Effect of hyperammonemia on brain amino acids in young and adult ferrets

Summary Effects of arginine deficiency and hyperammonemia on the brain concentrations of amino acids and urea cycle enzyme activities in young and adult ferrets were investigated. Only young ferrets developed hyperammonemia and encephalopathy immediately after consuming the arginine-free diet. Brain ornithine and citrulline concentrations in young ferrets fed arginine containing diet were significantly lower than those in adult ferrets. Compared to rats and other animals, young and adult ferrets had lower concentrations of brain glutamic acid and glutamine. Unlike in other species, brain glutamine was not elevated in young, hyperammonemic ferrets. Brain arginase and glutamate dehydrogenase activities were significantly increased in young ferrets fed arginine-free diet. Young ferrets provide a useful animal model for investigating the neurotoxicity of acute hyperammonemia.Abbreviations ACD Arginine-containing diet - AFD Arginine-free diet This work was presented, in part, at the annual meeting of the Midwest Society for Pediatric Research, Chicago, IL, 1991.     

Guanidino compound metabolism in arginine-free diet induced hyperammonemia.

Guanidino compounds, intermediates of arginine metabolism, are altered in many pathological conditions especially those involving the urea cycle. Arginine and creatine play an important role in nitrogen metabolism whereas other guanidino compounds such as guanidinosuccinic acid and N-acetylarginine are toxins. Our objective was to investigate the relationship between guanidino compounds and hyperammonemia. Young and adult ferrets were fed a single meal of either an arginine-containing diet (ACD) or an arginine-free diet (AFD). Guanidino compounds were determined by HPLC in the plasma, liver, kidney and brain 3 h after feeding the specified diet. Only young ferrets fed AFD developed hyperammonemia. Plasma and kidney arginine was decreased whereas guanidinosuccinic acid was increased in young ferrets fed AFD. Hepatic creatine and kidney and brain guanidinoacetic acid were significantly decreased in this group. These results indicate that AFD-induced hyperammonemia produced decreased methylation activity in the liver and transamidination activity in kidney. Elevated guanidinosuccinate levels coupled with deficient hepatic creatine synthesis may play a role in the pathophysiology of hyperammonemia.     

Arginine deficiency, hyperammonemia and Reye's syndrome in ferrets

Young male ferrets developed hyperammonemia and encephalopathy shortly after eating a diet lacking in arginine. The dietary supplementation of arginine or intraperitoneal injection of ornithine prevented hyperammonemia and shortened the duration of encephalopathy. Therefore, young ferrets were assumed to be unable to meet their ornithine needs from sources other than arginine. Adult ferrets did not develop hyperammonemia and encephalopathy after eating arginine-free diet. Because young ferrets are also susceptible to human influenza infections, they were further tested as animal model of Reye's syndrome. Reye's syndrome is a serious childhood disorder that develops following influenza infections and is characterized in part by an encephalopathy, hyperammonemia and elevated serum transaminases. In young ferrets, concurrent administration of aspirin with human influenza inoculation and an arginine-free diet produced symptoms similar to those seen in humans with Reye's syndrome. The ferret model appears to be useful for studying the roles of various etiologic agents and their interactions in producing Reye's syndrome-like disorders. The ammonia metabolism in ferrets is reviewed and the ferret model for Reye's syndrome and its applications for the better understanding of this disorder in humans are discussed.     

Plasma arginine and ornithine levels and selected enzyme activities in uremic rats fed various amounts of arginine

