Changes in Creatine and Urea Formation in Rats Fasted and Fed Low Protein Diets

Changes in the time course of the urinary excretion of creatinine, creatine and urea, and the activities of kidney transamidinase and liver urea-cycle enzymes were investigated in rats fasted and fed on a 10% casein diet and 10% casein diets supplemented with 10% glycine and/or 1.4% arginine.

The urinary total-creatinine of the fasted rats increased extremely during fasting for 7 days, while that of the animals given the 10% casein diet supplemented with glycine and arginine rose exceedingly on the 3rd day and thereafter no significant change was observed. Most of the increase of total-creatinine could be accounted for by the increase of creatine. The activity of kidney transamidinase in the fasted rats decreased in the 3rd day and thereafter kept nearly constant. The transamidinase activity of rats fed on the 10% casein diet after giving a protein-free diet for 5 days increased in the 3rd day. An inverse relation was observed between the urinary creatine and the transamidinase activity. The urinary urea increased in the rats fasted or fed on the 10% casein diets with the supplement of glycine and/or arginine. In fasting, the activities of liver urea-cycle enzymes, except arginase, had a tendency of increasing with the lapse of time. The arginase activity remained more or less constant. The reason of the extreme increase of urinary creatine during starvation was discussed.     

Contribution of dietary arginine to nitrogen utilisation and excretion in juvenile sea bass (Dicentrarchus labrax) fed diets differing in protein source

The role of dietary arginine in affecting nitrogen utilisation and excretion was studied in juvenile European sea bass (Dicentrarchus labrax) fed for 72 days with diets differing in protein sources (plant protein-based (PM) and fish-meal-based (FM)). Fish growth performance and nitrogen utilisation revealed that dietary Arg surplus was beneficial only in PM diets. Dietary Arg level significantly affected postprandial plasma urea concentrations. Hepatic arginase activity increased (P<0.05) in response to dietary Arg surplus in fish fed plant protein diets; conversely ornithine transcarbamylase activity was very low and inversely related to arginine intake. No hepatic carbamoyl phosphate synthetase III activity was detected. Dietary arginine levels did not affect glutamate dehydrogenase activity. A strong linear relationship was found between liver arginase activity and daily urea-N excretion. Dietary Arg excess reduced the proportion of total ammonia nitrogen excreted and increased the contribution of urea-N over the total N excretion irrespective of dietary protein source. Plasma and excretion data combined with enzyme activities suggest that dietary Arg degradation via hepatic arginase is a major pathway for ureagenesis and that ornithine-urea cycle is not completely functional in juvenile sea bass liver.     

Activities of ornithine aminotransferase and ornithine decarboxylase in chronically uremic rats

The activities of two enzymes mediating different pathways of ornithine catabolism were measured in liver and kidney of chronically uremic rats and their pair-fed controls. Two months following partial nephrectomy hepatic ornithine aminotransferase (OAT) activity tended to be lower in uremic rats and was correlated with urea clearance and with carbamoyl phosphate synthetase activity. Renal OAT activity in uremic rats was also correlated with urea clearance. When uremic rats were maintained for five months, OAT activity was significantly decreased in liver but not in kidney and the activity of ornithine decarboxylase (ODC), the enzyme regulating polyamine biosynthesis, was reduced in both liver and kidney. In cross-over experiments, evidence was obtained for a factor in uremic kidney cytosol which inhibited renal ODC activity.     

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 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.     

