy and using allopurinol may have some protective effects in ammonia toxicity. Copyright © 2010 John Wiley & Sons, Ltd.     

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 L-carnitine on urea synthesis following acute ammonia intoxication in mice

L-Carnitine protects mice against acute ammonia intoxication. The effect of L-carnitine on in vivo incorporation of [14C] bicarbonate into urea has been investigated in mice given large doses of ammonium acetate. The hepatic content of N-acetylglutamate has been measured. Following ammonia administration the animals given L-carnitine have much higher production of urea than the unprotected mice. The marked protective effect of L-carnitine on ammonium acetate-induced hyperammonemia and on the increase in urea synthesis is not due primarily to activation of N-acetylglutamate synthetase.     

Ornithine aminotransferase activity, liver ornithine concentration and acute ammonia intoxication

5-Fluoromethylornithine (5FMOrn) is a specific inactivator of L-ornithine:2-oxoacid aminotransferase (OAT). Inactivation of OAT causes the enhancement of L-ornithine (Orn) concentrations in all tissues. Intraperitoneal or oral administration of 10-50 mg/kg of 5FMOrn per day to albino mice rendered partial protection against lethal intoxication with 26 mmol/kg of ammonium acetate. The protective effect was maximal around 16 h after 5FMOrn administration, at the time when endogenous Orn concentrations were maximal. At this time protection by 5FMOrn against acute ammonia intoxication was comparable to that observed 1 h after the intraperitoneal administration of 10 mmol/kg of L-arginine. Pretreatment with 5FMOrn prevented the enhancement of excessive urinary excretion of orotic acid by ammonia intoxicated mice, and it enhanced urea formation in the liver. These biochemical effects demonstrate that 5FMOrn shifts Orn into the urea cycle, Orn which normally would be transaminated. Since even long-term treatment of mice with 5FMOrn did not reveal toxic effects, this compound may be considered for the treatment of certain conditional deficiencies of Orn or arginine.     

Prevention of ammonia toxicity byl-carnitine: metabolic changes in brain

l-Carnitine when injected in mice 30 min before an LD₁₀₀ of ammonium acetate (12 mmol/kg body weight, intraperitoneal) reduced mortality (100% survival with 16 mmoll-carnitine/kg) and prevented the appearance of symptoms of ammonia toxicity. Brain ammonia decreased in the animals givenl-carnitine. Ammonia decreased the levels of glutamate in brain; they were partially restored byl-carnitine, which also reduced the increase in brain glutamine in animals given only ammonia. The redox state of the brain was altered following ammonia intoxication. The ratio of lactate to pyruvate in the cytosol increased while that of glutamate to -ketoglutarate in the mitochondria decreased. These ratios were partially restored byl-carnitine. The implications of these findings are discussed relative to the mechanism of ammonia toxicity.     

Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol. Role of acetyl CoA synthetase in anaerobic ATP synthesis

Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.     

NMDA Receptor antagonists prevent acute ammonia toxicity in mice

We proposed that acute ammonia toxicity is mediated by activation of NMDA receptors. To confirm this hypothesis we have tested whether different NMDA receptor antagonists, acting on different sites of NMDA receptors, prevent death of mice induced by injection of 14 mmol/Kg of ammonium acetate, a dose that induces death of 95% of mice. MK-801, phencyclidine and ketamine, which block the ion channel of NMDA receptors, prevent death of at least 75% of mice. CPP, AP-5, CGS 19755, and CGP 40116, competitive antagonists acting on the binding site for NMDA, also prevent death of at least 75% of mice. Butanol, ethanol and methanol which block NMDA receptors, also prevent death of mice. There is an excellent correlation between the EC₅₀ for preventing ammonia-induced death and the IC₅₀ for inhibiting NMDA-induced currents. Acute ammonia toxicity is not prevented by antagonists of kainate/AMPA receptors, of muscarinic or nicotinic acetylcholine receptors or of GABA receptors. Inhibitors of nitric oxide synthase afford partial protection against ammonia toxicity while inhibitors of calcineurin, of glutamine synthetase or antioxidants did not prevent ammonia-induced death of mice. These results strongly support the idea that acute ammonia toxicity is mediated by activation of NMDA receptors.     

Ammonium Injection Induces an N-Methyl-d-Aspartate Receptor-Mediated Proteolysis of the Microtubule-Associated Protein MAP-2

Abstract: We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule-associated protein 2 (MAP-2). Activation of the N -methyl- d -aspartate (NMDA) receptor dephosphorylates MAP-2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP-2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP-2. Also, the amount of MAP-2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP-2 was decreased by ∼75% 1-2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP-2 was prevented completely by injection of 2 mg/kg MK-801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor. l -Carnitine, which protects rats against ammonia toxicity, also prevented MAP-2 degradation. Because activation of the NMDA receptor increases [Ca²⁺]_i, we determined whether rat brain contains a Ca²⁺-dependent protease that selectively degrades MAP-2. We show that there is a cytosolic Ca²⁺-dependent protease that degrades MAP-2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca²⁺]_i, which activates calpain I. This, in turn, selectively degrades MAP-2. Possible implications in chronic hyperammonemic states and in the mechanism of ammonia toxicity are discussed.     

