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Ammonia removal using hepatoma cells in mammalian cell cultures.

It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2-3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells.

Ammonia↗

PubMed2006

Active ammonia transport and excretory nitrogen metabolism in the climbing perch, Anabas testudineus, during 4 days of emersion or 10 minutes of forced exercise on land.

The climbing perch, Anabas testudineus, inhabits large rivers, canals, stagnant water bodies, swamps and estuaries, where it can be confronted with aerial exposure during the dry season. This study aimed to examine nitrogen excretion and metabolism in this fish during 4 days of emersion. Contrary to previous reports, A. testudineus does not possess a functional hepatic ornithineurea cycle because no carbamoyl phosphate synthetase I or III activity was detected in its liver. It was ammonotelic in water, and did not detoxify ammonia through increased urea synthesis during the 4 days of emersion. Unlike many air-breathing fishes reported elsewhere, A. testudineus could uniquely excrete ammonia during emersion at a rate similar to or higher than that of the immersed control. In spite of the fact that emersion had no significant effect on the daily ammonia excretion rate, tissue ammonia content increased significantly in the experimental fish. Thus, it can be concluded that 4 days of emersion caused an increase in ammonia production in A. testudineus, and probably because of this, a transient increase in the glutamine content in the brain occurred. Because there was a significant increase in the total essential free amino acid in the experimental fish after 2 days of emersion, it can be deduced that increased ammonia production during emersion was a result of increased amino acid catabolism and protein degradation. Our results provide evidence for the first time that A. testudineus was able to continually excrete ammonia in water containing 12 mmol l(-1) NH4Cl. During emersion, active ammonia excretion apparently occurred across the branchial and cutaneous surfaces, and ammonia concentrations in water samples collected from these surfaces increased to 20 mmol l(-1). It is probable that the capacities of air-breathing and active ammonia excretion facilitated the utilization of amino acids by A. testudineus as an energy source to support locomotor activity during emersion. As a result, it is capable of wandering long distance on land from one water body to another as reported in the literature.

Amino Acids↗

PubMed1963

THE METABOLISM OF THE VOLATILE AMINES: III. A MICROAERATION TECHNIQUE FOR THE MEASUREMENT OF THE TWO COMPONENTS OF THE BLOOD AMMONIA LEVEL.

A microaeration technique for the measurement of the ammonia content of blood is described by means of which the amount of ammonia present in blood ("free" ammonia) can be differentiated from that which is liberated during the testing procedure by deamidating action of the alkali reagents employed. Blood is mixed with saturated potassium carbonate solution in the microaeration chamber (a bent 10-ml. pipette) and a stream of nitrogen carries the released ammonia into a collecting acid where the total ammonia liberated can be estimated at frequent intervals.A blood ammonia diffusion curve can be drawn by plotting the total ammonia diffused against the microaeration time. This is a composite curve in that it consists of the sum of an exponential curve which corresponds to the diffusion of "free" ammonia, and a relatively straight line which corresponds to the formation of ammonia by the breakdown of blood constituents. By extrapolating the linear segment back to the ordinate the quantity of ammonia initially present in the blood can be estimated. This technique is easy to learn, requires inexpensive equipment and small volumes of blood, and gives reproducible results.

Amines↗

PubMed2005

The interplay of increased urea synthesis and reduced ammonia production in the African lungfish Protopterus aethiopicus during 46 days of aestivation in a mucus cocoon.