Rats weighing 100 g were made chronically uremic by partial left renal artery ligation and contralateral nephrectomy. Rats with urea clearances below 0.30 ml/min and sham-operated controls were pair-fed arginine-free diets, diets containing normal amounts of arginine or diets with high levels of arginine. After 4 to 8 weeks, rats were killed and plasma levels of arginine, ornithine and lysine were measured. In addition, activities of various urea cycle enzymes in liver and kidney and renal transamidinase were determined. Plasma amino acid levels and enzyme activities of the urea cycle remained constant in control rats fed diets differing in arginine content. However, renal transamidinase activity was elevated in control rats fed arginine-free diets. In plasma of uremic as compared with control rats, arginine levels varied with the arginine intake, and lysine levels were elevated when arginine supplements were fed. With all diets, plasma ornithine remained constant in uremic rats at slightly but not significantly increased levels. Hepatic carbamoyl phosphate synthetase activity and renal arginine synthetase activity were reduced in uremic as compared to control rats. Renal transamidinase activity, expressed per g of kidney, was elevated in uremic rats with all diets except arginine-free. When amino acid diets were fed, hepatic arginase activity was higher in uremic rats and this increase was enhanced by arginine-free diets. Other enzyme activities in uremic rats were not affected by the amount of arginine in the diet.     

Effect of Individual Essential Amino Acid-Devoid Diets on the Activities of Liver Urea Cycle Enzymes in Rats

The activities of all urea cycle enzymes (carbamyl phosphate synthetase, ornithine trans- carbamylase, argininosuccinate synthetase, argininosuccinase and arginase) have been determined in the liver of rats forcibly fed diets lacking in individual essential amino acids from amino acid mixture simulating to a casein. In general, these enzyme activities (units/g liver and total units/body wt) in rats fed the single essential amino acid-devoid diet decreased as compared with those activities in animals fed complete diet, but their decreases were not as large as those observed in group of all amino acid-devoid diet. The degree of decrease in these enzyme activities differed somewhat from each other in individual enzymes and each essential amino acie-devoid groups. In contrast, in rats fed the arginine devoid diet, the activities (total units/body wt) of all enzymes expect the case of arginase increased more than those in the group of complete diet.     

Growth hormone and rat liver mitochondria effects on urea cycle enzymes

Effects of hypophysectomy and subsequent growth hormone administration on mitochondrial enzymes of the urea cycle were investigated in rat liver. Hypophysectomy increased the activities of the two mitochondrial enzymes, carbamyl phosphate synthetase and ornithine transcarbamylase but not of the cytosolic enzyme, argininosuccinate synthetase. The activity of mitochondrial phosphate dependent glutaminase was not affected. Administration of bovine growth hormone (100 μg/100 g body weight) for two weeks decreased the activities of carbamyl phosphate synthetase and ornithine transcarbamylase almost to the normal level. These results suggest a specific effect of growth hormone on mitochondrial enzymes of the urea cycle and serve to explain the increased urea formation in hypopituitarism.     

Fibrin membrane endowed with biological function. VII. An approach to an artificial liver; conversion of ammonia to urea in vitro

As an “artificial liver” for the conversion of ammonia to urea, a group of enzymes in ornithine cycle together with carbamyl phosphate synthetase I and inorganic pyrophosphatase were embedded in a single fibrin membrane. The immobilized enzyme system thus prepared had an ability to convert ammonia to urea not only in a buffer solution but also in human plasma.     

Influence of Forced-Feeding of Individual Essential Amino Acids on the Activities of All Urea Cycle Enzymes in Rat Liver

The response of all urea cycle enzymes, i.e. carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase and arginase, has been determined in the liver of protein-depleted young rats which were forcibly fed individual essential l-amino acids along with or without caloric sources. The feeding of individual amino acids produced different effects on the level of each of the enzymes, and generally the response of carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase was greater than that of ornithine transcarbamylase. Of all the essential amino acids tested tryptophan was most effective on the elevation of these enzymes. Several amino acids, phenylalanine, leucine, threonine and methionine had also somewhat effect on the increase of some enzyme activities, but other amino acids had little or no effect on the response of these enzymes. On the contrary, histidine and lysine caused appreciable decrease of arginase activity. These enzyme activities in rats fed tryptophan alone were extremely higher than those of animals fed it along with caloric sources. The response level of the enzymes was essentially dependent on the tryptophan content in diets under the proper conditions. Tryptophan feeding did not produce any increase in both levels of urine and plasma urea despite the elevation of all urea cycle enzyme activities occured.     