Effects of dietary salt intake on plasma arginine

Because L-arginine is degraded by hepatic arginase to ornithine and urea and is transported by the regulated 2A cationic amino acid y(+) transporter (CAT2A), hepatic transport may regulate plasma arginine concentration. Groups of rats (n = 6) were fed a diet of either low salt (LS) or high salt (HS) for 7 days to test the hypothesis that dietary salt intake regulates plasma arginine concentration and renal nitric oxide (NO) generation by measuring plasma arginine and ornithine concentrations, renal NO excretion, and expression of hepatic CAT2A, and arginase. LS rats had lower excretion of NO metabolites and cGMP, lower plasma arginine concentration (LS: 83 +/- 7 vs. HS: 165 +/- 10 micromol/l, P < 0.001), but higher plasma ornithine concentration (LS: 82 +/- 6 vs. HS: 66 +/- 4 micromol/l, P < 0.05) and urea excretion. However, neither the in vitro hepatic arginase activity nor the mRNA for hepatic arginase I was different between groups. In contrast, LS rats had twice the abundance of mRNA for hepatic CAT2A (LS: 3.4 +/- 0.4 vs. HS: 1.6 +/- 0.5, P < 0.05). The reduced plasma arginine concentration with increased plasma ornithine concentration and urea excretion during LS indicates increased arginine metabolism by arginase. This cannot be ascribed to changes in hepatic arginase expression but may be a consequence of increased hepatic arginine uptake via CAT2A.     

Dietary arginine degradation is a major pathway in ureagenesis in juvenile turbot (Psetta maxima)

Recent studies indicate that urea excretion is responsive to protein intake and that turbot, Psetta maxima, appear to differ from other species by their urea excretion pattern and levels. This study was undertaken to evaluate the influence of dietary nitrogen and arginine on ureagenesis and excretion in turbot. Juvenile turbot (29 g) were fed semi-purified diets containing graded levels of nitrogen (0-8% dry matter) and arginine (0-3% dry matter) for 6 weeks. Growth data showed that turbot have high dietary nitrogen (123 mg/kg metabolic body weight/day) and very low dietary arginine (9.3 mg/kg metabolic body weight/day) requirements for maintenance. Requirements for unit body protein accretion were 0.31 g and 0.15 g for nitrogen and arginine respectively. Post-prandial plasma urea levels and urea excretion rates showed that urea production was significantly (P<0.05) influenced by dietary arginine levels. While hepatic arginase (EC 3.5.3.1) activity increased significantly (P<0.05) with increasing dietary arginine levels, activities of other enzymes of the ornithine urea cycle were very low. Our data strongly suggest that the ornithine urea cycle is not active in the turbot liver and that dietary arginine degradation is a major pathway of ureagenesis in turbot.     

STUDIES ON FUNCTIONAL AND METABOLIC ROLE OF UREA CYCLE INTERMEDIATES IN BRAIN

Abstract— The distribution of argininosuccinate synthetase, argininosuccinase and arginase, and the synthesis of urea in cerebullum. cerebral cortex and brain stem have been studied. Cerebral cortex had high levels of argininosuccinate synthetase and argininosuccinase. and a high ability to synthesize urea from aspartic acid and citrulline. Of the three regions, cerebullum had the highest arginase activity. The activities of the enzymes transamidinase and ornithine aminotransferase in the metabolism of arginine and ornithine in pathways other than urea formation have been studied in the three regions of the rat brain. The activity of creatine phosphokinase in all regions was the same: carbamylphosphatase activity was highest in cerebullum. Cerebral cortex had a high activity of aspartic acid transcarba-mylase. The brain stem, among the three regions, had the lowest activities of glutamine synthetase and glutaminase. The activities of these enzymes in the different regions are discussed in relation to urea production and the utilization of the urea cycle intermediates.
Intraperitoneal injection of high amounts of citrulline brought about a rise in the glutamine synthetase activity of cerebellum and brain stem and a rise in ornithine aminotransferase in cerebral cortex and liver. These results are discussed in relation to the mechanism of action of citrulline in alleviating the toxicity in hyperammonaemic states.     

