Short Term Regulation of Ureagenesis*

In order to examine the mechanism of the acute response of ureagenesis to amino acid loads, rats were injected intraperitoneally with various doses of a mixture of 20 amino acids. Blood ammonia rose only slightly with doses of 0.5 to 2.0 g/kg, but increased sharply at doses of 3 to 5 g/kg. Carbamyl phosphate synthetase I (EC 2.7.2.5) activity, assayed in intact mi- tochondria isolated from livers removed 15 min after injection of amino acids, with N-acetylglutamate at its endogenous levels, rose up to &fold with increasing doses u p to 2 g/kg; no further activation occurred with larger doses. This maximal activity was the same as the activity measured in disrupted mitochondria. He- patic levels of glutamate and N-acetylglutamate increased approximately linearly with dose of amino acids. The time course of these changes following a dose of 1.5 g/kg was studied. Glutamate, N-acetylglu- tamate, and carbamyl phosphate synthetase I activity all peaked 5 to 15 min after injection. All of these results were virtually unaltered by omission of arginine from the injected mixture, indicating that the increase in N-acetylglutamate was not attributable to activation by arginine of N-acetylglutamate synthetase. These results indicate that moderate loads of amino acids acti- vate ureagenesis via a rapid increase in N-acetylgluta-mate levels, secondary to increased mitochondrial glu- tamate, and independently of injected arginine. This autoregulatory mechanism becomes saturated at

In order to examine the mechanism of the acute response of ureagenesis to amino acid loads, rats were injected intraperitoneally with various doses of a mixt u r e of 20 amino acids. Blood ammonia rose only slightly with doses of 0.5 to 2.0 g/kg, but increased sharply at doses of 3 to 5 g/kg. Carbamyl phosphate synthetase I (EC 2.7.2.5) activity, assayed in intact mitochondria isolated from livers removed 15 min after injection of amino acids, with N-acetylglutamate at its endogenous levels, r o s e u p to &fold with increasing doses u p to 2 g/kg; no further activation occurred with larger doses. This maximal activity was the same as the activity measured in disrupted mitochondria. Hepatic levels of glutamate and N-acetylglutamate increased approximately linearly with dose of amino acids. The time course of these changes following a dose of 1.5 g/kg was studied. Glutamate, N-acetylglutamate, and carbamyl phosphate synthetase I activity all peaked 5 to 15 min after injection. All of these results were virtually unaltered by omission of arginine from the injected mixture, indicating that the increase in Nacetylglutamate was not attributable to activation b y arginine of N-acetylglutamate synthetase. These results indicate that moderate loads of amino acids activate ureagenesis via a rapid increase in N-acetylglutamate levels, secondary to increased mitochondrial glutamate, and independently of injected arginine. This autoregulatory mechanism becomes saturated at large doses of amino acids, and hyperammonemia then supervenes.
Adaptive changes in the activity of enzymes of the urea cycle play an important role in the response to variations in the protein content of the diet (1-3). However, enzyme adaptations cannot occur sufficiently rapidly to play a role in facilitating urea synthesis following a single protein load (4).
No mechanism whereby such facilitation might occur has yet been identified. Indeed, experiments designed to reveal such a mechanism apparently have not been reported.
The existence of such a mechanism could in theory attenuate, both in magnitude and duration, the increase in circulating concentrations of amino acids and ammonia which would otherwise occur following a large protein load. High concentrations of blood ammonia, especially if they are prolonged, are known to induce cerebral dysfunction (5).
The activation of carbamyl phosphate synthetase I (EC 2.7.2.5) by N-acetylglutamate is a potential site for rapid activation of the urea cycle following a protein load. Since Nacetylglutamate is required for carbamyl phosphate synthetase I activity, it is conceivable that the mitochondrial level of this substance could increase rapidly in response to protein ingestion, thereby activating carbamyl phosphate synthetase I. In this way, the activity of the urea cycle could exhibit short term activation, thus reducing the fluctuations in blood ammonia and amino acid concentrations with protein content of meals.