This study was undertaken to test the hypothesis that the rate of urea synthesis in Protopterus aethiopicus was up-regulated to detoxify ammonia during the initial phase of aestivation in air (day 1-day 12), and that a profound suppression of ammonia production occurred at a later phase of aestivation (day 35-day 46) which eliminated the need to sustain the increased rate of urea synthesis. Fasting apparently led to a greater rate of nitrogenous waste excretion in P. aethiopicus in water, which is an indication of increases in production of endogenous ammonia and urea probably as a result of increased proteolysis and amino acid catabolism for energy production. However, 46 days of fasting had no significant effects on the ammonia or urea contents in the muscle, liver, plasma and brain. In contrast, there were significant decreases in the muscle ammonia content in fish after 12, 34 or 46 days of aestivation in air when compared with fish fasting in water. Ammonia was apparently detoxified to urea because urea contents in the muscle, liver, plasma and brain of P. aethiopicus aestivated for 12, 34 or 46 days were significantly greater than the corresponding fasting control; the greatest increases in urea contents occurred during the initial 12 days. There were also significant increases in activities of some of the hepatic ornithine-urea cycle enzymes from fish aestivated for 12 or 46 days. Therefore, contrary to a previous report on P. aethiopicus, our results demonstrated an increase in the estimated rate of urea synthesis (2.8-fold greater than the day 0 fish) in this lungfish during the initial 12 days of aestivation. However, the estimated rate of urea synthesis decreased significantly during the next 34 days. Between day 35 and day 46 (12 days), urea synthesis apparently decreased to 42% of the day 0 control value, and this is the first report of such a phenomenon in African lungfish undergoing aestivation. On the other hand, the estimated rate of ammonia production in P. aethiopicus increased slightly (14.7%) during the initial 12 days of aestivation as compared with that in the day 0 fish. By contrast, the estimated rate of ammonia production decreased by 84% during the final 12 days of aestivation (day 35-day 46) compared with the day 0 value. Therefore, it can be concluded that P. aethiopicus depended mainly on increased urea synthesis to ameliorate ammonia toxicity during the initial phase of aestivation, but during prolonged aestivation, it suppressed ammonia production profoundly, eliminating the need to increase urea synthesis which is energy-intensive.

Amino Acids↗

PubMed2007

Distinct fluctuations of ammonia levels during asparaginase therapy for childhood acute leukemia.

BACKGROUND: The enzyme asparaginase (L-Asp) catalyses the hydrolysis of the non-essential amino acids asparagine and glutamine to aspartic and glutamic acid and ammonia. Ammonia therefore represents a direct metabolite of the biochemical reaction induced by this enzyme. However, data regarding the dynamics and clinical relevance of ammonia levels during L-Asp therapy are lacking. PROCEDURE: We prospectively followed the dynamics of ammonia levels during L-Asp containing induction therapy according to the ALL-BFM 2000 protocol in 10 pediatric patients with acute lymphoblastic leukemia (ALL), in order to assess the possible relevance of ammonia levels for clinical practice and its use as a possible surrogate parameter of L-Asp enzyme activity. RESULTS: We observed a significant elevation of ammonia levels 1 day after intravenous L-Asp administration with ammonia levels reaching up to the seventh fold of normal values, followed by a steep decline to basal values within another 2 days, resulting in an undulating course of ammonia concentrations during L-Asp containing induction treatment. CONCLUSIONS: Although there are potential neurotoxic properties of ammonia, central nervous system (CNS) toxicity has not been observed in our study and is generally not seen as a common side effect of L-Asp therapy. Furthermore, due to the characteristic fluctuation profile, ammonia levels may represent a suitable surrogate parameter of L-Asp enzyme activity and may enable the monitoring of silent inactivation of L-Asp.

Adolescent↗

PubMed1994

Effects of atmospheric ammonia on pulmonary hemodynamics and vascular permeability in pigs: interaction with endotoxins.