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.     

Anaerobic nitrate reduction to ammonium in two strains isolated from coastal marine sediment: A dissimilatory pathway

Abstract: A total of 28 nitrate-reducing bacteria were isolated from marine sediment (Mediterranean coast of France) in which dissimilatory reduction of nitrate to ammonium (DRNA) was estimated as 80% of the overall nitrate consumption. Thirteen isolates were considered as denitrifiers and ten as dissimilatory ammonium producers. ¹⁵N ammonium production from ¹⁵N nitrate by an Enterobacter sp. and a Vibrio sp., the predominant bacteria involved in nitrate ammonification in marine sediment, was characterized in pure culture studies. For both strains studied, nitrate-limited culture (1 mM) produced ammonium as the main product of nitrate reduction (> 90%) while in the presence of 10 mM nitrate, nitrite was accumulated in the spent media and ammonia production was less efficient. Concomitantly with the dissimilation of nitrate to nitrite and ammonium the molar yield of growth on glucose increased. Metabolic products of glucose were investigated under different growth conditions. Under anaerobic conditions without nitrate, ethanol was formed as the main product; in the presence of nitrate, ethanol disappeared and acetate increased concomitantly with an increased amount of ammonium. These results indicate that nitrite reduction to ammonium allows NAD regeneration and ATP synthesis through acetate formation, instead of ethanol formation which was favoured in the absence of nitrate.     

Ammonia Intoxication: Effects on Cerebral Cortex and Spinal Cord

The effect of an acute systemic ammonia intoxication on the metabolic states of the cerebral cortex and the spinal cord of the same animal was studied in the cat. The intravenous infusion of ammonium acetate (2 and 4 mmol/kg body weight/30 min) increased the gross levels of tissue NH4+, glutamine, glutamine/glutamate ratio, lactate, and the lactate/pyruvate ratio in the cerebral cortex and the spinal cord. Pyruvate increased, but significantly only in the spinal cord; aspartate decreased, but significantly only in the cerebral cortex. The infusion of ammonium acetate did not significantly change the levels of phosphocreatine, ATP, ADP, AMP, total adenine nucleotides, adenylate energy charge, glucose, glutamate, alpha-ketoglutarate, and malate in either tissue. The changes of NH4+, glutamine, and lactate levels as well as glutamine/glutamate and lactate/pyruvate ratios in the spinal cord correlated significantly with the corresponding changes of these metabolites in the cerebral cortex. Thus, cerebral cortex and spinal cord show certain specific and comparable metabolic changes in response to a systemic ammonia intoxication. The effect of ammonia intoxication on the increases of glutamine and lactate levels is discussed.     

Postprandial exchange of urea and ammonia nitrogen between the digestive tract and portal blood in the conscious pig after ingestion of a diet with or without urea

The concentrations of urea and ammonia were measured in the portal and arterial blood simultaneously to the blood flow rate in the portal vein during the postprandial period (8 hrs.) after ingestion of a normal protein diet with 3% urea (10 meals) or without urea (12 meals) in conscious pigs (mean body weight: 55.5 +/- 2.3 kg). When no urea was present in the diet, there was a slight and permanent uptake of blood urea by the gut (570 mg/h, i.e. 9,5 mmoles/h) as well as a permanent appearance of ammonia in the portal vein (258 mg/h i.e. 15.2 mmoles/h), increasing with time (P less than 0.05). The absorbed ammonia nitrogen represented a maximum of 70% of urea nitrogen taken up. 2. Addition of urea to the diet brought about a large absorption of that substance (73% of the ingested amount) followed by a rather large excretion (960 mg/h, i.e. 16 mmoles/h), 5-6 hrs. after the meal and led to an increase (P less than 0.05) in the absorption of ammonia.     

Superoxide Production and Antioxidant Enzymes in Ammonia Intoxication in Rats

Injection of large doses of ammonium salts lead to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of injecting 7 mmol/kg of ammonium acetate on the production of superoxide and on the activities of some antioxidant enzymes in rat liver, brain, erythrocytes and plasma. Glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver and brain (both in cytoso-lic and mitochondrial fractions) and also in blood red cells, while glutathione reductase activity remained unchanged. Superoxide production in submitochon-drial particles from liver and brain was increased by more than 100% in both tissues. Both diminished activity of antioxidant enzymes and increased superoxide radical production could lead to oxidative stress and cell damage, which could be involved in the mechanism of acute ammonia toxicity.     