Since carbamyl phosphate synthetase I is the first committing step in ureagenesis, this enzyme is a likely site for regulatory control (6). Furthermore, it is clear that the rate of ureagenesis cannot exceed the rate of carbamyl phosphate synthesis in the steady state. If the rate of ureagenesis were less than the rate of carbamyl phosphate formation, then urea cycle intermediates would accumulate in the liver.
The present experiments were designed to examine the response of hepatic amino acid concentrations, including urea cycle intermediates, N-acetylglutamate content, and carbamyl phosphate synthetase I activity to a complete load of amino acids administered to the intact animal. In view of the known effect of large doses of arginine in activating N-acetylglutamate synthesis (7,8), experiments were conducted with and without arginine as a component of the injected mixture, so as to clarify the role of arginine (or ornithine) in the response.

EXPERIMENTAL PROCEDURES
Preparation of Animals-Female rats of the Sprague-Dawley strain weighing 180 to 200 g were purchased from Gibco, Madison, Wisc. The animals were maintained four to a cage in a controlled environment with a 12-h light-dark cycle and with free access to water and commercial chow, RMH 1O00, containing 14% protein, obtained from Agway, Syracuse, N. Y. Animals were kept under these conditions for at least 6 days before use. On the day of the experiment, food was removed 1 h before commencement. Because of the known diurnal variations in metabolism, specifically in urea synthesis (9), all experiments were commenced between 9 and 10 a.m. Amino acid loads varying from 0.5 g/kg to 5 g/kg were administered by intraperitoneal injection in a volume of 30 ml/kg. Controls received 30 ml/kg of 0.15 M saline (0.9% NaCl solution). Solutions were administered at pH 7.2 and 37°C. With the high dose injections, the amino acids were administered as part suspension and part solution, because of the limited solubility of some of the components. The amino acid mixture was similar to that of Rogers  28. An arginine-free mixture was also prepared with identical proportions except that arginine was replaced by an equal weight of glutamine. Groups of animals (three to six in each group) were killed at 15 min after each dose, and at intervals varying from 5 to 180 min after a dose of 1.5 g/kg.
Preparation of Mitochondria-Liver mitochondria were prepared by differential centrifugation as described by Chappell and Hansford (ll), with the modification of replacing sucrose with 250 m~ mannitol because sucrose interfered with the colorimetric determination of citrulline. Assay of Carbamyl Phosphate Synthetase I-Carbamyl phosphate using the following incubation conditions: NRCI, 10 m~; KHCOa20 synthetase I activity in intact mitochondria was measured at 30°C mM; ornithine, 10 m~; phosphate, 5 mM; Tris, 50 mM; ATP, 5 mM; KCI, 50 mM; carbonyl cyanide p-tritluorophenylhydrazone, 1.0 pM; and oligomycin, 5 pg/ml (the latter two were added as ethanolic solutions). Final pH was 7.2. Incubation was carried out in a volume of loo0 4. The reaction was commenced by the addition of mitochondria, 100 pl, as a 5 to 10 mg/ml suspension in preparative medium. All assays were performed in triplicate. The reaction was terminated after 12 min by the addition of an equal volume of 10% trichloroacetic acid solution. Linearity of assay was confiied for time intervals in excess of 20 min. After centrifugation, citrulline formation was determined (see below) in the supernatant. Under these assay conditions, rates of citrulline synthesis obtained were lower than has been obtained by some other authors (12-15) although similar to those of McGivan (16,17). This may be related in part to differences in dietary protein content and to whether the animal was fed or fasted. When certain alternative incubations are employed, for example, when glutamate is used as an endogenous substrate (15), the rate of citrulline production increases with time, presumably due to N-acetylglutamate synthesis within the mitochondria (18 therefore, carbamyl phosphate synthetase I should be limiting under the conditions of this assay. Further evidence for this is that carbamyl phosphate as substrate in this system augments citrulline synthesis above that achieved from ammonia (16). It has also been demonstrated that there exists a correlation