Showing papers on "Urea cycle published in 1996"
TL;DR: Analysis of the structure of trimeric11 rat liver arginase reveals that this unique metal cluster resides at the bottom of an active-site cleft that is 15 Å deep, and indicates that arginine hydrolysis is achieved by a metal-activated solvent molecule which symmetrically bridges the two Mn2+ ions.
Abstract: Each individual excretes roughly 10 kg of urea per year, as a result of the hydrolysis of arginine in the final cytosolic step of the urea cycle. This reaction allows the disposal of nitrogenous waste from protein catabolism, and is catalysed by the liver arginase enzyme. In other tissues that lack a complete urea cycle, arginase regulates cellular arginine and ornithine concentrations for biosynthetic reactions, including nitric oxide synthesis: in the macrophage, arginase activity is reciprocally coordinated with that of NO synthase to modulate NO-dependent cytotoxicity. The bioinorganic chemistry of arginase is particularly rich because this enzyme is one of very few that specifically requires a spin-coupled Mn2+-Mn2+ cluster for catalytic activity in vitro and in vivo. The 2.1 angstrom-resolution crystal structure of trimeric rat liver arginase reveals that this unique metal cluster resides at the bottom of an active-site cleft that is 15 angstroms deep. Analysis of the structure indicates that arginine hydrolysis is achieved by a metal-activated solvent molecule which symmetrically bridges the two Mn2+ ions.
402 citations
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340 citations
TL;DR: Biochemical evaluation extended to the first-degree relatives is consistent with a familial form of hypoglycemia due to a leucine-sensitive hyperinsulinism, and an unexpected hyperammonemia, which was also detected in the mother, might be related toLeucine hypersensitivity.
Abstract: A case of a child with recurrent episodes of severe hypoglycemia since the age of 6 months is reported. Biochemical evaluation extended to the first-degree relatives is consistent with a familial form of hypoglycemia due to a leucine-sensitive hyperinsulinism. In addition, this patient has a persistent elevation of serum ammonia levels of uncertain etiology that is more pronounced after meals. Urea cycle defects, organic acidurias, and β-oxidation defects have been ruled out, as well as a possible excessive deamination of glucogenetic amino acids. This unexpected hyperammonemia, which was also detected in the mother, might be related to leucine hypersensitivity.
84 citations
TL;DR: The findings suggested that H. pylori has a urea cycle which acts as an effective mechanism to extrude excess nitrogen from cells.
Abstract: The presence and activities of the enzymes of the urea cycle in the bacterium Helicobacter pylori were investigated employing one- and two-dimensional NMR spectroscopy and radioactive tracer analysis. Cell suspensions, lysates and membrane preparations generated L-ornithine and ammonium at high rates in incubations with L-arginine, indicating the presence of arginase activity. Anabolic ornithine transcarbamoylase (OTCase) activity was identified by the formation of heat-stable products in incubations of cell-free extracts with ornithine and radiolabelled carbamoyl phosphate. The heat-labile product that resulted from incubations of cell-free extracts with citrulline radiolabelled in the guanidino moiety revealed the presence of catabolic OTCase activity. Argininosuccinate formation and catalysis indicated the presence of argininosuccinate synthetase and argininosuccinase activities. The findings suggested that H. pylori has a urea cycle which acts as an effective mechanism to extrude excess nitrogen from cells.
81 citations
TL;DR: Aiming the selection of possible candidates for the reactor cells in a hybrid artificial liver, the activities of 7-ethoxycoumarin deethylation and ammonia removal in 31 hepatocytes cell lines established from the human, rat, mouse, and chicken, compared with freshly isolated rat hepatocytes were investigated.
38 citations
TL;DR: The results show that, within the liver, all mRNAs of the ornithine cycle pathway are zonated, suggesting that, in the rat, common regulatory factors are involved in the establishment of the zonation pattern.
30 citations
TL;DR: The data suggest that biotin deficiency decreases OTC activity and the amount of OTC mRNA, which is well known as a cause of hyperammonemia, on hepatic ureagenesis.
