Decreasing urea∶trimethylamine N-oxide ratios with depth in chondrichthyes: a physiological depth limit?

In marine osmoconformers, cells use organic osmolytes to maintain osmotic balance with seawater. High levels of urea are utilized in chondrichthyans (sharks, rays, skates, and chimaeras) for this purpose. Because of urea's perturbing nature, cells also accumulate counteracting methylamines, such as trimethylamine N-oxide (TMAO), at about a 2∶1 urea∶methylamine ratio, the most thermodynamically favorable mixture for protein stabilization, in shallow species. However, previous work on deep-sea teleosts (15 species) and chondrichthyans (three species) found an increase in muscle TMAO content and a decrease in urea content in chondrichthyans with depth. We hypothesized that TMAO counteracts protein destabilization resulting from hydrostatic pressure, as is demonstrated in vitro. Chondrichthyans are almost absent below 3,000 m, and we hypothesized that a limitation in urea excretion and/or TMAO retention might play a role. To test this, we measured the content of major organic osmolytes in white muscle of 13 chondrichthyan species caught with along-contour trawls at depths of 50-3,000 m; the deepest species caught was from 2,165 m. Urea and TMAO contents changed significantly with depth, with urea∶TMAO declining from 2.96 in the shallowest (50-90 m) groups to 0.67 in the deepest (1,911-2,165 m) groups. Urea content was 291-371 mmol/kg in the shallowest group and 170-189 mmol/kg in the deepest group, declining linearly with depth and showing no plateau. TMAO content was 85-168 mmol/kg in the shallowest group and 250-289 mmol/kg in the deepest groups. With data from a previous study for a skate at 2,850 m included, a second-order polynomial fit suggested a plateau at the greatest depths. When data for skates (Rajidae) were analyzed separately, a sigmoidal fit was suggested. Thus, the deepest chondrichthyans may be unable to accumulate sufficient TMAO to counteract pressure; however, deeper-living specimens are needed to fully test this hypothesis.     

Lamprey parasitism of sharks and teleosts: high capacity urea excretion in an extant vertebrate relic

We observed 10 sea lampreys (Petromyzon marinus) parasitizing basking sharks (Cetorhinus maximus), the world's second largest fish, in the Bay of Fundy. Due to the high concentrations of urea in the blood and tissues of ureosmotic elasmobranchs, we hypothesized that sea lampreys would have mechanisms to eliminate co-ingested urea while feeding on basking sharks. Post-removal urea excretion rates (J(Urea)) in two lampreys, removed from separate sharks by divers, were initially 450 ( approximately 9000 micromol N kg-1 h-1) and 75 times ( approximately 1500 micromol N kg-1 h-1) greater than basal (non-feeding) rates ( approximately 20 micromol N kg-1 h-1). In contrast, J(Urea) increased by 15-fold after parasitic lampreys were removed from non-ureosmotic rainbow trout (Oncorhynchus mykiss). Since activities of the ornithine urea cycle (OUC) enzymes, carbamoyl phosphate synthetase III (CPSase III) and ornithine carbamoyl transferase (OCT) were relatively low in liver and below detection in intestine and muscle, it is unlikely that the excreted urea arose from de novo urea synthesis. Measurements of arginase activity suggested that hydrolysis of dietary arginine made a minor contribution to J(Urea.). Post-feeding ammonia excretion rates (J(Amm)) were 15- to 25-fold greater than basal rates in lampreys removed from both basking sharks and rainbow trout, suggesting that parasitic lampreys have a high capacity to deaminate amino acids. We conclude that the sea lamprey's ability to penetrate the dermal denticle armor of sharks, to rapidly excrete large volumes of urea and a high capacity to deaminate amino acids, represent adaptations that have contributed to the evolutionary success of these phylogenetically ancient vertebrates.     

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.     

Urea formation in the green vegetable bug Nezara viridula

Urea comprises 7·7 per cent of the total nitrogen excretion of Nezara viridula. The bug is capable of oxidizing uric acid to allantoin, which is also excreted, but the uricolytic pathway is not active beyond this point. Of the enzymes of the ornithine cycle, arginase and ornithine transcarbamalase are active, but there is no evidence for the arginine synthetase system. Carbamyl phosphate synthetase has a low activity detectable only by the use of radioactive substrates. Confirmation of the operation of only part of the ornithine cycle is seen in the incorporation of bicarbonate carbon into citrulline, but not into arginine or urea, by homogenates of bug tissue. It is concluded that urea in the excreta is derived from excess arginine in the diet by the action of the enzyme arginase. Free arginine is present in the cell sap of the bean pods on which the bugs feed in amounts sufficient to account for the urea excreted.     

