Control of ureogenesis

Control of urea synthesis was studied in rat hepatocytes incubated with physiological mixtures of amino acids in which arginine was replaced by equimolar amounts of ornithine. The following observations were made. Intramitochondrial carbamoyl phosphate was always below 0.1 mM. Only when ornithine was absent and when, in addition, the concentration of amino acids was higher than four times their plasma concentration, intramitochondrial carbamoyl phosphate rose up to about 3 mM; under these conditions ammonia accumulated in the medium. The relationship between ornithine-cycle flux and the concentration of the cycle intermediates at varying amino acid concentration indicated that under near-physiological conditions the ornithine-cycle enzymes are far from being saturated with their subsidiaries. Moderate concentrations of norvaline had no effect on the rate of urea synthesis unless the cells were severely depleted of ornithine. Activation of carbamoyl-phosphate synthetase (ammonia) by addition of N-carbamoylglutamate only slightly stimulated urea production at all amino acid concentrations. However, in the presence of the activator the curve relating ornithine-cycle flux to the steady-state ammonia concentration was shifted to lower concentrations of ammonia. The intramitochondrial concentration of carbamoyl phosphate in rat liver in vivo was below 0.1 mM. This value is far below the concentration required for substantial inhibition of carbamoyl-phosphate synthetase. It is concluded that in vivo the function of activity changes in carbamoyl-phosphate synthetase, via the well-documented alterations in the intramitochondrial concentration of N-acetylglutamate, is to buffer the intrahepatic ammonia concentration rather than to affect urea production per se. At constant concentration of ammonia the rate of urea production is entirely controlled by the activity of carbamoyl-phosphate synthetase.     

Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures

Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a key role in synthesis of urea. Because aging affects some liver functions, and because there is no information on the levels of carbamoyl phosphate synthetase I during aging, we assayed the activity of this enzyme and determined immunologically the level of carbamoyl phosphate synthetase I in liver homogenates from young (4 months) and old (18 or 26 months) rats. In addition, we used electron microscopic immunogold procedures to locate and measure the amount of the enzyme in the mitochondrial matrix. There is no significant change in enzyme activity or enzyme protein content with age, although there is a higher concentration of the enzyme in the mitochondria (c. 1.5 times greater) from old rats, which is compensated by a decrease in the fractional volume of the mitochondrial compartment during aging.     

Arginine-specific carbamoyl phosphate metabolism in mitochondria of Neurospora crassa. Channeling and control by arginine

Citrulline is synthesized in mitochondria of Neurospora crassa from ornithine and carbamoyl phosphate. In mycelia grown in minimal medium, carbamoyl phosphate limits citrulline (and arginine) synthesis. Addition of arginine to such cultures reduces the availability of intramitochondrial ornithine, and ornithine then limits citrulline synthesis. We have found that for some time after addition of excess arginine, carbamoyl phosphate synthesis continued. Very little of this carbamoyl phosphate escaped the mitochondrion to be used in the pyrimidine pathway in the nucleus. Instead, mitochondrial carbamoyl phosphate accumulated over 40-fold and turned over rapidly. This was true in ornithine- or ornithine carbamoyltransferase-deficient mutants and in normal mycelia during feedback inhibition of ornithine synthesis. The data suggest that the rate of carbamoyl phosphate synthesis is dependent to a large extent upon the specific activity of the slowly and incompletely repressible synthetic enzyme, carbamoyl-phosphate synthetase A. In keeping with this conclusion, we found that when carbamoyl-phosphate synthetase A was repressed 2-10-fold by growth of mycelia in arginine, carbamoyl phosphate was still synthesized in excess of that used for residual citrulline synthesis. Again, only a small fraction of the excess carbamoyl phosphate could be accounted for by diversion to the pyrimidine pathway. The continued synthesis and turnover of carbamoyl phosphate in mitochondria of arginine-grown cells may allow rapid resumption of citrulline formation after external arginine disappears and no longer exerts negative control on ornithine biosynthesis.     