Effects in chicks of arsenic,arginine, and zinc and their interaction on body weight,plasma uric acid,plasma urea,and kidney arginase activity

The interaction among arsenic, zinc, and arginine was studied in chicks using two fully crossed, three-way, two-by-two-by-two experiments. Arsenic at levels of 0 and 2 μg/g zinc at levels of 2.5 (zinc-deficient) and 25 (zinc-adequate) μg/g, and arginine at levels of 0 and 16 mg/g were supplemented to the diet. After 28 d in both experiments, growth was depressed in chicks fed diets either supplemented with arginine or deficient in zinc. Arsenic deprivation depressed growth of chicks fed diets containing the basal level of arginine and 25 μg supplemental Zn/g. Arsenic deprivation had little or no effect on growth of zinc-deprived chicks fed diets containing the basal level of arginine, or in zinc-deprived or zinc-adequate chicks fed supplemental arginine. Zinc-deficiency elevated urea in plasma and arginase activity in kidney. Those elevations, however, were more marked in arsenic-supplemented than in arsenic-deprived chicks. Also, plasma urea and kidney arginase activity were markedly elevated in chicks fed supplemental arginine; the elevations were more marked in zinc-deficient chicks. These findings support the concept that arsenic has a physiological role, associated with zinc, that can influence arginine metabolism in the chick.     

Effects of arginine-free diet on urea cycle enzymes in young and adult ferrets

Young ferrets develop hyperammonemia soon after eating an arginine-free diet, whereas adult ferrets do not develop hyperammonemia after an identical treatment. Earlier reports indicate that young or adult rats do not develop hyperammonemia and encephalopathy after a single meal of an arginine-free diet. The effects of a single feeding of an arginine-free diet on the urea cycle enzyme activities in the liver of young and adult ferrets is reported. Ornithine carbamyl transferase, carbamyl phosphate synthetase and ornithine aminotransferase activities in the livers of adult ferrets were significantly higher than those in the livers of young ferrets. A single meal of an arginine-free diet did not alter the urea cycle enzyme activities in the liver of young or adult ferrets. The levels of urea cycle enzymes in the liver and kidney of young ferrets were comparable to those in rat liver and kidney. The results suggest that the hyperammonemia observed in young ferrets following a single meal of an arginine-free diet may not be due to the deficiency of enzyme activities.     

Repression of rat kidney L-arginine:glycine amidinotransferase synthesis by creatine at a pretranslational level

The first committed reaction in the biosynthesis of creatine is catalyzed by the enzyme L-arginine:glycine amidinotransferase, commonly called transamidinase. Creatine, the end product of the biosynthetic pathway, is known to alter the levels of kidney transamidinase activity. Rats fed a diet containing 0.3% creatine had 26% of the kidney transamidinase activity of the rats fed a creatine-free diet. This reduction in transamidinase activity was correlated with a decrease in transamidinase protein in the creatine-fed rats. The relative synthetic rates and mRNA functional activities of transmidinase were measured in control and creatine-fed rats. The relative synthetic rate of transamidinase in creatine-fed rats was 21% of that found in the control animals. The functional transamidinase mRNA in creatine-fed rats was correspondingly reduced to 37% of the amount in the control animals. Thus, creatine affects transamidinase activity by altering its rate of synthesis at a pretranslational step and represents an example of end-product repression in a higher eukaryote.     

Competitive interrelationships between lysine and arginine in rat liver under normal conditions and in experimental hyperammonemia

The effects of lysine administration on arginine and ornithine liver levels were studied in normal and urease-treated rats. L-Arginine injections produced a rise in liver arginine with a parallel increase in liver ornithine. Pretreatment with L-lysine resulted in an elevation in liver arginine. Administration of lysine to urease treated rats induced a significant increase in liver arginine content with a parallel drop in ornithine/arginine ratio. A similar decrease in ornithine/arginine ratio due to lysine administration was observed in animals, in which arginine and ornithine levels had been raised by loading with arginine. The mechanism of the lysine effect is most likely by inhibition of liver arginase activity $\text{in vivo}$.     

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.     