The influence of atmospheric ammonia on the somatic growth, the plasma cortisol and ammonia concentrations, and cell blood counts was investigated in pigs exposed to four concentrations (0, 25, 50, and 100 ppm) for 6 days in a specifically designed air-pollutants exposure chamber. The effects of this gas on pulmonary vascular hemodynamics and permeability and on the endotoxin-induced vascular response were also assessed using an isolated perfused lung preparation. The total pulmonary blood flow resistance (Rt) was partitioned into four components: arterial (Ra), pre-(Ra') and post-(Rv') capillary and venous (Rv). The capillary filtration coefficient (Kf,c) was evaluated by using a gravimetric technique. None of the concentrations of ammonia significantly modified the plasma cortisol and ammonia concentrations or the differential leukocyte percentages and total white blood cell count, suggesting an absence of stress related to ammonia. In exposed animals, lethargy and a concentration-related depression of the somatic growth were observed. The equation of the regression line plotted relating the mean values of the changes in body weight gain recorded over the exposure period expressed as percentages of the initial body weight (y) and ammonia concentrations (x) was: y = 3.204 - 0.177x + 0.001x2 (r = 0.99; p < or = 0.013). Endotoxin infused in the perfusion liquid of lungs from unexposed animals for 180 min induced a significant 208% increase in Rt (p < 0.001) which can be ascribed to a 338 and 180% increase in Ra' and Rv', respectively. Endotoxin infusion also induced a 62% (p < or = 0.001) increase in the Kf,c. Exposure of pigs to ammonia at any concentration did not modify the baseline values of any hemodynamic or permeability parameters. However, the hemodynamic response to endotoxins in lungs from pigs exposed to 100 ppm was significantly altered. The increase in Rt, Ra', and Rv' observed in unexposed pigs was completely abolished as shown by the limited changes in Rt (+34.9%). An intermediate reaction (+131.7%) was obtained in pigs exposed to 50 ppm. This inhibiting effect of ammonia was closely correlated with gas concentration by a linear regression (r = 0.99; p < or = 0.037). The changes in the Kf,c recorded in the control group were not modified by exposure to ammonia. It was concluded that exposure of pigs to aerial ammonia concentrations from 0 to 100 ppm for 6 days has no direct effect on the pulmonary microvascular hemodynamics and permeability and induces no stress response.(ABSTRACT TRUNCATED AT 400 WORDS)

Air Pollutants↗

PubMed1975

High pH ammonia toxicity, and the search for life on the Jovian planets.

Jovian plants have enviroments apparently suitable for the evolution of life, but nevertheless, present severe challenges to organisms. One such challenge arises from the presence of ammonia. Ammonia is an efficient biocide, its effect being dependent on pH as well as on concentration. The effects of pH and ammonia concentration were studied separately, where possible, on a variety of organisms, including some isolated from natural enviornments of high pH and/or ammonia concentration. Escherichia coli and Bacillus subtilis are both extremely sensitive to ammonia. An aerobic organism (growth up to pH 11.4) from an alkaline spring is more resistant, but exhibits a toxic response to ammonia at a pH much lower than its maximum for growth. The greatest ammonia resistance has been found in an unidentified organism growing at near neutral pH. Even in this case, however, survival at ammonia concentrations reasonably expected on the Jovian planets is measured in hours. This is, nevertheless, two to three orders of magnitude longer than for E. coli. Our data support the tentative conclusion that contamination of the Jovian planets with terrestrial organisms that can grow is unlikely. However, the range of toxic response noted, coupled with the observation that terrestrial life has not been exposed to high ammonia concentrations for millions of years, suggests that adaptation to greater ammonia tolerance may be possible.

Ammonia↗

PubMed2004

Increased production and excretion of urea in the kuruma shrimp (Marsupenaeus japonicus) exposed to combined environments of increased ammonia and nitrite.

Marsupenaeus japonicus (6.37 +/- 1.29 g) individually exposed to 9 different combined solutions of ambient ammonia (C) and nitrite (C') ammonia at 0.003 [control], 0.39, and 1.49 mmol/L combined with nitrite at 0.001 [control], 0.38, and 1.49 mmol/L in 30 ppt were examined for nitrogenous excretion accumulations of ammonia, nitrite, urea, and uric acid in tissues after 48 hours. M. japonicus exposed to 0.39 mmol/L ammonia-0.38 mmol/L nitrite displayed higher levels of urea-nitrogen (UNE) and organic-N (ONE) excretion by a factor of 2.2 and 5.7, respectively, compared with shrimp exposed only to 0.39 mmol/L ammonia. Exposure to 0.39 mmol/L ammonia-0.38 mmol/L nitrite resulted in lower levels of hemolymph uric acid (HUA), gill ammonia (GAM), gill urea (GUE), gill uric acid (GUA), hepatopancreas ammonia (HPAM), hepatopancreas urea (HPUE), and hepatopancreas uric acid (APUA), respectively, compared with shrimp exposed only to 0.39 mmol/L ammonia. We concluded that M. japonicus exposed to combined environments of ammonia and nitrite display increased nitrogen metabolism and production of urea-N and other organic-N.