Increases in urea synthesis and the ornithine-urea cycle capacity in the giant African snail, Achatina fulica, during fasting or aestivation, or after the injection with ammonium chloride

The objectives of this study are to determine whether a full complement of ornithine-urea cycle (OUC) enzymes is present in the hepatopancreas of the giant African snail Achatina fulica, and to investigate whether the rate of urea synthesis and the OUC capacity can be up-regulated during 23 days of fasting or aestivation, or 24 hr post-injection with NH(4)Cl (10 micromol g(-1) snail) into the foot muscle. A. fulica is ureotelic and a full complement of OUC enzymes, including carbamoyl phosphate synthetase III (CPS III), was detected from its hepatopancreas. There were significant increases in the excretion of NH(4)(+), NH(3) and urea in fasting A. fulica. Fasting had no significant effect on the tissue ammonia contents, but led to a progressive accumulation of urea, which was associated with an 18-fold increase in the rate of urea synthesis. Because fasting took place in the presence of water and because there was no change in water contents in the foot muscle and hepatopancreas, it can be concluded that the function of urea accumulation in fasting A. fulica was unrelated to water retention. Aestivation in arid conditions led to a non-progressive accumulation of urea in A. fulica. During the first 4 days and the last 3 days of the 23-day aestivation period, experimental snails exhibited significantly greater rates of urea synthesis compared with fasted snails. These increases were associated with significant increases in activities of various OUC enzymes, except CPS III, in the hepatopancreas. However, the overall urea accumulation in snails aestivated and snails fasted for 23 days were comparable. Therefore, the classical hypothesis that urea accumulation occurred to prevent water loss through evaporation during aestivation in terrestrial pulmonates may not be valid. Surprisingly, there were no accumulations of ammonia in the foot muscle and hepatopancreas of A. fulica 12 or 24 hr after NH(4)Cl was injected into the foot muscle. In contrast, the urea content in the foot muscle of A. fulica increased 4.5- and 33-fold at hour 12 and hour 24, respectively, and the respective increases in the hepatopancreas were 4.9- and 32-fold. The exogenous ammonia injected into A. fulica was apparently detoxified completely to urea. The urea synthesis rate increased 148-fold within the 24-hr experimental period, which could be the greatest increase known among animals. Simultaneously, there were significant increases in activities of glutamine synthetase (2.5-fold), CPS III (3.1-fold), ornithine transcarbamoylase (2.3-fold), argininosuccinate synthetase+lyase (13.6-fold) and arginase (3.5-fold) in the hepatopancreas 12 hr after the injection of NH(4)Cl. Taken altogether, our results support the view that the primary function of urea synthesis through the OUC in A. fulica is to defend against ammonia toxicity, but suggest that urea may have more than an excretory role in terrestrial pulmonates capable of aestivation.     

Effects of exercise on nitrogen excretion, carbamoyl phosphate synthetase III activity and related urea cycle enzymes in muscle and liver tissues of juvenile rainbow trout (Oncorhynchus mykiss).

The purpose of this study was to determine if carbamoyl phosphate synthetase III (CPSase III) and related urea cycle enzyme activities in skeletal muscle tissue of juvenile rainbow trout (Oncorhynchus mykiss) increase during short- or long-term exercise, in parallel with changes in whole-body urea excretion rates. Urea excretion was elevated by 65% in fish that swam at high-speed (50 cm/s) vs. low-speed (20 cm/s) over a 2-h period, with no significant changes in CPSase III, ornithine transcarbamoylase or glutamine synthetase activities in muscle tissue. Fish that swam for 4 days at high-speed had higher rates of ammonia excretion and GSase activity in muscle and liver tissue relative to low-speed swimmers. Calculations showed that 47-53% of excreted urea, theoretically could be accounted for by total muscle CPSase III activity in juvenile and adult trout. The data indicate that increases in the rate of urea excretion during short-term high intensity exercise are not linked to higher activities of urea cycle enzymes in muscle tissue, but this does not rule out the possibility of increased flux through muscle CPSase III and related enzymes. Furthermore, these results indicate that urea cycle enzyme activities in skeletal muscle tissue can account for a significant portion of total urea excretion in juvenile and adult trout.     