Abstract: Biotin deficiency is well known as a cause of hyperammonemia, but there has been no report on the effect of biotin deficiency on hepatic ureagenesis. In this study, we examined the changes in the activities and gene expression of urea cycle enzymes using rats fed raw egg white as a model of biotin deficiency. All rats were made biotin-deficient by feeding them an avidin-containing diet for 6 wk. The rats were divided into two groups at the beginning of this experiment: biotin-deficient rats (BD rats) and biotin-supplemented rats (BS rats) which were treated with biotin once a day at a dose of 1 mg per rat intraperitoneally. The plasma ammonia concentration of the BD rats (92.8 +/- 12 mumol/L) was significantly higher than that of BS rats (63.9 +/- 16 mumol/L, P < 0.05). The activities of ornithine transcarbamylase (OTC) was significantly lower in the liver of the BD (110.2 +/- 5.5) rats than in the BS rats (154 +/- 3.8 U/mg protein, P < 0.01). Activities of the other urea cycle enzymes were not significantly different in the two groups. OTC gene expression in the liver of BD rats was 40% lower than in BS rats (P < 0.05). These data suggest that biotin deficiency decreases OTC activity and the amount of OTC mRNA.
27 citations
TL;DR: Treatment with CG in a kinetic variant or a partial deficit of CPS has never been clinically proven until now, and the first use has been in N-acetylglutamate synthetase deficiency.
Abstract: Ammonia is channelled into the urea cycle by means of carbamoyl-phosphate synthetase (CPS I). This initial step of urea synthesis takes place in the mitochondria of the liver cell and is catalysed by N-acetylglutamate (NAGA), the physiological allosteric activator of CPS 1I(Grisolia and Cohen 1953). Carbamylglutamate (CG) has a structural analogy with NAGA. Exogenously administered NAGA is ineffective because it cannot enter the mitochondria and is rapidly deacylated in the cytosol (Reglero et al 1977). CG is able to cross the mitochondrial membranes and, in addition, is not inactivated by cytosolic acylases (Kim et al 1953). So far, the first (Schubiger et al 1991) and only use of carbamylglutamate has been in N-acetylglutamate synthetase deficiency. Treatment with CG in a kinetic variant or a partial deficit of CPS has been discussed (Guffon et al 1995 ; O'Conner et al 1985), but has never been clinically proven until now.
26 citations
TL;DR: It is shown that urea synthesis is controlled in chronic HCl acidosis by amino acid uptake by the liver and/or intrahepatic degradation and that the ornithine cycle per se has only minor control of acid-base homeostasis.
Abstract: In a previous paper, we showed that an inhibition of amino acid transport across the liver plasma membrane is responsible for the decrease in urea synthesis in acute metabolic acidosis. We have now studied the mechanism responsible for the decline in urea synthesis in chronic acidosis. Chronic metabolic acidosis and alkalosis were induced by feeding three groups of rats HCl, NH4Cl, and NaHCO3 (8 mmol/day) for 7 days. Amino acids and NH4+ were measured in portal vein, hepatic vein, and aortic plasma, and arteriovenous differences were calculated. The rates of urinary urea and NH4+ excretion were also determined. Hepatic amino acid consumption was lower in both HCl and NH4Cl acidosis compared with NaHCO3-fed rats. Glutamine release was not different in the three conditions. Because intrahepatic concentrations of amino acids and intracellular protein degradation were similar under these conditions, it can be concluded that at low blood pH amino acid catabolism may be inhibited and might explain the observed decrease in urea excretion in HCl, but not NH4Cl, acidosis; urea excretion was comparable in the NH4Cl and NaHCO3 groups presumably because the increased NH4+ load in the former group was processed, uninhibited, to urea. Amino acids not used by the liver in acidosis could account for the 25-fold increase in NH4+ excretion in HCl and NH4Cl compared with alkalosis (P < 0.05). These findings indicate that urea synthesis is decreased in chronic HCl acidosis. They show that urea synthesis is controlled in chronic, as in acute, acidosis by amino acid uptake by the liver and/or intrahepatic degradation and that the ornithine cycle per se has only minor control of acid-base homeostasis.