Ammonia↗

PubMed2005

High ambient ammonia promotes growth in a ureogenic goby, Mugilogobius abei.

Mugilogobius abei has the ability to produce large amounts of urea when exposed to high ambient ammonia. Despite this metabolically costly approach, and reports of growth inhibition effects of ammonia on fish, M. abei exposed to ammonia shows no adverse effects on growth. To investigate this observation the growth of M. abei was measured at room temperatures for 8 weeks at a constant ration level under solitary and grouped conditions, in 20% SW with or without (control) 2 mM NH(4)Cl. Furthermore, pituitary mRNA levels of growth hormone, oxygen consumption, incorporation of external (15)N-ammonia into amino acid and protein fractions as well as behavioral activities were also examined. The specific growth rates of ammonia-exposed fish under grouped condition over the 8 weeks were significantly higher than those of control, while those rates under solitary condition were not significantly different between the treatments. The pituitary of ammonia-exposed fish had higher growth hormone mRNA than in control fish. The use of (15)N isotope revealed that M. abei can actively use external ammonia as a supplementary nitrogen source. Oxygen consumption of ammonia-exposed fish was significantly lower than that of control fish. Locomotor activity and aggressive behavior under grouped condition were significantly reduced in ammonia-exposed fish as compared to those of control. These combined alterations in the ammonia-exposed fish may result in the higher growth rates.

Amino Acids↗

PubMed1999

Helicobacter pylori and ammonia concentrations of whole, parotid and submandibular/sublingual saliva.

The aim of this study was to determine the ammonia concentration in whole, parotid and submandibular/sublingual saliva of healthy volunteers using the indophenol direct method. It also investigated the hypothesis that higher saliva ammonia concentrations are associated with the presence of Helicobacter pylori (H. pylori) in the oral cavity. In healthy volunteers, the mean ammonia concentration of whole saliva (2574 mumol/l) was significantly higher (P < 0.0001) than the mean ammonia concentration of both parotid (238 mumol/l) and submandibular/sublingual (355 mumol/l) saliva. In whole saliva, no difference in ammonia concentration was found between healthy controls and dyspeptic patients (mean ammonia values 2574 and 2489 mumol/l respectively, P = 0.7). In addition, no significant differences were observed in the salivary ammonia concentration between dyspeptic patients with and without H. pylori carriage. It is concluded that the ammonia concentration in parotid and submandibular/sublingual saliva does not differ, but is significantly lower than the ammonia concentration of whole saliva. This difference is not due to carriage of H. pylori with its strong urease activity. Therefore, the determination of ammonia in whole saliva is an inappropriate screening test for patients being at risk for (chronic) gastritis and peptic ulcer disease.

Adult↗

PubMed2004

Combined effects of ammonia and manganese on astrocytes in culture.

Ammonia has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and astrocytes appear to be the primary target of ammonia neurotoxicity. Recent work has shown that manganese also plays a role in the pathogenesis of HE and causes astrocyte morphologic and functional changes similar to ammonia. We therefore investigated whether a combination of these compounds could produce additive/synergistic effects. Cultured astrocytes treated with 5 mM ammonia (NH4Cl) along with 100 microM manganese acetate (MnAc) for 3 h showed a 55-65% increase in free radical production over ammonia or manganese alone (P < 0.05). There was also a 50% decrease in the mitochondrial membrane potential (beta psi(m)) at 24 h following treatment with NH4Cl (5 mM) plus MnAc (50 microM) (P < 0.05), as compared to ammonia or manganese alone. Astrocytes treated with ammonia or manganese alone for 24 h showed no cell death, as determined by LDH release and light microscopic examination. However, cultures treated with ammonia plus manganese showed 80-90% necrotic cell death as estimated by light microscopy and 59% cell death as determined by LDH release. LDH release by ammonia plus manganese was blocked by the antioxidant superoxide dismutase (25 units/ml) as well as by the nitric oxide synthase inhibitor N(omega)-nitro-L-argininemethyl ester (500 microM). In conclusion, ammonia plus manganese exert additive/synergetic effects on the induction free radicals, mitochondrial inner membrane depolarization and cellular integrity, which may contribute to the tissue injury associated with chronic forms of HE.

