Interorgan ammonia metabolism in liver failure

In the post-absorptive state, ammonia is produced in equal amounts in the small and large bowel. Small intestinal synthesis of ammonia is related to amino acid breakdown, whereas large bowel ammonia production is caused by bacterial breakdown of amino acids and urea. The contribution of the gut to the hyperammonemic state observed during liver failure is mainly due to portacaval shunting and not the result of changes in the metabolism of ammonia in the gut. Patients with liver disease have reduced urea synthesis capacity and reduced peri-venous glutamine synthesis capacity, resulting in reduced capacity to detoxify ammonia in the liver.The kidneys produce ammonia but adapt to liver failure in experimental portacaval shunting by reducing ammonia release into the systemic circulation. The kidneys have the ability to switch from net ammonia production to net ammonia excretion, which is beneficial for the hyperammonemic patient. Data in experimental animals suggest that the kidneys could have a major role in post-feeding and post-haemorrhagic hyperammonemia.During hyperammonemia, muscle takes up ammonia and plays a major role in (temporarily) detoxifying ammonia to glutamine. Net uptake of ammonia by the brain occurs in patients and experimental animals with acute and chronic liver failure. Concomitant release of glutamine has been demonstrated in experimental animals, together with large increases of the cerebral cortex ammonia and glutamine concentrations. In this review we will discuss interorgan trafficking of ammonia during acute and chronic liver failure. Interorgan glutamine metabolism is also briefly discussed, since glutamine synthesis from glutamate and ammonia is an important alternative pathway of ammonia detoxification. The main ammonia producing organs are the intestines and the kidneys, whereas the major ammonia consuming organs are the liver and the muscle.     

The "peripheral-type" benzodiazepine (omega 3) receptor in hyperammonemic disorders

Increased levels of brain ammonia occur in both congenital and acquired hyperammonemic syndromes including hepatic encephalopathy, fulminant hepatic failure, Reye's syndrome and congenital urea cycle disorders. In addition to its effect on neurotransmission and energy metabolism, ammonia modulates the expression of various genes including the astrocytic "peripheral-type" benzodiazepine (or omega 3) receptor (PTBR). Increased expression of the isoquinoline carboxamide binding protein (IBP), one of the components of the PTBR complex, is observed in brain and peripheral tissues following chronic liver failure as well as in cultured astrocytes exposed to ammonia. Increased densities of binding sites for the PTBR ligand [3H]-PK11195 are also observed in these conditions as well as in brains of animals with acute liver failure, congenital urea cycle disorders and in patients who died in hepatic coma. The precise role of PTBR in brain function has not yet fully elucidated, but among other functions, PTBR mediates the transport of cholesterol across the mitochondrial membrane and thus plays a key role in the biosynthesis of neurosteroids some of which modulate major neurotransmitter systems such as the gamma-aminobutyric acid (GABA(A)) and glutamate (N-methyl-D-aspartate (NMDA)) receptors. Activation of PTBR in chronic and acute hyperammonemia results in increased synthesis of neurosteroids which could lead to an imbalance between excitatory and inhibitory neurotransmission in the CNS. Preliminary reports suggest that positron emission tomography (PET) studies using [11C]-PK11195 may be useful for the assessment of the neurological consequences of chronic liver failure.     

Toxic impact of phosphamidon on protein degradation in phasic and tonic muscles of marine prawn, Penaeus indicus (H. Milne Edwards).

Levels of various protein fractions, (sarcoplasmic, myosin, actin, non-collagen and collagen) and the rate of their degradation by proteases were studied in phasic and tonic muscles of marine prawn, Penaeus indicus following acute (2 d) and chronic (15 d) exposure to sublethal concentration of phosphamidon. During exposure, greater decrease in sarcoplasmic protein fraction was observed in phasic muscle as compared to other myofibrillar proteins. But the sarcoplasmic protein content showed an elevation in tonic muscle. The changes in protein fractions were more pronounced during acute exposure than chronic exposure both in phasic and tonic muscles. These changes were correlated with the elevation of the acidic, neutral and basic protease activities during acute and chronic exposure. Free amino acids were increased during acute exposure, while they showed a significant decrease during chronic exposure in both the muscles. These results indicate that protein metabolism in both phasic and tonic muscles was significantly altered following phosphamidon exposure. These differential responses observed at acute and chronic exposure indicate the operation of compensatory mechanisms to mitigate the phosphamidon toxic stress.     