21 citations
TL;DR: Results suggested that the activities of amino acid synthetase localized in the small intestinal flora were lost and infusion limited to the essential amino acids to SBS patients will cause a deficiency of the urea cycle intermediates, ornithine, citrulline, and arginine and may lead to hyperammonemia.
Abstract: A patient with short bowel syndrome (SBS) and renal failure developed a disturbance of consciousness with hyperammonemia. Abnormally low concentrations of ornithine, citrulline, and arginine were observed on the plasma aminogram. These results suggested that the activities of amino acid synthetase localized in the small intestinal flora were lost. The small intestine is required for arginine synthesis; thus, infusion limited to the essential amino acids to SBS patients will cause a deficiency of the urea cycle intermediates, ornithine, citrulline, and arginine and may lead to hyperammonemia. In addition, the renal insufficiency may have caused decreased excretion of ammonia. In this patient, supplemental arginine improved the symptoms.
20 citations
TL;DR: There are a number of human illnesses that are associated with increased levels of ammonia in blood, including liver cirrhosis, fulminant hepatic failure and congenital defects of urea cycle enzymes.
Abstract: Ammonia is a product of the degradation of proteins and of other compounds ; however, when it is in excess, ammonia is a toxic compound . A fiveto ten-fold increase in blood ammonia levels leads to alterations in the function of the central nervous system, and can lead to coma and death . To prevent these toxic effects, ureotelic animals have developed the urea cycle, which is mainly located in liver, and eliminates ammonia by incorporating it into urea, which is eliminated in urine . This maintains safe levels of ammonia in blood and tissues. However, when this process fails due to a congenital defect in the urea cycle enzymes or by impairment of liver function, the levels of ammonia in blood rise and can lead to altered brain function. This syndrome, known as hepatic encephalopathy, can lead to coma and death. Ammonia interferes with neurotransmission and with electrophysiological processes (Fan et al., 1990; Szerb and Butterworth, 1992 ; Raabe and Lin, 1984 ; Raabe, 1992 and 1994, Butterworth, 1994) . There are a number of human illnesses that are associated with increased levels of ammonia in blood, including liver cirrhosis, fulminant hepatic failure and congenital defects of urea cycle enzymes . Independently of its origin, in these situations the levels of ammonia in blood increase 5 to 10-fold, leading to hepatic encephalopathy, and high mortality . In fact, hepatic encephalopathy is one of the main causes of death in occidental countries . In Spain 12,000 people die every year for this reason, which represents about
TL;DR: Forty children with Reye syndrome (RS) or Reye‐like illnesses were investigated to elucidate the underlying aetiologies and the underlying inherited metabolic disorders confirmed.
Abstract: Forty children with Reye syndrome (RS) or Reye-like illnesses were investigated to elucidate the underlying aetiologies. Extensive biochemical studies including patterns of organic acids and amino acids, liver histopathology, and, if available, a DNA approach were performed. In addition to classical RS (n = 10), the causes of Reye-like conditions included hereditary organic acidaemias (n = 13), urea cycle defects (n = 4), mitochondrial disorders (n = 3), fulminant hepatitis (n = 2), tyrosinaemia (n = 1), valproate-associated hepatotoxicity (n = 1), and other non-specific generalized organic acid disorders (n = 6). It is important to collect specimens when encephalopathy with liver dysfunction of unknown causes is noted. When the underlying inherited metabolic disorders are confirmed, the prevention of the recurrence by adequate diet control and medications, and genetic counselling become possible.
TL;DR: Three children with transaminase elevations and hepatic insufficiency who were given the diagnosis of cryptogenic hepatitis after the more common viral and metabolic diseases of the liver had been excluded showed hyperammonemia, low blood urea levels, elevated plasma glutamine levels, and low citrulline levels, suggesting ornithine transcarbamylase deficiency.