Ammonia↗

PubMed1981

Ammonia and disinhibition in cat motor cortex by ammonium acetate, monofluoroacetate and insulin-induced hypoglycemia.

Ammonia intoxication abolished the suppression of action potential generation by cortical postsynaptic inhibition due to the inactivation of neuronal Cl- extrusion. The disinhibition by ammonia intoxication occurred when ammonia concentrations in the cerebral cortex were increased to 320% of normal. Fluoroacetate poisoning and insulin-induced hypoglycemia, which are known to increase ammonia concentrations in the CNS and previously have been shown to inactivate Cl- extrusion in spinal motoneurons, abolished the suppression of action potential generation by cortical postsynaptic inhibition like ammonia intoxication. This disinhibition occurred at unchanged cerebral ammonia concentrations. The effect of fluoroacetate and insulin induced hypoglycemia on cortical postsynaptic inhibition is either due to a direct, i.e. not ammonia mediated, inactivation of neuronal Cl- extrusion or due to a disturbance of the synaptic mechanisms mediated by the transmitter of cortical inhibition, GABA. Toxic-metabolic encephalopathies which increase cerebral ammonia concentrations beyond 320% of normal may produce a dysfunction of the CNS due to inactivation of neuronal Cl- extrusion leading to ineffective cortical inhibition. However, in fluoroacetate poisoning and insulin-induced hypoglycemia increased ammonia concentrations in the CNS have only a secondary role in initiating a dysfunction of the CNS since disinhibition occurs before ammonia concentrations increase.

Acetates↗

PubMed1987

The H-reflex in the encephalopathy due to ammonia intoxication.

Ammonia intoxication has been shown to decrease excitatory synaptic transmission in several regions of the central nervous system. To investigate the relation between an effect of ammonia on excitatory synaptic transmission and the behavioral depression in the encephalopathy due to ammonia intoxication, this study examined in the rat the effects of ammonia intoxication on the H-reflex, the behavioral and neurological signs of the encephalopathy due to ammonia intoxication, and correlated the effects on the H-reflex with the signs of encephalopathy. Ammonia intoxication abolished the H-reflex without affecting the M-response. This indicated that ammonia intoxication decreased spinal excitatory synaptic transmission without affecting neuromuscular excitatory synaptic transmission. In the encephalopathy due to ammonia intoxication, the H-reflex disappeared only during a very advanced stage of behavioral depression, i.e., coma. During early stages of behavioral depression, i.e., during a decrease of reactions to sensory stimuli, the H-reflex was not affected by ammonia intoxication. Therefore, mechanisms other than a decrease of excitatory synaptic transmission in the central nervous system may be responsible for the behavioral depression seen in early stages of the encephalopathy due to ammonia intoxication.

Action Potentials↗

PubMed1985

Elevated plasma ammonia level in hepatic cirrhosis: role of glucagon.