Rats fed prolonged high protein diets show an increase in nitrogen metabolism and liver megamitochondria

Rats were fed diets containing 20, 50 and 80% protein for 14 months. The urea excreted by the rats fed diets containing 50 and 80% protein when compared to rats fed diets containing 20% protein increased ca. 2- and 3-fold, respectively, in ca. 2 days; this increase was maintained essentially unchanged through the experimental period. The serum levels of urea increased 2.5- and 4-fold, respectively, in the first days and were also maintained during the experiment. Glutamate dehydrogenase activity of liver remained unchanged. The five urea cycle enzymes increased with respect to the control values. Orotic acid excretion increased as well as orotidylate decarboxylase and orotate phosphoribosyltransferase, but aspartate transcarbamylase did not. The key amino acids involved in the urea and pyrimidine pathways in liver were also measured; aspartic and glutamic acids and citrulline were increased, and ornithine and arginine did not change with the higher protein intake. In general, no differences were observed between animals fed 50 and 80% protein in their diets. Protein synthesis did not increase with the increase of protein content of the diet. Stereological analysis of ultrathin sections showed that the high protein diet induced a significant increment in the volumetric density, numerical density and size of hepatocyte mitochondria. Moreover, the presence of giant mitochondria, a hundred times larger than normal, was also observed in some periportal hepatocytes of rats fed the 80% protein diet.     

Effect of Dietary Addition of Amino Acids and Proteins on the Activities of Some Urea Cycle Enzymes in Rat Liver

The activities of ornithine transcarbamylase, arginine synthetase and arginase in the liver of rats receiving basal diets containing 25% casein supplemented respectively with arginine, aspartic acid, glutamic acid, glycine, a mixture of arginine, aspartic acid and glutamic acid, egg albumin, casein, wheat gluten and gelatin have been determined.

These urea cycle enzymes in rats receiving diets supplemented with the various nitrogen sources were generally increased, but the increments were due to the increase of the ingested amount of nitrogen, and not the specific effect of the individual amino acids or proteins. The excretion of urinary urea in general was increased proportionally with the elevations of these enzyme activities, independent of the nature of the dietary nitrogen.     

Supplemental Effects of Arginine and Methionine on Growth,and on Formations of Urea and Creatine of Adrenalectomized Rats Fed High Glycine Diets

The effects of adrenalectomy on growth, some enzyme activities in the liver and kidney, and urinary excretion of urea, creatinine and creatine were investigated in rats fed the 10% casein diets containing 7% glycine with or without l-arginine and l-methionine (10C, 10C7G and 10C7ArgMet).

Body weight gains of the intact 10C and 10C7GArgMet groups were almost same as the corresponding adrenalectomized groups. The body weight of the adrenalectomized 10C7G group was extremely decreased though that of the intact 10C7G group was maintained almost constant; but the decrease was recovered by the administration of hydrocortisone. The activities of liver arginase and carbamylphosphate synthetase were not affected by those diets. Liver serine dehydratase and ornithine δ-aminotransferase activities were increased in the intact 10C7G and 10C7GArgMet groups, but these increases were depressed by adrenalectomy. Glutamate-pyruvate transminase activities in the liver of intact 10C7G and 10C7GArgMet groups were also enhanced, but were extremely decreased in the corresponding adrenalectomized groups. Kidney transamidinase activity was not affected by adrenalectomy. The amount of urinary excreted urea was almost unchanged by adrenalectomy, but was increased by hydrocortisone administration. The amounts of excreted creatine of the adrenalectomized groups were generally larger than the corresponding intact groups, but slightly decreased by the administration of hydrocortisone. The amount of excreted creatinine was not generally affected by adrenalectomy.     

Monoamine and diamine oxidase activities in uremic rats

The activities of monoamine and diamine oxidases in various organs and tissues and the amine levels in plasma and urine were determined in chronically uremic and pair-fed control rats. Plasma amine levels were elevated in uremic animals while the urinary excretion of amines was decreased. In uremic as compared to control animals, monomaine oxidase activity was decreased in kidney and muscle, increased in heart and plasma and not altered in liver and cerebrum. Diamine oxidase activity in uremic rats was decreased in kidney, increased in plasma and unchanged in liver and muscle. These alterations of amine oxidase activities in renal failure may affect the metabolism of many amines and thus contribute to the pathogenesis of the uremic syndrome.     