Interorgan ammonia, glutamate, and glutamine trafficking in pigs with acute liver failure

Ammonia reduction is the target for therapy of hepatic encephalopathy, but lack of quantitative data about how the individual organs handle ammonia limits our ability to develop novel therapeutic strategies. The study aims were to evaluate interorgan ammonia metabolism quantitatively in a devascularized pig model of acute liver failure (ALF). Ammonia and amino acid fluxes were measured across the portal drained viscera (PDV), kidneys, hind leg, and lungs in ALF pigs. ALF pigs developed hyperammonemia and increased glutamine levels, whereas glutamate levels were decreased. PDV contributed to the hyperammonemic state mainly through increased shunting and not as a result of increased glutamine breakdown. The kidneys were quantitatively as important as PDV in systemic ammonia release, whereas muscle took up ammonia. Data suggest that the lungs are able to remove ammonia from the circulation during the initial stage of ALF. Our study provides new data supporting the concept of glutamate deficiency in a pig model of ALF. Furthermore, the kidneys are quantitatively as important as PDV in ammonia production, and the muscles play an important role in ammonia removal.     

Accumulation of large neutral amino acids in the brain of sparse-fur mice at hyperammonemic state

Sparse-fur mice which are deficient in ornithine transcarbamylase, the second-step enzyme in the urea cycle, were examined for hyperammonemia and its relationship with encephalopathy. We compared amino acid concentrations in the serum and brain of spf mice with those of control mice. Unlike hepatic encephalopathy we could not find marked amino acid changes in the serum of spf mice besides low levels of citrulline and arginine. But in the brain of spf mice, glutamine was increased strikingly during hyperammonemia, and a concomitant accumulation of large neutral amino acids such as tyrosine, phenylalanine, methionine, and histidine was observed. The accumulation of these large neutral amino acids in the brain was not influenced by 24-hr fasting which caused increases in branched chain amino acids in the serum. From these results, we conclude that the accumulation of the large neutral amino acid in the brain of hyperammonemic state is caused by uptake of ammonia in the brain and the subsequent accumulation of glutamine, but is not influenced by a decreased ratio of branched chain amino acids to aromatic amino acids in the serum.     

Effects of congenital hyperammonemia on the cerebral and hepatic levels of the intermediates of energy metabolism in spf mice.

Sparse-fur (spf) mutant mice with X-linked ornithine transcarbamylase (OTC) deficiency were examined for hyperammonemia and its effect on energy metabolism. We compared the levels of ammonia, glutamine, glutamate and some of the intermediates of energy metabolism in the brain and liver of spf mice with those of control mice. In spf mice we observed significant increases in ammonia, glutamine, alpha-ketoglutarate and glucose with a significant decrease in ATP, glutamate and pyruvate in both brain and liver. The redox states of the brain and liver were also altered in spf mice. The results suggest that many of the metabolic alterations seen in spf mice could be due to the elevated ammonia levels. The spf mouse may, therefore, be an ideal model for the study of the neurotoxic effects of ammonia in chronic hyperammonemic syndromes.     

Muscle cell growth in protein deficient rats following administration of sheep red blood cells.