Abstract: We describe three children with transaminase elevations and hepatic insufficiency who were given the diagnosis of cryptogenic hepatitis after the more common viral and metabolic diseases of the liver had been excluded. However, further laboratory investigations showed hyperammonemia, low blood urea levels, elevated plasma glutamine levels, and low citrulline levels. Urinary excretion of orotic acid was higher than normal, with absent urinary homocitrulline and normal fractional tubular reabsorption of lysine, ornithine, and arginine. These findings suggest the diagnosis of ornithine transcarbamylase deficiency. We emphasize the importance of investigating possible urea cycle disorders by determining ammonia plasma levels, both at baseline and after a protein load; urinary and plasma amino acids; and urinary orotic acid in all patients with liver disease of indeterminate etiology.
TL;DR: Early changes in plasma amino acid pool characteristics after wounds were of systemic origin, not related to the type of injured tissues, and suggested disturbance in urea cycle activity, although urea level was not altered.
Abstract: Plasma amino acid profiles in patients during the early period (first 18 hours) following military gunshot/missile wounds were investigated. Patients (n = 29) were casualties from the war in the former Yugoslavia with injury severity scores ranging from 4 to 18. They were divided into three groups : soft tissue (muscle) damage, wounds with fractures, and vital structures injured. Controls were normal blood donors (n = 17). Free amino acids were analyzed in venous plasma. Increased concentrations of phenylalanine and glutamine associated with increased molar phenylalanine/tyrosine ratio in plasma indicated increased net protein catabolism in the peripheral tissues, regardless of the type of injured tissues. Decreased plasma arginine, ornithine and citrulline levels, accompanied with increased molar glutamine/valine ratio, suggested disturbance in urea cycle activity, although urea level was not altered. We concluded that early changes in plasma amino acid pool characteristics after wounds were of systemic origin, not related to the type of injured tissues.
TL;DR: The cloned cDNA for human AI was inserted into an expression vector downstream from the mitochondrial targeting leader sequence for the mitochondrial enzyme ornithine transcarbamylase and transfected into a variety of recipient cell types, suggesting that the mitochondrial matrix space may be a preferred site for delivery of enzymes in gene replacement therapy.
Abstract: As a toxic metabolic byproduct in mammals, excess ammonia is converted into urea by a series of five enzymatic reactions in the liver that constitute the urea cycle. A portion of this cycle takes place in the mitochondria, while the remainder is cytosolic. Liver arginase (L-arginine ureahydrolase, A1) is the fifth enzyme of the cycle, catalyzing the hydrolysis of arginine to ornithine and urea within the cytosol. Patients deficient in this enzyme exhibit hyperargininemia with episodic hyperammonemia and long-term effects of mental retardation and spasticity. However, the hyperammonemic effects are not so catastrophic in arginase deficiency as compared to other urea cycle defects. Earlier studies have suggested that this is due to the mitigating effect of a second isozyme of arginase (AII) expressed predominantly in the kidney and localized within the mitochondria. In order to explore the curious dual evolution of these two isozymes, and the ways in which the intriguing, aspects of AII physiology might be exploited for gene replacement therapy of AI deficiency, the cloned cDNA for human AI was inserted into an expression vector downstream from the mitochondrial targeting leader sequence for the mitochondrial enzyme ornithine transcarbamylase and transfected into a variety of recipient cell types. AI expression in the target cells was confirmed by northern blot analysis, and competition and immunoprecipitation studies showed successful translocation of the exogenous AI enzyme into the transfected cell mitochondria. Stability studies demonstrated that the translocated enzyme had a longer half-life than either native cytosolic AI or mitochondrial AII. Incubation of the transfected cells with increasing amounts of arginine produced enhanced levels of mitochondrial AI activity, a substrate-induced effect that we have previously seen with native AII but never AI. Along with exploring the basic biological questions of regulation and subcellular localization in this unique dual-enzyme system, these results suggest that the mitochondrial matrix space may be a preferred site for delivery of enzymes in gene replacement therapy.
TL;DR: Long‐term exposure of cultured rat hepatocytes to 6.67 μM arsenite decreases urea production, cellular and total arginase activity and protein content and increases the release ofArginase into the culture medium, which could be useful markers of hepatotoxicity in in vitro assays.