Elevated plasma ammonia level in hepatic cirrhosis has been attributed to a lack of conversion of enteric ammonia into urea or to its entry into systemic circulation via portasystemic shunting, or to both. It is exaggerated by excessive protein intake. Because hyperglucagonemia is well documented in cirrhosis and a protein meal is an effective glucagon secretagogue, plasma glucose, insulin, glucagon, and ammonia levels were determined in 50 cirrhotic patients after an overnight fast. Effects of a protein meal were also assessed in 20 of these patients. Plasma glucose was normal and remained unaltered after a protein meal. Insulin, glucagon, and ammonia levels were elevated, but only in patients with advanced liver dysfunction. Ammonia levels correlated significantly with glucagon (r = 0.61, p less than 0.001), but not with insulin or glucose levels. Insulin and glucagon levels rose after a protein meal in all patients and controls; whereas a significant rise in the ammonia level occurred only in decompensated cirrhotics. Elevation of the ammonia level was significantly correlated with fasting glucagon (r = 0.54, p less than 0.05), as well as with glucagon response (r = 0.65, p less than 0.01), but not with basal insulin or insulin response. Furthermore, the rise in ammonia level occurred too early to be accounted for by enteric generation. Finally, direct effects of glucagon administration on plasma glucose and serum ammonia were examined in 15 cirrhotic patients. Glucose response was significantly blunted in cirrhotic patients as compared with normal subjects, whereas serum ammonia rose promptly but only in cirrhotics, with maximum rise being noted in cirrhotic patients with advanced liver dysfunction. This study, therefore, suggests that hyperglucagonemia may contribute significantly to hyperammonemia in hepatic cirrhosis.

Adult↗

PubMed2005

Emission of ammonia from indoor concrete wall and assessment of human exposure.

Addition of urea-based antifreeze admixtures during cement mixing can make it possible to produce concrete cement in construction of buildings in cold weather; this, however, has led to increasing indoor air pollution due to continuous transformation and emission from urea to gaseous ammonia in indoor concrete wall. It is believed that ammonia is harmful to human body and exposure to ammonia can cause some serious symptoms such as headaches, burns, and even permanent damage to the eyes and lungs. In order to understand the emission of ammonia from indoor concrete wall in civil building and assess the health risk of people living in these buildings, the experimental pieces of concrete wall were first prepared by concreting cement and urea-based antifreeze admixtures to simulate the indoor wall in civil building in this work. Then environmental chamber was adopted for studying the effect of temperature, relative humility and air exchange rate on emission of ammonia from experimental pieces of concrete wall. Also the field experiment was made at selected rooms in given civil buildings. Exposure and potential dose of adult and children exposed to indoor/outdoor ammonia in summer and in winter are calculated and evaluated by using Scenario Evaluation Approach. The results indicated that high air exchange rate leads to decreased ammonia concentration, and elevation of temperature causes increasing ammonia concentration and volatilizing rate in chamber. The complete emission of ammonia from the wall containing urea-based antifreeze admixtures needs more than 10 years in general. Ventilating or improving air exchange can play a significant role in reducing ammonia concentration in actual rooms in field experiments. Urea-based antifreeze admixtures in concrete wall can give rise to high exposure and potential dose, especially in summer. Generally, adults have a high potential dose than children, while children have personal average dose rate beyond adults in the same conditions.

Adult↗

PubMed2005

Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo.

We investigated ammonia-oxidizing bacteria in activated sludge collected from 12 sewage treatment systems, whose ammonia removal and treatment processes differed, during three different seasons. We used real-time PCR quantification to reveal total bacterial numbers and total ammonia oxidizer numbers, and used specific PCR followed by denaturing gel gradient electrophoresis, cloning, and sequencing of 16S rRNA genes to analyze ammonia-oxidizing bacterial communities. Total bacterial numbers and total ammonia oxidizer numbers were in the range of 1.6 x 10(12) - 2.4 x 10(13) and 1.0 x 10(9) - 9.2 x 10(10)cellsl(-1), respectively. Seasonal variation was observed in the total ammonia oxidizer numbers, but not in the ammonia-oxidizing bacterial communities. Members of the Nitrosomonas oligotropha cluster were found in all samples, and most sequences within this cluster grouped within two of the four sequence types identified. Members of the clusters of Nitrosomonas europaea-Nitrosococcus mobilis, Nitrosomonas cryotolerans, and unknown Nitrosomonas, occurred solely in one anaerobic/anoxic/aerobic (A2O) system. Members of the Nitrosomonas communis cluster occurred almost exclusively in association with A2O and anaerobic/aerobic systems. Solid residence time mainly influenced the total numbers of ammonia-oxidizing bacteria, whereas dissolved oxygen concentration primarily affected the ammonia-oxidizing activity per ammonia oxidizer cell.