In order to observe the effects of sheep red blood cells (SRBC) administration on the muscle cell growth in malnourished states, adult male Wistar rats (135 +/- 10 g 10 animals per group) subjected during 30 days to 1% and 10% protein diets, were injected (i.v.) either 15.5 x 10(8) sheep red blood cells or 0.5 ml saline/100 g b.w. after 20 days of experiment. On the 10th day after injection the animals were sacrificed and the gastrocnemius muscle was removed, weighed and homogenized. The supernatant fluids were used to evaluate muscle protein, DNA and RNA rates and acid DNase activity. All parameters were depleted in malnourished rats, indicating a muscle cellular atrophy as well as a decrease in muscle protein synthesis per DNA-unit. Muscle hyperplasia and hypertrophy were found in antigenically stimulated rats fed 10% protein against non-stimulated control. In contrast, muscle growth in protein-deficient rats SRBC-treated was unmodified when compared to non-stimulated malnourished muscle, although RNA functionality seems to be enhanced (RNA/DNA). These data suggest that a redistribution of essential nutrients occurred for muscle growth adaptation rather than for defensive mechanism.     

Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine

To elucidate the mechanism of hydrolysis of fish muscle proteins by fish proteinases in fish sauce production, each pure preparation of three alkaline proteinases and two acid proteinases from sardine was tested for its ability to hydrolyze various proteins and its stability in the presence of 0 to 25% of NaCl. Each of the alkaline proteinases hydrolyzed casein more rapidly than other proteins. A major alkaline proteinase (III) hydrolyzed sarcoplasmic protein from sardine 5-times faster than other alkaline proteinases. Each of two acid proteinases hydrolyzed hemoglobin and myoglobin more rapidly than the other proteins. After preincubation with 25% NaCl, an alkaline proteinase (III) and an acid proteinase (II) were stable although the other proteinases became unstable. The two proteinases, alkaline proteinase III and acid proteinase II, were also stable for three months after the beginning of fish sauce production. The proteolytic activity of each of alkaline and the acid proteinases was strongly inhibited by more than 15% NaCl; however, minimum inhibition was observed when sardine muscle proteins were used as the substrate.     

Brain energy metabolism and mitochondrial dysfunction in acute and chronic hepatic encephalopathy

One proposed mechanism for acute and chronic hepatic encephalopathy (HE) is a disturbance in cerebral energy metabolism. It also reviews the current status of this mechanism in both acute and chronic HE, as well as in other hyperammonemic disorders. It also reviews abnormalities in glycolysis, lactate metabolism, citric acid cycle, and oxidative phosphorylation as well as associated energy impairment. Additionally, the role of mitochondrial permeability transition (mPT), a recently established factor in the pathogenesis of HE and hyperammonemia, is emphasized. Energy failure appears to be an important pathogenetic component of both acute and chronic HE and a potential target for therapy.     

Urea cycle disorders, hyperammonemia and neurotransmitter changes

In congenital urea cycle disorders, detoxification of ammonia is impaired, leading to hyperammonemia. Ammonia is the major component causing the acute neurological disturbances. It may influence the supply of substrate and its transport at the blood-brain barrier (BBB) which results in alterations in the synthesis and catabolism of neurotransmitters in the brain. In hyperammonemic rats, the uptake of tryptophan into the brain is increased with an augmented flux through the serotonin pathway. In the forebrain, glutamine as well as amino acids transported with the same L-carrier system, such as phenylalanine, tyrosine and tryptophan, are elevated. It is postulated that the increased transport of tryptophan at the BBB occurs in exchange with glutamine. Methionine sulfoximine (MSO) inhibits glutamine synthetase in the cerebral cortex. The activity drops from 5.85 +/- 0.38 to 1.07 +/- 0.37 mumol/min/g wet weight. Under MSO, the brain tryptophan uptake also decreased to 64.2 +/- 4.5% in hyperammonemic rats, to 54.1 +/- 8.0% in untreated hyperammonemic rats, whereas without MSO an increase of tryptophan uptake was observed. An effect of glutamine on tryptophan transport could also be demonstrated using brain microvessel preparations as a model for the BBB. Our findings indicate that preloading isolated microvessels with L-glutamine increases tryptophan uptake into the endothelia when L-glutamine is at concentrations found in brain homogenates under hyperammonemia. Since brain microvessels do not contain glutamine synthetase activity, enzymes from the gamma-glutamyl cycle may be involved in the glutamine-mediated tryptophan transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA,peroxidase profile,and biochemical composition changes during the treatment in vitro of pollen of Argemone mexicana L. with SO 2