Effect of growth hormone on rat liver N-acetyl-L-glutamate

Earlier studies have revealed, upon hypophysectomy, a specific increase in mitochondrial urea cycle enzymes, namely carbamyl phosphate synthetase and ornithine transcarbamylase. Administration of growth hormone to hypophysectomized rats brought these enzyme activities back to normal. Since growth hormone plays a role in the formation of citrulline and ultimately urea, in the present study its effect on the levels of N-acetyl-L-glutamate, an allosteric activator of carbamyl phosphate synthetase has been investigated. A significant increase in N-acetyl-L-glutamate concentration in rat liver on hypophysectomy and its reversal back to normal levels on growth hormone administration was reported. These results suggest that the lack of growth hormone tends to amplify urea production by the liver.     

Effect of dietary protein on purine nucleoside phosphorylase and xanthine dehydrogenase activities of liver and kidney in chicken and pigeon

Comparative studies were made on the effects of diets of different protein contents on the activities of purine nucleoside phosphorylase and xanthine dehydrogenase of avian livers and kidneys. In chicken liver and kidney, both enzyme activities were increased with high protein diet, confirming the previous results. In pigeon liver, only purine nucleoside phosphorylase was increased but xanthine dehydrogenase activity was not detected after feeding a high protein diet, while both enzyme activities were increased in the pigeon kidney. The increase in the levels of plasma oxypurines in pigeon serum was consistent with the result that the xanthine dehydrogenase activity of pigeon was not detected in the liver but in the kidney.     

Urea synthesis in the liver and kidney of developing sheep

In order to establish if the urea found in foetal fluids in sheep could be of foetal origin and whether there are changes in the ability of ovine liver to synthesise urea during foetal and postnatal development, the rates of urea production from ammonium and bicarbonate ions have been measured in liver and kidney slices from animals aged from 50 days conceptual age to 16 weeks after birth, and in pregnant and non-pregnant ewes. The activities of five enzymes directly involved in the biosynthesis of urea have also been determined.Urea was found to be synthesised by foetal liver from at least 50 days conceptual age at rates similar to those observed in adult ewes. Highest rates of urea synthesis per unit weight of liver were found immediately after birth. In the liver there were significant positive correlations between the rates of urea synthesis by slices and the activities of carbomoyl phosphate synthase (ammonia) (EC 2.7.2.5), argininosuccinate synthetase (EC 6.3.4.5) and argininosuccinate lyase EC 4.3.2.1). Ornithine carbomoyl transferase (EC 2.1.3.3) activity was highest in the livers of ruminating animals. Hepatic arginase activity (EC 3.5.3.1) was highest during the late foetal life and in the mature foetuses the activity was ten-fold greated than that in maternal liver.Urea was not synthesised from ammonia and bicarbonate in kidney slices and neither ornithine carbomoyl transferase activity nor argininosuccinate synthetase activity could be detected. The activity of renal arginase was at least 70 times less than that found in the liver and the highest activity was found in ruminating lambs.The changes observed in the activities of the urea cycle enzymes during development have been contrasted with those reported to occur in other species. It is concluded that there is no single factor regulating the activities of the five enzymes directly concerned with urea synthesis during development. The results support the hypothesis that in mammals the ability of the liver to synthesise urea in foetal life is related to renal development.     

Immortalization of normal liver functions in cell culture: rat hepatocyte-hepatoma cell hybrids expressing ornithine carbamoyltransferase activity.

Normal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver-specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H-ornithine into protein arginine, and can be selectively grown in arginine-free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue-specific phenotypes.     