Changes in urea cycle-related metabolites in the mouse after combined administration of valproic acid and an amino acid load

Increased blood ammonia was induced in fasting mice by ip administration of 200 mg/kg Na-valproate followed 1 h later by 13 and 4 mmol/kg alanine and ornithine, respectively. When valproate was not used blood or liver ammonia was not increased, but increases were observed in liver glutamate (5-fold), glutamine (2-fold), aspartate (5-fold), acetylglutamate (15-fold), citrulline (35-fold), argininosuccinate (11-fold), arginine (11-fold), and urea (3-fold). The level of carbamoyl phosphate (less than 2 nmol/g) was, by far, the lowest of all urea cycle intermediates. The large increase in citrulline indicates that argininosuccinate synthesis was limiting, and that the increase in acetylglutamate induced a considerable activation of carbamoyl phosphate synthetase, which agrees with theoretical expectations, irrespective of the actual KD value for acetylglutamate. Pretreatment with valproate resulted in lower hepatic levels of glutamate, glutamine, aspartate, acetyl-CoA, and acetylglutamate. At the level found of acetylglutamate the activation of carbamoyl phosphate synthetase would be expected to be similar to that without valproate. Indeed, the levels of citrulline were similar with or without valproate. Argininosuccinate, arginine, and urea levels exhibited little if any change. Although the model used may not replicate exactly the situation in patients, from our results it appears that changes in citrullinogenesis or in other steps of the urea cycle do not account for the increase in blood ammonia induced by valproate, and it is proposed that valproate may alter glutamine metabolism.     

从大肠杆菌中表达、纯化宁乡猪N-乙酰谷氨酸合成酶并制备其多克隆抗体

N-乙酰谷氨酸合成酶催化生成的N-乙酰谷氨酸(NAGS)对于哺乳动物尿素循环第一个酶—氨基甲酰磷酸合成酶I变象异构激活是必需的。N-乙酰谷氨酸合成酶定位于肝脏和小肠线粒体基质中,通过提供N-乙酰谷氨酸调节氨基甲酰磷酸合成酶I的活性来调节尿素合成。我们用RT-PCR方法从宁乡猪肝脏中扩增了N-乙酰谷氨酸合成酶的开发阅读框,并将此基因连接到原核表达载体上,构建了pET-NAGS质粒。将重组质粒转化到Ec.oliBL21(DE3),在IPTG诱导下表达His-NAGS融合蛋白。通过SDS-PAGE,得出NAGS分子量约为40kDa。一步亲和层析纯化后,我们将纯化后的NAGS蛋白注射到新西兰大白兔中制备多克隆抗体。通过免疫组化和免疫印迹测试抗体,结果表明此抗体有较好的抗原性和特异性。据我们所知,这是第一次在大肠杆菌中表达来源于宁乡猪的NAGS。     

Factors influencing the development of urea-synthesizing enzymes in rat liver

1. The activities of enzymes of the urea cycle, carbamoyl phosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinase (the last two comprising the arginine synthetase system) and arginase, were measured in the liver during development of the rat. All five enzymes exhibited relatively low activities in foetal liver and a rapid postnatal increase was found. The rate-limiting enzyme of urea synthesis in the rat, the condensing enzyme of the arginine synthetase system, showed the lowest activity at birth and the most rapid postnatal increase, a fivefold increase within 24hr. after birth. A second increase of activity was noted after the tenth day. These results suggest that the postnatal increase of arginine synthetase activity initiates the ability for urea synthesis in the rat. 2. Some factors influencing the development of the rate-limiting arginine synthetase system were studied in more detail. (a) Intraperitoneal administration of puromycin inhibited the postnatal increaseof the enzyme activity. (b) Starvation of newborn animals for 24hr. after birth had no effect on the postnatal development of the enzyme. (c) Bilateral adrenalectomy at birth caused a marked diminution in the postnatal increase of the enzyme activity and injections of triamcinolone were effective in preventing the effect of adrenalectomy. (d) Administration of triamcinolone alone had a marked stimulatory effect on the postnatal development of this enzyme. (e) Premature and postmature birth had virtually no effect on the developmental pattern of the arginine synthetase activity, suggesting that the increase of this enzyme activity after birth is not initiated by the birth process.     