Ammonia↗

PubMed2026

Proteomic analysis of ammonia-induced stress in Chinese hamster ovary (CHO) cell cultures.

Ammonia (NH₃) and its ionic form ammonium (NH₄+) are both metabolic waste products and essential nitrogen sources within Chinese hamster ovary (CHO) cell cultures. Although necessary for amino acid synthesis, excessive accumulation in the extracellular environment can exert stress, reducing cell proliferation and impairing the efficiency of recombinant protein production. Proper endoplasmic reticulum (ER) function is critical for CHO cells as biotherapeutic producers. Previous work has linked elevated ammonia concentrations to reduced productivity via altered N-glycosylation pathways, but its broader effects on ER biology remain unclear. In this study, we applied high-resolution mass spectrometry to perform a comprehensive analysis of changes in the ER proteome in CHO cells exposed to two ammonia concentrations, 10 mM and 30 mM, 48 and 120 h after supplementation. Both conditions suppressed cell growth and reduced product titre; however, the 10 mM supplementation resulted in a minor increase in specific cell productivity. Gene Ontology analysis revealed that ammonia strongly affected the tricarboxylic acid cycle, as well as key metabolic, catabolic and biogenetic processes. Several ER membrane proteins, including HMGCR and PREB, were consistently downregulated. In extended cultures, transmembrane proteins linked to Golgi-transport were upregulated, while vesicle transport associated proteins were downregulated, indicating altered intracellular trafficking. SIGNIFICANCE: This study provides a novel perspective on CHO cell biology under environmental stress by investigating the impact of ammonia accumulation in culture. Despite its presence in CHO culture, ammonia has been relatively under-investigated, compared to other culture conditions. Using high-throughput mass spectrometry for comprehensive proteomic profiling, we characterise the cellular response to ammonia build-up with a level of depth not previously applied to the study of this biological stressor. By specifically analysing proteins localised to the ER, we identify candidate pathways and molecular mechanisms that contribute to reduced CHO cell growth and productivity, offering insights directly relevant to industrial bioprocessing conditions. The link between ammonia concentration and a decrease in productivity has previously been linked to genes involved in N-glycosylation of the recombinant biotherapeutic, but the full extent of ammonia stress on ER function has not yet been investigated. These methods were applied to two IgG producing CHO cell lines to allow for comparison of cell line specific stress adaptations, as well as comparing the short- and long-term effects of excess ammonia.

Proteomics↗

PubMed2005

Mechanisms of ammonia-induced cell death in rat cortical neurons: roles of NMDA receptors and glutathione.

The occurrence, nature and prevention of ammonia-induced cell death were assayed in cultured primary cortical neurons from newborn rats. Treatment with 1-10 mM ammonium chloride for 24 or 48 h, dose-dependently decreased neuronal survival (MTT assay) and GSH/GSSG ratio in the cultures, whereas total GSH content was significantly reduced only with 10mM ammonia. Treatment with a glutathione synthesis inhibitor, buthionyl sulfoximine (BSO) (10 microM), decreased the GSH content and GSH/GSSG ratio to a degree similar to that of 10 mM ammonia, but it did not decrease cell survival in control cells. This indicates that glutathione depletion per se is not a cause of ammonia-induced neuronal death. However, ammonia-induced decrease of cell viability was attenuated by incubation with glutathione diethyl ester (GEE), which transiently increased the intracellular GSH level in both control and ammonia-treated cells. Neuronal survival in the presence of ammonia was partly improved by the NMDA receptor antagonists MK-801 and APV. Morphological analysis revealed that ammonia treatment causes both apoptotic and non-apoptotic neuronal death, the former not being inhibited by MK-801. Apoptosis was the dominant type of cell death at 10mM ammonia, as concluded both from morphologic examination and the absence of survival improvement in the presence of GABA+nipecotic acid or taurine, model anti-excitotoxic treatments of cortical neurons. The mechanism underlying apoptosis may include inhibition of a survival kinase, Akt, whose activatory phosphorylation at Ser473 is reduced in neurons treated with 10 mM, but not 1 mM ammonia.

Ammonia↗