Compensatory changes in enzymes of arginine metabolism during renal hypertrophy in mice

The present study investigates enzyme activities of the urea cycle, transamidinase and ornithine–proline inter-conversion in the hypertrophied kidney after unilateral nephrectomy in mice. Surgical removal of the left kidney in mice led to compensatory enlargement of the right kidney after 1 and 14 days. This renal growth was associated with an increase in glomerular volume (but not number) and enlargement of the proximal convoluted tubules. The total renal protein content increased in proportion to the increase in kidney weight, but the protein per gram weight of kidney did not change. The specific activity of only ornithine aminotransferase (OAT), the rate-limiting enzyme in the conversion of ornithine to proline, increased in 2 weeks of hypertrophy. The specific activity of all other enzymes was unchanged. However, the total enzyme activity per kidney of all the enzymes, without exception, was elevated in the hypertrophied kidney. While the increase in total OAT activity was much more than the increase in kidney weight, all other enzymes increased more or less in proportion to the increase in renal mass. The results suggest that compensation in OAT activity to chronic reduction in renal mass was complete, but only partial in the case of other enzymes.     

Organization and control in the arginine biosynthetic pathway of Neurospora.

Eight enzymes involved in the conversion of acetylglutamate to arginine in Neurospora crassa were studied. The data indicate that of three enzymes early in the sequence, only the first, acetylglutamate kinase, is a nonorganellar enzyme. The next two, N-acetyl-gamma-glutamyl-phosphate reductase and acetylornithine aminotransferase, are in the mitochondrion, which was previously shown to contain the subsequent enzymes: acetylornithine-glutamate acetyltransferase, ornithine carbamyltransferase, and carbamyl-phosphate synthetase A (arginine specific). The last two enzymes of the pathway, argininosuccinate synthetase and argininosuccinate lyase, were previously shown to be cytosolic. All enzymes but one have low amplitudes or repression. Their levels respond little to arginine excess and are about twofold elevated (threefold for ornithine carbamyltransferase) as a result of arginine limitation in the arg-12-8 strain. No restriction of the incorporation of mitochondrial enzymes into mitochondria could be detected when the levels of these enzymes were elevated. Two enzymes, acetylglutamate kinase and carbamyl-phosphate synthetase A, which initiate the synthesis of the ornithine and guanidino moieties of arginine, respectively, show the lowest specific activities in crude extract. These enzymes display special regulatroy features. Acetylglutamate kinase, which has a typically low amplitude of repression, is subject to feedback inhibition. Carbamyl-phosphate synthetase A is wholly insensitive to arginine or citrulline in vitro or in vivo, but displays a very large amplitude of repression (about 60-fold). It is unique in that it can be almost completely repressed by growth of mycelia in excess arginine. These data suggest that mitochondrial localization may be incompatible with a mechanism of feedback inhibition by a cytosolic effector, arginine. Further, they suggest that the high repressibility of carbamyl-phosphate synthetase A compensates for its feedback insensitivity.     

Regulation of polyamine synthesis by dietary alpha-aminoisobutyric acid and ornithine

An experiment was conducted to determine the effect of feeding ornithine in combination with alpha-aminoisobutyric acid (AIB), an inhibitor of arginase, on the regulation of polyamine synthesis in chicks. A total of 48 chicks with genetically elevated renal arginase activity was fed diets containing crystalline amino acids and 1% AIB with or without 2% ornithine. Feeding AIB reduced renal arginase activity, while renal and hepatic ornithine decarboxylase (ODC) activity increased. Feeding AIB plus ornithine caused no further reduction in renal arginase activity compared with that in chicks fed the AIB-supplemented diet. Renal and hepatic ODC activities, however, fell to below control levels. Renal, hepatic, and breast muscle ornithine concentrations increased substantially when ornithine was fed. AIB plus ornithine increased renal putrescine and spermidine concentrations. It was concluded that AIB could partially overcome the ornithine-induced inhibition of ODC activity. These findings support the hypothesis that dietary manipulation of precursor amino acids of polyamines in the presence of metabolites that induce ODC activity can influence tissue polyamine concentrations.