The Effect of Ouabain and External Potassium on the Ion Transport of Rabbit Red Cells

Na efflux of rabbit RBC is approximately 10 mmoles/kg wet weight. hr. One-half of this consists of a ouabain-insensitive exchange diffusion component. Ouabain inhibits 2.5 mmoles/kg.hr of Na efflux. K influx is 3.0 mmoles/kg.hr; 2.2 mmoles/kg.hr are inhibited by ouabain. In contrast with human RBC, ouabain inhibition of Na efflux and K influx of rabbit RBC is easily reversible. After 2 hr, ouabain inhibition of Na efflux is completely compensated for by increased internal Na concentration and Na efflux returns to initial levels. Removal of ouabain at this stage results in stimulation of the efflux by 4.3 mmoles/kg.hr. Na influx is initially not affected by ouabain but is increased by 2.4 mmoles/kg.hr after 2 hr incubation with the drug. Removal of K from normal Ringer does not affect Na efflux and increases Na influx by 1.6 mmoles/kg.hr. Addition of ouabain to K-free Ringer inhibits Na efflux and influx to the same extent (1.6 mmoles/kg.hr). Removal of Na from K-free Ringer has an inhibitory effect on efflux similar to that of ouabain. These findings suggest that the fraction of Na efflux inhibited by removal of external K is completely replaced by a new, ouabain-sensitive exchange diffusion of Na ions.     

Na and K in outer segments of retinal rods

The amount of Na⁺ and K⁺ in isolated bovine retina outer segments and slices of outer segments obtained from frozen and freeze-dried bovine and frog retinas was established. It is shown that during the conventional procedure of isolation nearly 75% of the Na⁺ and K⁺ present in native structures was lost.

The average amount of K⁺ in bovine outer segments is 158 mmoles/kg dry wt.; Na⁺, 136 mmoles/kg dry wt. In frog outer segments there is: K⁺, 133 mmoles/kg dry wt.; Na⁺, 91 mmoles/kg dry wt.

With the help of the electron microscopic technique Na⁺ was shown to be located predominantly in the sacs of the outer segments. As for K⁺, it is, in all probability, in the extrasaccular space which agrees with some experimental biochemical data obtained.     

Quantification of ammonia release from fruit fly (Diptera: Tephritidae) attractants using infrared spectroscopy

Ammonia is the primary attractant for tephritid fruit flies, and traps baited with synthetic attractants using ammonia formulations have been highly successful in capturing these pests. However, difficulties in quantifying release rates of ammonia have limited abilities to make comparisons among field tests of different species by using different formulations. Therefore, Fourier transform infrared (FTIR) spectroscopy was evaluated as a method to quantify ammonia from synthetic lures. Analysis of the headspace from commercial ammonium bicarbonate and ammonium acetate lures indicated that there is a large burst of ammonia liberated upon initial exposure of the lures, but after 5-7 d the release rates stabilize and remain steady for at least 60 d under laboratory conditions. During the period of steady release, FTIR st atic measurements showed a n average of 0.12 and 0.21 microg of ammonia per 50-ml sample from ammonium bicarbonate and ammonium acetate lures, respectively. FTIR dynamic measurements from ammonium acetate lures indicated a steady release rate of approximately 200 microg/h. Ammonia release rate from ammonium acetate lures could be reduced by decreasing the surface area of the release membrane, and the presence of crystal formations on the membrane seemed to decrease the longevity of the ammonium acetate lures.     

Efficacy of wax matrix bait stations for Mediterranean fruit flies (Diptera: Tephritidae)

Tests were conducted that evaluated efficacy of wax matrix bait stations for Ceratitis capitata (Wiedemann) adults in Guatemala. Bait stations were exposed to outdoor conditions to determine effect of weathering on longevity as indicated by bait station age. Results of laboratory tests found that bait stations with spinosad and ammonium acetate remained effective for at least 31 d compared with pesticide-free controls, although there was some loss of efficacy over time. Percentage mortality for bait station strips with 2% spinosad and 1% ammonium acetate decreased from 100 +/- 0.0% on day 0 to 70 +/- 7.1% after 31 d. Ammonia concentration had little effect on percentage mortality although there was some indication that ammonia concentration affected number of flies observed on the bait stations. Bait station strips (one per cage) were more effective than controls for 6-8 wk when tests were conducted in field cages (3 m diameter x 2 m), but only 2-3 wk when tests were conducted in large (2.5 m high and 6.0 m wide and 7.5 m long) field cages. Longevity was restored when multiple bait stations (3, 6, or 12) were deployed per cage. Bait stations containing methomyl were used for field tests of efficacy for wild flies. Dipped lure bait stations, which were made by coating two edges of commercial ammonium acetate and trimethylamine lures, killed six times more flies than corn cob bait stations dipped into a Nulure/malathion solution. They also killed more flies than pesticide-free controls for 8 wk.     

Energy and reducing equivalent potential of C2-compounds for microbial growth

Yeasts were chemostatically cultivated on C₂-substrates. Experimental yield figure were 0.77 g/g and 0.57 g/g on ethanol, 0.37 g/g and 0.29 g/g on acetate and ammonium and nitrate, respectively, as the nitrogen sources. Growth on hexadecane/ammonium yielded 0.94 g/g. Simultaneous utilization of formate lead to an increase of growth yield on acetate and hexadecane but not on ethanol. The experimental data were compared to theoretical figures. These results show that growth yield on ethanol/ammonium is carbon-limited, on ethanol/nitrate is NADPH-limited and on acetate with both nitrogen sources is energy-limited. With hexadecane/ammonium growth yield is energy-limited too. According to these results ethanol seems to be the only substrate from which an excess in energy can be obtained.