Excretory nitrogen metabolism in the juvenile axolotl Ambystoma mexicanum: differences in aquatic and terrestrial environments

The fully grown but nonmetamorphosed (juvenile) axolotl Ambystoma mexicanum was ureogenic and primarily ureotelic in water. A complete ornithine-urea cycle (OUC) was present in the liver. Aerial exposure impeded urea (but not ammonia) excretion, leading to a decrease in the percentage of nitrogen excreted as urea in the first 24 h. However, urea and not ammonia accumulated in the muscle, liver, and plasma during aerial exposure. By 48 h, the rate of urea excretion recovered fully, probably due to the greater urea concentration gradient in the kidney. It is generally accepted that an increase in carbamoyl phosphate synthetase activity is especially critical in the developmental transition from ammonotelism to ureotelism in the amphibian. Results from this study indicate that such a transition in A. mexicanum would have occurred before migration to land. Aerial exposure for 72 h exhibited no significant effect on carbamoyl phosphate synthetase-I activity or that of other OUC enzymes (with the exception of ornithine transcarbamoylase) from the liver of the juvenile A. mexicanum. This supports our hypothesis that the capacities of OUC enzymes present in the liver of the aquatic juvenile axolotl were adequate to prepare it for its invasion of the terrestrial environment. The high OUC capacity was further supported by the capability of the juvenile A. mexicanum to survive in 10 mM NH(4)Cl without accumulating amino acids in its body. The majority of the accumulating endogenous and exogenous ammonia was detoxified to urea, which led to a greater than twofold increase in urea levels in the muscle, liver, and plasma and a significant increase in urea excretion by hour 96. Hence, it can be concluded that the juvenile axolotl acquired ureotelism while submerged in water, and its hepatic capacity of urea synthesis was more than adequate to handle the toxicity of endogenous ammonia during migration to land.     

The crab-eating frog, Rana cancrivora, up-regulates hepatic carbamoyl phosphate synthetase I activity and tissue osmolyte levels in response to increased salinity

The crab-eating frog Rana cancrivora is one of only a handful of amphibians worldwide that tolerate saline waters. They typically inhabit brackish water of mangrove forests of Southeast Asia, but live happily in freshwater and can be acclimated to 75% seawater (25 ppt) or higher. We report here that after transfer of juvenile R. cancrivora from freshwater (1 ppt) to brackish water (10 -->20 or 20 -->25 ppt; 4-8 d) there was a significant increase in the specific activity of the key hepatic ornithine urea cycle enzyme (OUC), carbamoyl phosphate synthetase I (CPSase I). At 20 ppt, plasma, liver and muscle urea levels increased by 22-, 21-, and 11-fold, respectively. As well, muscle total amino acid levels were significantly elevated by 6-fold, with the largest changes occurring in glycine and beta-alanine levels. In liver, taurine levels were 5-fold higher in frogs acclimated to 20 ppt. There were no significant changes in urea or ammonia excretion rates to the environment. As well, the rate of urea influx (J(in) (urea)) and efflux (J(out) (urea)) across the ventral pelvic skin did not differ between frogs acclimated to 1 versus 20 ppt. Taken together, these findings suggest that acclimation to saline water involves the up-regulation of hepatic urea synthesis, which in turn contributes to the dramatic rise in tissue urea levels. The lack of change in urea excretion rates, despite the large increase in tissue-to-water gradients further indicates that mechanisms must be in place to prevent excessive loss of urea in saline waters, but these mechanisms do not include cutaneous urea uptake. Also, amino acid accumulation may contribute to an overall rise in the osmolarity of the muscle tissue, but relative to urea, the contribution is small.     

African sharptooth catfish Clarias gariepinus does not detoxify ammonia to urea or amino acids but actively excretes ammonia during exposure to environmental ammonia

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and exhibits high tolerance of environmental ammonia. This study aimed at elucidating the strategies adopted by C. gariepinus to defend against ammonia toxicity during ammonia exposure. No carbamoyl phosphate synthetase (CPS) I or III activities were detected in the liver or muscle of the adult C. gariepinus. In addition, activities of other ornithine-urea cycle (OUC) enzymes, especially ornithine transcarbamylase, were low in the liver, indicating that adult C. gariepinus does not have a "functional" hepatic OUC. After being exposed to 50 or 100 mM NH4Cl for 5 d, there was no induction of hepatic OUC enzymes and no accumulation of urea in tissues of the experimental animals. In addition, the rate of urea excretion remained low and unchanged. Hence, ammonia exposure did not induce ureogenesis or ureotely in C. gariepinus as suggested elsewhere for another obligatory air-breathing catfish of the same genus, Clarias batrachus, from India. Surprisingly, the local C. batrachus did not possess any detectable CPS I or III activities in the liver or muscle as had been reported for the Indian counterpart. There were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus exposed to 50 or 100 mM NH4Cl for 5 d; neither were there any changes in the glutamine levels in these tissues. Yet even after being exposed to 100 mM NH4Cl for 5 d, there was no significant increase in the level of ammonia in the muscle, which constitutes the bulk of the specimen. In addition, the level of ammonia accumulated in the plasma was relatively low compared to other tropical air-breathing fishes. More importantly, for all NH4Cl concentrations tested (10, 50, or 100 mM), the plasma ammonia level was maintained relatively constant (2.2-2.4 mM). These results suggest that C. gariepinus was able to excrete endogenous ammonia and infiltrated exogenous ammonia against a very steep ammonia gradient. When exposed to freshwater (pH 7.0) with or without 10 mM NH4Cl, C. gariepinus was able to excrete ammonia continuously to the external medium for at least 72 h. This was achieved while the plasma NH4+ and NH3 concentrations were significantly lower than those of the external medium. Diffusion trapping of NH3 through boundary layer acidification can be eliminated as the pH of the external medium became more alkaline instead. These results represent the first report on a freshwater fish (C. gariepinus) adopting active excretion of ammonia (probably NH4+) as a major strategy to defend against ammonia toxicity when exposed to environmental ammonia.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