Abstract: Urea cycle is a hepatic metabolic pathway involving five enzymes and several intermediary metabolites and can be altered by different chemicals. To investigate the effect of arsenic, an ubiquitous hepatotoxic agent, on urea production we exposed long-term cultures of adult rat hepatocytes, which produce urea, to 1.33 and 6.67 microM arsenite for 2 weeks. In cultures exposed to 6.67 microM, urea production decreased 60-70% and cellular arginase activity decreased 30, 70 and 85% after 4, 7 and 14 days of exposure, respectively. The arginase activity released to the medium increased significantly after 4, 7 and 14 days, with a maximum value after 7 days of exposure that was 27-fold higher than that of the untreated controls. The total arginase activity also decreased 35, 52 and 82% after 4, 7 and 14 days of exposure and protein content decreased 57 and 65% after 7 and 14 days of exposure, respectively. Exposure to 6.67 microM arsenite also produced accumulation of intracytoplasmic lipid droplets, vacuolizations and enlargement of the intercellular spaces. On the other hand, exposure of hepatocytes to 1.33 microM arsenite caused an initial decrease of 20% in urea production, did not change cellular, released and total arginase activity and cellular protein content and produced accumulation of intracytoplasmic lipid droplets. These results show that long-term exposure of cultured rat hepatocytes to 6.67 microM arsenite decreases urea production, cellular and total arginase activity and protein content and increases the release of arginase into the culture medium. These alterations could be useful markers of hepatotoxicity in in vitro assays.
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TL;DR: This work describes a case of fulminant citrullinemia in an infant in whom the major pathologic findings included diffuse cerebral edema and a lack of overt metabolic derangement characteristic of neonates with a urea cycle defect.
Abstract: Citrullinemia, a rare inborn error of metabolism, is characterized by a deficiency of argininosuccinic acid synthetase that results in large increases in plasma ammonia, citrulline, and glutamine, with normal acid-base balance. The neurologic symptoms vary from poor feeding, vomiting, and irritability to hypotonia, apnea, and death. The most common pathologic findings at autopsy are cerebral edema and focal neuronal necrosis. We describe a case of fulminant citrullinemia in an infant in whom the major pathologic findings included diffuse cerebral edema and a lack of overt metabolic derangement characteristic of neonates with a urea cycle defect. Our case differs from the classic presentation of citrullinemia in that subarachnoid hemorrhage was identified early in the clinical course. We report the first observation of subarachnoid hemorrhage in an infant with a urea cycle defect.
TL;DR: Conversion of "C arginine to 14C02 would be impossible in retrograde perfusions if the perivenous, OAT-containing cells, lacked an arginase, so "CO, production, from U-14CArginine in livers perfused in antegrade and retrograde directions" was examined.
Abstract: Arginine catabolism in liver, requires arginase to convert arginine to ornithine and ornithine aminotransferase (OAT) to initiate ornithine catabolism. OAT is restricted to a few cells of the hepatic aanus, the perivenous hepatocytes (1). The question arises as to whether these cells contain an arginase or whether arginase in the periportal cells, where the urea cycle occurs, must cleave arginine and release ornithine downstream to the OAT-containing hepatocytes. To discriminate between these two hypotheses we examined "CO, production from U-14C arginine in livers perfused in antegrade and retrograde directions. Rat liver contains no arginine decarboxylase and only very low activity of ornithine decarboxylase (2). Furthermore, l4CO, production from U-"C arginine in our perfusions is inhibited by gabaculine, an inhibitor of OAT (3). Therefore, we used "CO, production, from U-"C arginine as a measure of arginine catabolism through the OAT pathway. Conversion of "C arginine to 14C02 would be impossible in retrograde perfusions if the perivenous, OAT-containing cells, lacked an arginase. Figure 1. represents data for the catabolism of U-"C arginine to "CO, in livers perfused in both antegrade and retrograde directions. The maximum rate of "CO, production in the
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TL;DR: This is the first report in a Chinese family confirmed by biochemical and DNA analyses of OTC deficiency, which has been described as the most common inborn error of ureagenesis in humans.