Effect of Urease-Induced Hyperammonemia on Metabolism of Guanidino Compounds

We previously reported that guanidino compounds produced by the catabolism of arginine play an important role in the pathophysiology of acute hyperammonemia. In order to understand the metabolism of guanidino compounds during sustained hyperammonemia, we investigated the effect of intraperitoneal urease injection (800 IU/kg) on the levels of guanidino compounds in blood, liver, kidney, and brain of rats. Control rats received an equal volume of saline. Eight hours following injection, rats were sacrificed and blood and tissues were removed. Ammonia and urea were determined by enzymatic and colorimetric assays, respectively. Guanidino compounds were analyzed by high-performance liquid chromatography. Blood and tissue ammonia were significantly increased and urea decreased in urease-treated animals. Blood and kidney arginine levels were significantly decreased although hepatic arginine was increased following urease injection. Elevated hepatic arginine may be due to the rapid conversion of urea to ammonia by urease and the development of a futile urea cycle. Catabolites produced by the transamidination of arginine were significantly decreased in the blood, liver, kidney, and brain of urease-treated rats, whereas acetylation of hepatic arginine to α-N-acetylarginine was increased. Blood and tissue guanidinosuccinic acid levels were not elevated during urease induced hyperammonemia, supporting the hypothesis that urea is a precursor for the synthesis of guanidinosuccinic acid.     

Null mutations in the N-acetylglutamate synthase gene associated with acute neonatal disease and hyperammonemia

N-acetylglutamate synthase (NAGS) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate, an essential allosteric activator of carbamyl phosphate synthetase I, the first enzyme of the urea cycle. Liver NAGS deficiency has previously been found in a small number of patients with hyperammonemia. The mouse and human NAGS genes have recently been cloned and expressed in our laboratory. We searched for mutations in the NAGS gene of two families with presumed NAGS deficiency. The exons and exon/intron boundaries of the NAGS gene were sequenced from genomic DNA obtained from the parents of an infant from the Faroe Islands who died in the neonatal period and from two Hispanic sisters who presented with acute neonatal hyperammonemia. Both parents of the first patient were found to be heterozygous for a null mutation in exon 4 (TGG-->TAG, Trp324Ter). Both sisters from the second family were homozygous for a single base deletion in exon 4 (1025delG) causing a frameshift and premature termination of translation. The finding of deleterious mutations in the NAGS gene confirms the genetic origin of NAGS deficiency. This disorder can now be diagnosed by DNA testing allowing for carrier detection and prenatal diagnosis.     

Regulation and expression of carbamyl phosphate synthetase I mRNA in developing rat liver and Morris hepatoma 5123D

A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.     

Effects of dithiothreitol and thioredoxines on the in vitro activity of enzymes of the urea cycle in rats

The five urea cycle enzymes were studied in desactivated extracts of rat liver. After reduction by dithiothreitol (DTT) and in presence of Mg2+ ions, thioredoxines isolated from rat liver were able to activate carbamyl phosphate synthetase-I (CPS-I) and argininosuccinate synthetase (ASS) respectively by 468% and by 370%. Thioredoxines were purified from adult rat liver and an antiserum was raised to these proteins. After immunologic quantitation, their level in adult rat was 0.103 mg/g liver.     

Lack of hepatic enzymatic adaptation to low and high levels of dietary protein in the adult cat.

The activities of three urea cycle enzymes, several nitrogen catabolic, gluconeogenic, and lipogenic enzymes were measured in the liver of adult cats fed: a commercial kibble; a 17.5 or 70% protein purified diet, or starved for 5 days. Except for an increase in tyrosine transaminase (EC 2.6.1.5) after feeding the high protein diet, there were no changes in the activities of the hepatic enzymes as influenced by dietary protein level. Likewise, starvation had a minimal effect on the activities of these enzymes as compared to that found in similar experiments in rats. These results indicate that the cat may have only minimal capabilities for enzyme adaptation as compared to that found in many herbivores and omnivores and may provide an explanation as to why cats have an unusually high protein requirement as compared to many other mammals.     