between N-acetylglutamate content and citrulline synthesis in intact mitochondria under similar assay conditions (16, 20). Measurement of total carbamyl phosphate synthetase I activity in disrupted mitochondria was performed as follows. Permeability barriers in the intact mitochondria were destroyed by multiple freezing and thawing of the suspension. The reaction was performed at 30°C with the concentrations of reactants as follows: KCI, 150 m~; NH&I, 10 m~; KHCOJ, 20 m~; ornithine, 20 m~; MgCl2, 15 mM; Tris, 100 r n~; N-acetylglutamate, 10 m~; ATP, 5 m~; oligomycin, 5 pg/ml; pH, 7.2. The protocol was then identical with that used to assay mitochondrial citrulline production. Addition of ornithine carbamyltransferase (EC 2.1.3.3) to the reaction did not augment citrulline synthesis, confiiing that carbamyl phosphate synthetase I activity is ratelimiting under these conditions. Citrulline was measured by the Archibald method (21) as modified by Nuzum and Snodgrass (22) and calculated with the molar extinction coefficient. The method gave excellent reproducibility of standards with an interassay variation of less than 1%. Mitochondrial protein was measured by a biuret procedure (23). Activity of carbamyl phosphate synthetase I was calculated as nanomoles of citrulline formed min-l mg" of mitochondrial protein.
Carbamyl phosphate synthetase I activity in a distilled water homogenate of liver from these animals using the method of Schimke Assay of N-Acetylglutamate-N-Acetylglutamate was isolated from the liver by the method of Shigesada et al. (27) with minor modifications. A quantity of neutralized extract of perchloric acidtreated liver equivalent to 0.3 to 0.5 g of wet liver was applied to a Dowex 1-formate column (1 ml), 100 to 200 mesh, which was washed with 20 ml of HZ0 followed by 20 ml of pyridine formate, 0.1 M, pH 4.0. The N-acetylglutamate fraction was eluted with 20 ml of pyridine formate, 0.4 M, pH 3.0, lyophilized, and redissolved in 300 m~ imidazole buffer, pH 7.2. N-Acetylglutamate was then assayed by the activation of carbamyl phosphate synthetase I (using disrupted rat mitochondria as the source of both carbamyl phosphate synthetase I and ornithine carbamyltransferase) (16). Final concentrations of sub-strates were as described above, except that N-acetylglutamate was omitted and replaced by the unknowns. The absolute values were determined by the use of a standard curve in the range of 0 to 24 nmol of N-acetylglutamate, taken through the same procedures as the unknowns, including column chromatography. Within this range of standards, the standard curve was linear. Product formation was determined by using ['4C]bicarbonate as the substrate (0.6 pCi/ml) and measuring the acid-stable I4C remaining in the incubation mixture after terminating the reaction with perchloric acid added to achieve a final concentration of 5% centrifuging, and allowing the supernatant to stand overnight before the addition of PCS scintillant, obtained from Amersham/Searle, Arlington Heights, Ill. [I4C]Bicarbonate was obtained in minivials and used fresh, because acid-stable radioactivity was noticed to increase significantly with storage. The standards carried through the procedure gave recoveries averaging 89 +-7% (mean +-S.E.). N-Acetylglutamate content of mitochondria was assayed using the same method.
Assay of N-Acetylglutamate Synthetase-N-Acetylglutamate synthetase activity was determined in mitochondria disrupted by freezing and thawing by a modification of the method of McGivan el al. (16). The enzyme was found to be unstable at incubation temperatures of 30°C or higher; consequently, an incubation temperature of 25°C was employed. Very little activity could be detected at pH 7.2, so the enzyme was assayed at what was found to be its optimal pH, 8.5, using Tris buffer ( 5 0 mM). Substrate concentrations were glutamate, 10 mM; CoASAc, 2.0 m~; arginine, 1 m~. These were selected on the basis of measurements of the following parameters: K , for glutamate, 2.1 mM; K , for CoASAc, 0.6 mM; KO for arginine, 30 p~. These values are confirmatory of previous reports (28). The assay was linear for at least 10 min at 25'C. The product N-acetylglutamate was purified as described above and measured using similar conditions as used to measure N-acetylglutamate content. Activity was expressed as nanomoles min" mg" of mitochondrial protein.