The allergenic pollen of Argemone mexicana L. a common road-side weed was subjected to artificial SO 2 fumigation at a concentration of 100ppm for 24 hours, 48 hours and 72 hours and its effect on the total carbohydrate, lipid, free amino acid, DNA and RNA content as well as peroxidase isozyme and DNA profile studied. In comparison to the control (pollen exposed to charcoal-filtered air), the carbohydrate, lipid, DNA and RNA content decreased with the increase in the time of exposure to SO 2 , where as the percentage composition of free amino acid increased. Contrary, to this, the activity of peroxidase was found to increase with a change in the iso-peroxidase zymogram showing three new anionic isozyme bands. SO 2 affected the total DNA profile leading to the gradual breakdown of DNA with several bands observed on agarose gel electrophoresis with an increase in the time of fumigation.     

Cerebral Ammonia Metabolism in Hyperammonemic Rats

The short-term metabolic fate of blood-borne [13N]ammonia was determined in the brains of chronically (8- or 14-week portacaval-shunted rats) or acutely (urease-treated) hyperammonemic rats. Using a "freeze-blowing" technique it was shown that the overwhelming route for metabolism of blood-borne [13N]ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of [13N]ammonia to L-[amide-13N]glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14-week portacaval-shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight-matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.     

Cellular effects of adrenosterone feeding to juvenile carp, Cyprinus carpio L., effect on liver, kidney, brain and muscle protein and nucleic acids

Different groups of juvenile common carp (3–4 g) were fed diets supplemented with adrenosterone at doses of 1.0, 2.5 and 10.0 mg kg^-1 for 60 days and the effect on food conversion efficiency and cellular growth responses of liver, kidney, brain and muscle were investigated. At the end of the hormone feeding phase an increase of 40–75% was observed in food conversion efficiency. No change in hepato-somatic and viscero-somatic index was observed, but brain and kidney weights in relation to body weight was decreased compared with the controls. Variable changes in protein, RNA/DNA, protein/RNA and protein/DNA were seen in liver, kidney, brain and muscle. Feeding of the steroid for 60 days increased the water content of the muscle but ash contents of the muscle were decreased. No change in the total muscle lipids and total nitrogen content were observed. After the removal of the drug from the feed, some of the changes reported were ameliorated.     

Effect of short-term treatment of eugenol on the seminal vesicles of adult albino rats

The structure and biochemical content of adult albino rat seminal vesicles, were studied, after administration of two different doses of eugenol for 10 days (0.2 and 0.3 mg/kg/day, i.m.). Marked decreases in the concentrations of nucleic acids, fructose and total protein as well as RNA/DNA ratio (61%) and protein/DNA ratio (27%) were observed. A remarkable increase in phospholipid concentration was noted with a corresponding decrease in neutral lipids. Histologically, eugenol treated animals showed degeneration of the secretory columnar cells and well developed myofibrillar connective tissues when compared to control animals.     

Protein and RNA synthesis in the skeletal muscle of hereditary dystrophic mouse.

Protein and RNA contents in muscle of normal and hereditary dystrophic mice C57BL/6J-dy/dy were reexamined on the basis of DNA. It was observed that protein and RNA contents in dystrophic muscle decreased at the early stage of the disease, in disagreement with the reported results on a wet weight basis, in which RNA content in dystrophic muscle had been found to increase. Rates of protein and RNA systhesis in the early stage of the disease were also determined with a concomitant check of the specific activities of free amino acids and free nucleotides. The rates of both protein and RNA synthesis (i.e., specific activities of protein and RNA) were higher in the dystrophic muscle, but when they were expressed on a DNA basis, the total protein synthesis per cell was the same as that of normal muscle and the total RNA synthesis per cell showed a smaller increase in dystrophic muscle. These apparent increases of protein and RNA synthesis were discussed in connection with the decreased protein and RNA contents in the cells of dystrophic muscle. The synthesized RNAs seemed to contain mRNA on the basis of sedimentation character and Millipore filter binding ability. However, no particular RNA was mainly synthesized in dystrophic muscle.     