Modulation of intestinal urea cycle by dietary spermine in suckling rat

Argininosuccinate synthetase, an ubiquitous enzyme in mammals, catalyses the formation of argininosuccinate, the precursor of arginine. Arginine is recognised as an essential amino acid in foetuses and neonates, but also as a conditionally essential amino acid in adults. Argininosuccinate synthetase is initially expressed in enterocytes during the developmental period, it disappeared from this organ then appeared in the kidneys. Although the importance of both intestinal and renal argininosuccinate synthetases has been recognised for a long time, nutrients have not yet been identified as inducers of the gene expression. In the context of a proteomic screening of intestinal modifications induced by dietary spermine in suckling rats, we showed that argininosuccinate synthetase and carbamoyl phosphate synthase disappeared from enterocytes after this treatment. The disappearance of argininosuccinate synthetase in small intestine was confirmed by immunodetection. Expression of carbamoyl phosphate synthase and argininosuccinate synthetase coding genes decreased also after spermine administration. Expression of other urea cycle enzyme coding genes was modulated by spermine administration: argininosuccinate lyase decreased and arginase increased. Our results fit with the developmental variation of argininosuccinate synthetase and carbamoyl phosphate synthase. Modulation of the gene expression for several urea cycle enzymes suggests a coordination between all the pathway steps and switch toward polyamine (or proline and glutamate) biosynthesis from ornithine.     

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.     

Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines

Summary All the five enzymes of urea synthesis and the formation of urea in vitro can already be demonstrated in human liver as early as the 9th week of fetal development. At this stage the activity of carbamoyl phosphate synthetase is the highest, whereas that of ornithine carbamoyltransferase is the lowest as compared to those in the adult. The kinetic parameters of the urea cycle enzymes are the same in fetal liver as in adult liver, except that the Km values of ornithine carbamoyltransferase for L-ornithine are 3.5 mM and 0.42 mM in the fetus and in adult liver, respectively.Urea formation in vivo seems to begin in the second half of fetal life, and a gradual increase can be detected in the activity of the enzymes of urea synthesis. The activity of ortnithine decarboxylase, the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase, however, changes in the opposite direction.The concentration of carbamoyl phosphate and aspartate remains constant, but that of ornithine gradually decreases during ontogenesis. The ornithine, carbamoyl phosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.     

Effect of different dietary proteins on plasma and liver fatty acid compositions in growing rats

The activities of key glutamine and urea cycle enzymes were assayed in liver homogenates from control and chronically acidotic rats and compared with citrulline and urea productions by isolated mitochondria and intact liver slices, respectively. Glutamine-dependent urea and citrulline synthesis were increased significantly in isolated mitochondria and in liver slices; the activities of carbamoyl phosphate synthetase and arginase were unchanged and increased, respectively. Glutamine was not a precursor in the carbamoyl phosphate synthetase system, suggesting that the glutamine effect is an indirect one and that glutamine requires prior hydrolysis. Increased mitochondrial citrulline synthesis was associated with enhanced oxygen consumption, suggesting glutamine acts both as a nitrogen and fuel source. Hepatic phosphate-dependent glutaminase was elevated by chronic acidosis. The results indicate that the acidosis-induced reduction in ureagenesis and reversal from glutamine uptake to release observed in vivo are not reflections of corresponding changes in the hepatic enzyme content. Rather, when available, glutamine readily supports ureagenesis, suggesting a close coupling of hepatic glutaminase flux with citrulline synthesis.     

Insulin regulation of RNA synthesis

During periods of nitrogen exportation from the cell, mitochondrial carbamoyl phosphate is synthesized, thus initiating the urea cycle. During times of nitrogen conservation by the liver cell, carbamoyl phosphate is synthesized in the cytosol of the cell, whereupon the de novo pyrimidine synthesis pathway is initiated. The de novo pathway provides pyrimidines for increased ribonucleic acid synthesis. Formerly, it was believed that these two pathways functioned irrespective of one another. However, recent experimental evidence indicates that, when excess ammonia is present, mitochondrial carbamoyl phosphate passes from the mitochondria into the cell cytosol, where it is metabolized by the de novo pyrimidine synthesis pathway. When ornithine and excess ammonia are both present, mitochondrial carbamoyl phosphate no longer passes from the mitochondria into the cytosol to be metabolized by the de nova pathway. Thus the metabolic fate of mitochondrial carbamoyl phosphate, and that of excess nitrogen, is determined by the presence or absence of ornithine. In turn, this key molecule is the substrate for the cytoplasmic enzyme ornithine decarboxylase. When ornithine decarboxylase is stimulated by insulin, ornithine is metabolized to putrescine. The activated ornithine decarboxylase combines with ribonucleic acid polymerase, activating the later enzyme. When ornithine is acted upon by ornithine decarboxylase, it is no longer available for the perpetuation of the urea cycle and mitochondrial carbamoyl phosphate levels rise until the carbamoyl phosphate passes into the cytosol to be metabolized by the de novo pathway. Increased amounts of pyrimidines are available for the activated ribonucleic acid polymerase. Therefore insulin, through its stimulation of ornithine decarboxylase, achieves cellular nitrogen retention by regulating nitrogen incorporation into newly synthesized ribonucleic acid.     