Measurement of Amino Acids-For the measurement of amino acids, approximately 2 g of liver was removed while the animal was anesthetized with ether, immersed quickly in ice-cold 150 mM NaCl to rinse any residual amino acid solution and blood from the surface of the liver, then freeze-clamped and ground to a fine powder with mortar and pestle at the temperature of liquid nitrogen. Approximately one-half of this powder was mixed with cold 10% sulfosalicylic acid and the rest with 5% perchloric acid, each of known weight and volume. Calculation of dilution factors to express results per g wet weight was done by weight difference. The sulfosalicylic acid solution was centrifuged, and the supernatant passed through a microporous fdter (2 p~ pore size, Amicon, Lexington, Mass.) and used directly for neutralized to pH 7.0 with KOH, and the supernatant used for amino acid analysis. The perchloric acid solution was centrifuged, enzymatic analysis of amino acids and metabolites. Immediately after preparation, the supernatants were stored at -20°C until assayed.
Amino acids were measured on a Glenco model MM 70 amino acid analyzer (Glenco, Inc., Houston, Tex.). Under the conditions employed for the analyzer, glutamine and tryptophan were not quantitated, and aspartate was masked by a glutathione peak in liver samples. Cystine gave such low values that accurate quantitation was not possible. Argininosuccinate could not be identified in any samples, presumably because hepatic values were below the level of detection. Because of the large range of concentrations of amino acids, multiple runs were performed in differing dilutions so as to obtain accurate values at both high and low concentrations. Norleucine internal standards were used for calculation.
Glutamine was measured by hydrolysis to glutamate and was ase (29). Glutamate was quantitated both enzymatically and on the subsequently determined enzymatically with glutamate dehydrogenanalyzer, with results in close agreement. Aspartate was measured enzymatically (30).
Assay of a-Amino Nitrogen-Total a-amino nitrogen was measured using the dinitrofluorobenzene conjugate (31) on a supernatant of tungstate HCI-treated whole blood collected from the abdominal aorta under ether anesthesia. A standard curve with values in the range of the samples was performed for each batch of determinations. Values determined by the use of the standard curve and by use of the molar extinction coefficient were in ciose agreement.
Measurement of Ammonia-Ammonia was measured on the plasma of arterial blood by a modified Berthelot reaction using a Dowex 50W-X4,200 to 400 mesh, for extraction of ammonia (32).
Materials-Commercial sources of chemicals not mentioned above were: Dowex resins, N-acetylglutamate, oligomycin, ornithine carbamyltransferase, glutaminase, and amino acids from Sigma, St. Louis, Mo., other enzymes and carbonyl cyanidep-trifluorophenylhydzone from Boehringer Mannheim, Indianapolis, Ind. All other chemicals were of reagent grade.

RESULTS
Blood a-Amino Nitrogen-The time course (Fig. 1) and dose dependence (Fig. 2) of blood a-amino nitrogen in response to intraperitoneal injection of amino acids mixtures were scarcely different whether or not arginine was present in the injected mixture, except that the presence of arginine appeared to accelerate the absorption of the injected load (Fig. 1). The dose-response curves (Fig. 2) are convex upward.
Blood Ammonia-The dose dependence of blood ammonia ( Fig. 3) is strikingly curvilinear. With doses up to 2 g/kg of amino acids, blood ammonia rises only slightly. With doses of 3 to 5 g/kg, a steep increase occurs, which is about 30% higher when arginine is omitted from the injected mixture. These results suggest that some mechanism is facilitating ureagenic response to smaller loads, whether or not arginine is present, but that this mechanism fails at larger loads of amino acids. An independent and relatively minor effect of arginine appears to be present at all doses.