A smaller initial dose protects mice against several lethal doses of ammonium acetate

The synthesis of urea in the liver is the main mechanism for the elimination of excess ammonia. Rapid stimulation of the synthesis of urea (e.g. by administration of carbamyl glutamate, the analog of the physiological activator of carbamyl phosphate synthetase I) protects animals given lethal doses of ammonia. Since ammonia enhances the activity of the urea cycle, we tested and show here that administration of small doses of ammonium acetate supresses the mortality induced by a series of repeated LD100 of ammonium acetate separated by one hour, when the first LD100 is injected i.p. starting from 30 min to 5 hours after the initial smaller dose of ammonium acetate. Under these conditions, the levels of ammonia in blood are elevated more than ten times, but in spite of the greater amount of ammonia administered, the ammonemia is much lower than in mice dying after a single LD100. The enhanced synthesis of urea observed is correlated with an increase in the intramitochondrial content of N-acetyl glutamate. These findings are of interest as far as the short-term regulation of urea cycle, the mechanism of ammonia toxicity and have clinical implications.     

Normal N-acetylglutamate concentration measured in liver from a new patient with N-acetylglutamate synthetase deficiency: physiologic and biochemical implications.

N-Acetyl-L-glutamate synthetase (NAG synthetase) is a mitochondrial matrix enzyme which catalyzes the synthesis of N-acetyl-Lglutamate (NAG), a physiologic activator of the urea cycle enzyme carbamylphosphate synthetase I. Deficiency of NAG synthetase in humans has been reported only three times previously. Two cases presented with uncontrolable neonatal hyperammonemia leading to death, while a third child presented with hyperammonemia and a neurodegenerative picture at 15 months of age after previously being healthy. We report here a new case of NAG synthetase deficiency who presented at 4 years, 10 months of age with an episode of hyperammonemia. Diagnosis was made at age 5 years, 6 months when a liver biopsy showed 9.7% of normal activity. Urine orotic acid was low, and total NAG content in liver was normal. Liver pathology revealed micro- and macrovesicular fat and mitochondria of irregular size and shape with intracristae crystallizations. NAG content in liver in patients with NAG synthetase deficiency has not previously been reported. Its normal value in the face of NAG synthetase deficiency suggests an abnormal localization of NAG to the cytoplasm and the likelihood of aberrant cytoplasmic synthesis of this compound. Additional physiologic implications of this speculative abnormal compartmentalization are discussed.     

Regulation of messenger ribonucleic acid levels for five urea cycle enzymes in cultured rat hepatocytes. Requirements for cyclic adenosine monophosphate, glucocorticoids, and ongoing protein synthesis

In adult rat liver, amounts of the urea cycle enzymes are regulated by diet, glucocorticoids, and cAMP. Rat hepatocytes cultured in chemically defined medium were used to precisely define the roles of glucocorticoids and cAMP in regulation of these enzymes at the pretranslational level. With the exception of ornithine transcarbamylase mRNA, cultured rat hepatocytes retain the capacity to express mRNAs for the urea cycle enzymes at the same level observed for liver of intact rats. In the absence of added hormones, mRNAs for argininosuccinate synthetase and argininosuccinate lyase remained at or above normal in vivo levels, while mRNAs for the other three enzymes declined to very low levels. Messenger RNAs for carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinate lyase, and arginase increased in response to either dexamethasone or 8-(4-chlorophenylthio) cAMP (CPT-cAMP). Half-maximal responses occurred at 2-3 nM dexamethasone and at 2-7 microM CPT-cAMP. Cycloheximide abolished the response to dexamethasone but not to CPT-cAMP, suggesting that dexamethasone induced expression of an intermediate gene product required for induction of these mRNAs. The effects of a combination of both hormones were additive for argininosuccinate lyase mRNA and synergistic for carbamyl phosphate synthetase I, argininosuccinate synthetase, and arginase mRNAs. Messenger RNA for ornithine transcarbamylase showed little or no response to any condition tested. Depending on the particular mRNA and hormonal condition tested, increases in mRNA levels ranged from 1.4- to 70-fold above control values.(ABSTRACT TRUNCATED AT 250 WORDS)