Effect of streptozotocin diabetes on some urea cycle enzymes

Streptozotocin induced diabetes in rats increased the activities of the three mitochondrial enzymes, carbamylphosphate synthetase, ornithine transcarbamylase and N-acetylglutamate synthetase, but not of the cytosolic N-acetylglutamate deacylase. Levels of both N-acetylglutamate and arginine, which are activators of carbamylphosphate synthetase and N-acetylglutamate synthetase respectively, increased in diabetes. These results serve to explain the increase both of mitochondrial citrulline and urea formation in hepatocytes and the increased urea excretion in diabetes.     

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.     

Purification and properties of ornithine carbamoyl transferase from liver of Squalus acanthias

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.     

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway

N-Acetylglutamate synthase (AGS), N-acetylglutamate kinase (AGK), and glutamate N-acetyltransferase (GAT) are the key enzymes in the synthesis of arginine that serves as an important precursor for the synthesis of protein, polyamines, urea, and nitric oxide. Current assays available for these three enzymes are laborious and time-consuming and do not allow continuous monitoring of enzyme activities. Here we established continuous enzyme assays for AGS, AGK, and GAT based on the coupling of AGS and GAT reactions to AGK followed by coupling of the AGK reaction to N-acetylglutamate 5-phosphate reductase (AGPR). The rate of AGPR-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate was monitored continuously as a change in absorbance at 340 nm using spectrophotometry. These methods were applied to kinetic analyses for Escherichia coli AGK, E. coli AGS, and Saccharomyces cerevisiae GAT, and the kinetic parameters obtained in the coupling assays showed nearly the same values as those obtained previously using discontinuous assays. The specificity of these coupled assays was confirmed by the lack of enzyme activity from extracts of E. coli AGS-, E. coli AGK-, and S. cerevisiae GAT-deletion mutants. Moreover, the coupled assay enabled us to measure AGS activity from mammalian liver mitochondrial extracts, known to be an important regulatory enzyme for the urea cycle. These coupled enzyme assays are rapid, highly sensitive, and reproducible.     

Inhibition of ureagenesis by valproate in rat hepatocytes. Role of N-acetylglutamate and acetyl-CoA.

Valproate (0.5-5 mM) strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mM-alanine and 3 mM-ornithine. Valproate at the same concentrations markedly decreased concentrations of N-acetylglutamate, an essential activator of carbamoyl-phosphate synthetase I (EC 6.3.4.16), in parallel with the inhibition of urea synthesis by valproate. This compound also lowered the cellular concentration of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1); glutamate, aspartate and citrulline were similarly decreased. Valproate in a dose up to 2 mM did not significantly affect the cellular concentration of ATP and had no direct effect on N-acetylglutamate synthesis, carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) activities.     

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.     

Mitochondrial urea cycle enzymes in rats treated with sodium benzoate

Since sodium benzoate, which is widely used to treat hyperammonemia its effect on mitochondrial urea cycle enzymes was investigated. its effect on mitochondrial urea cycle enzymes was investigated. Sodium benzoate was administered to urease treated hyperammonemic rats and controls. In both groups no interference with the activity of carbamylphosphate synthetase, ornithine carbamyltransferase and N-acetylglutamate synthetase in the liver could be observed at concentrations of benzoate in plasma found in hyperammonemic patients. Careful monitoring of plasma levels reduces benzoate toxicity as shown in a patient with argininosuccinic aciduria.     

Regulation of urea synthesis by agmatine in the perfused liver: studies with 15N

Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of l-[guanidino-15N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15N-labeled glutamine and unlabeled ammonia or 15NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15N2]agmatine is formed in the liver from perfusate l-[guanidino-15N2]arginine ( approximately 90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15N-labeled NAG and [15N]urea from 15N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15N-labeled NAG and [15N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.     

The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.