Submitochondrial localization of arginase and other enzymes associated with urea synthesis and nitrogen metabolism, in liver of Squalus acanthias

The submitochondrial localization of the four mitochondrial enzymes associated with urea synthesis in liver of Squalus acanthias (spiny dogfish), a representative elasmobranch, was determined. Glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, ornithine carbamoyltransferase, glutamine synthetase, and arginase were all localized within the matrix of liver mitochondria. The subcellular and submitochondrial localization and activities of several related enzymes involved in nitrogen metabolism and gluconeogenesis in liver and dogfish are also reported. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase were localized in the mitochondrial matrix. Synthesis of citrulline by isolated mitochondria from ornithine proceeds at a near optimal rate at ornithine concentrations as low as 0.08 mM. The same stoichiometry and rates of citrulline synthesis are observed when ornithine is replaced by arginine. The mitochondrial location of arginase does not appear to reflect a mechanism for regulating ornithine availability.     

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.     

Function of arginase in lactating mammary gland

The potential for a considerable formation of ornithine exists in lactating mammary gland because of its arginase content. Late in lactation arginase reaches an activity in the gland higher than that present in any rat tissue except liver. Occurrence of the urea cycle can be excluded since two enzymes for the further reaction of ornithine in the cycle, carbamoyl phosphate synthetase I and ornithine carbamoyltransferase, are both absent from this tissue. Instead, carbamoyl phosphate synthetase II appears early in lactation, associated with accumulation of aspartate carbamoyltransferase and DNA, consistent with the proposed role of these enzymes in pyrimidine synthesis. The facts require another physiological role for arginase apart from its known function in the urea cycle. Significant activity of ornithine aminotransferase develops in mammary gland in close parallel with the arginase. By this reaction, ornithine can be converted into glutamic semialdehyde and subsequently into proline. The enzymic composition of the lactating mammary gland is therefore appropriate for the major conversion of arginine into proline that is known to occur in the intact gland.     

Urea biosynthesis in normal human fetuses

Normal human fetuses at different gestation periods were collected on ice after hysterotomy and the enzymes of the urea cycle were measured in the liver. The activity of all enzymes increased with increasing gestational age towards the adult value, however, in no case did the values reach the normal adult level. The bladder fluid of these fetuses contained urea and ammonia nitrogen at concentrations which were akin to the concentrations found in fetal blood. The ornithine transcarbamylase activity was the lowest when compared to the adult values and appeared to be the rate-limiting enzyme in the cycle, along with argininosuccinic acid synthetase activity, which was also very low. The activity of arginase was found to be the highest in the cycle. The very low ornithine transcarbamylase and argininosuccinic acid synthetase activities and the comparatively higher arginase activity migh lead to the channeling of ornithine into alternate metabolic pathways.     

Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias)

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).     

Regulation of genes for inducible nitric oxide synthase and urea cycle enzymes in rat liver in endotoxin shock

Arginine is an intermediate of the urea cycle in the liver. It is synthesized by the first four enzymes of the cycle, carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinate lyase, and is hydrolyzed to urea and ornithine by arginase I, forming the cycle. In endotoxemia shock, inducible nitric oxide (NO) synthase (iNOS) is induced in hepatocytes and arginine is utilized for NO production. Regulation of the genes for iNOS and the urea cycle enzymes was studied using lipopolysaccharide (LPS)-treated rat livers. When rats were injected intraperitoneally with LPS, iNOS mRNA was markedly induced. Cationic amino acid transporter-2 and C/EBPbeta mRNAs were also highly increased. In contrast, mRNAs for all the urea cycle enzymes except ornithine transcarbamylase were gradually decreased and reached 16-28% of controls at 12 h. However, all these enzymes remained unchanged at protein level up to 24 h. In light of these results, we suggest that synthesis of urea cycle enzymes is downregulated and that the protein synthetic capacity is directed to synthesis of proteins required for defense against endotoxemia.     

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.     

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.     

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.