The content of abscisic acid and the activities of proteinases and trypsin inhibitory proteins, in the germinating seed of common beans under water stress conditions

Specific features of changes in the contents of free and bound abscisic acid (ABA) and the activities of neutral and alkaline proteinases and trypsin inhibitory proteins were determined in the embryonic axis and cotyledons of the common bean (Phaseolus vulgaris L.) after drying. The changes in ABA content, observed following the loss of 5% seed weight, were regarded as an adaptive reaction to stress, whereas the corresponding changes after the loss of 10% of the seed’s weight, was regarded as a result of a pathological disturbance of ABA metabolism. Both drying modes had a negative effect on the state of the proteinase-inhibitory system, as was apparent from the disruption of the regular inverse correlation between the activities of proteinases and serine proteinase inhibitory proteins. A comparison of the dynamics of these characteristics with the buildup of water stress demonstrates an inverse correlation between the content of free ABA and the activity of the proteinases studied. This suggests a potential inhibitory effect of this hormone on the function of the hydrolases in question in the germinating seed.     

Changes in amino acid uptake and protein synthesis of bullfrog tadpole liver and tail tissues during triiodothyronine-induced metamorphosis

The effect of triiodothyronine (T₃′) on the uptake of several amino acids into the amino acid pools and into proteins of Rana catesbeiana tadpole liver and tail muscle and tail fin has been studied. Labeling of the alanine and glycine pool was stimulated in the liver more than the leucine pool. After exposure to T₃ for 3 days, uptake of α-aminoisobutyric acid (a transport model substrate) into liver was stimulated about 55%. In tail tissues uptake of leucine was stimulated but uptake of alanine was depressed by T₃. Incorporation of leucine and alanine into tissue protein was stimulated in the liver but inhibited in tail tissues after T₃ injection.Changes in other macromolecules and ATP and ADP levels in liver and tail muscle were also investigated during induced metamorphosis. In the liver, the total DNA content did not change, but the RNA and protein content per liver increased significantly. The increase in RNA/DNA and protein/DNA ratios, suggested that liver cells underwent hypertrophy during induced metamorphosis. The ATP level showed a transient decrease after 3 days of T₃ treatment. In tail muscle, protein and RNA content decreased as the muscle regressed, but the DNA content and ATP level remained unchanged throughout the experimental period.     

Seasonal patterns of nucleic acid concentrations and amino acid profiles of Parapenaeus longirostris (Crustacea, Decapoda): relation to growth and nutritional condition

The present work describes the seasonal changes in nucleic acid concentrations and amino acid profiles in the muscle of juvenile Parapenaeus longirostris and their relation to growth and nutritional condition. RNA content varied significantly between seasons, being the highest values attained in spring and the lowest in winter (p < 0.05). Similar results were obtained with RNA:protein and RNA:DNA ratios. In respect to total amino acid content (TAA), a significant increase from winter to spring was observed (p < 0.05) and the major essential amino acids (EAA) were arginine, histidine and leucine. Within non-essential amino acids (NEAA) glutamic acid, aspartic acid, glycine and proline were dominant. From winter to spring, a significant variation in NEAA content occurred (26.8; p < 0.05), mainly due to the significant increase of glutamic acid (79.1) and serine (66.7) (p < 0.05). EAA content did not vary significantly between seasons (p > 0.05). In opposition, during this period a significant decrease in the free amino acid content (FAA) was observed (p < 0.05); a higher percentage of decrease was attained in free non-essential (FNEAA – 42.9) in comparison to free essential amino acids (FEAA – 40.2). The significant increase in RNA and TAA contents from winter to spring may be related with protein synthesis. On the other hand, the lowest values obtained in winter may be due to a reduction in feeding activity; in this period the muscle protein must be progressively hydrolysed, which is evident with the higher FAA content. The liberated amino acids enter FAA pool and become available for energy production. In conclusion, it was evident that the seasonal cycle in activities such as feeding and growth with nucleic acids and amino acid analyses was noticed.     

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.