Carbamyl Phosphate Synthetase Z Activity-As can be seen in Figs. 4 and 5, whole mitochondrial carbamyl phosphate synthetase I rises within 15 min after a dose of 1.5 g/kg from 20% to 80% of the activity seen in disrupted mitochondria and assayed under optimal conditions. Thereafter, carbamyl phosphate synthetase I activity decreases rapidly as blood a-amino nitrogen returns to normal. With increasing amino acid dosage, carbamyl phosphate synthetase I activity reaches a maximum at 3 g/kg body weight, a value that approximates the value obtained in disrupted mitochondria. This further suggests that N-acetylglutamate content of these mitochondria is what is limiting for citrulline synthesis, as other substrates and cofactors are in concentrations that are not limiting. The changes in carbamyl phosphate synthetase I activity are indistinguishable between animals that received a complete amino acid mixture and those that received an arginine-free mixture, suggesting that neither arginine nor ornithine play a role in this response.
N-Acetylglutamate Content of Liver-As shown in Figs. 6 and 7, N-acetylglutamate also rises a t least as rapidly as does carbamyl phosphate synthetase I activity, and also increases in a dose-dependent manner. Again, essentially the Same results are seen whether or not arginine is a component of the injected mixture.
The relationship between carbamyl phosphate synthetase I in intact mitochondria isolated from the liver and N-acetylglutamate content of the mitochondria of these animals is approximately hyperbolic (Fig. 8), Half-maximal activation occurs at 0.8 nmol/mg of mitochondrial protein. Using presently accepted views that 1 mg of mitochondrial protein corresponds to about 1 pl of matrix volume, this means that half-maximal activation occurs at 0.8 mM. In these same experiments, whole liver N-acetylglutamate content was proportional to N-acetylglutamate content of isolated mitochondria (r = 0.92). The ratio of N-acetylglutamate content per g wet weight of liver to N-acetylglutamate content per mg of mitochondrial protein averaged 80 f 12 mg/g (S.D., n = 17). This value for mitochondrial protein per g of liver is higher than the usual estimate, namely, 60 mg/g. The difference could be attributed to an unusually high content of mitochondria, to some leakage of N-acetylglutamate out of the mitochondria during isolation, or to some impurities in the mitochondrial fraction.
Reported values for half-maximal activation of carbamyl phosphate synthetase I by N-acetylglutamate vary from 0.08 mM to 0.18 mM with the pur5ed enzyme (33, 34). The discrepancy between this value and the value estimated here has I P I FIG. 1 (left) tion, as a function of the injected dose of amino acids. Symbols  FIG. 2 (center). Dose dependence of blood a-amino nitrogen and vertical bars as in Fig. 1.  (left). Liver N-acetylglutamate concentration at inter-intact mitochondria isolated from rats given varying doses of vals following injection of 1.5 g/kg of mixtures of amino acids. amino acids intraperitoneally, calculated as per cent of the Symbols and vertical bars as in Fig. 1. value obtained from disrupted mitochondria, as n function of FIG. 7 (center). Liver N-acetylglutamate concentrations N-acetylglutamate concentration in these same mitochondria. measured 16 min after the injection of varying doses of amino Half-maximal activation is seen at an N-acetylglutamate concentraacids. Symbols and vertical bars as in Fig. 1. tion of 0.8 nmol/mg of mitochondrial protein. Each point is the mean  FIG. 8 (right). Carbamyl phosphate synthetase I activity of of three observations. been seen before. McGivan et al. (16) have shown that a value of 0.6 mM N-acetylglutamate gives a carbamyl phosphate synthetase I activity of 50% of maximum. The data of Meijer and Van Woerkom (20) are consistent with a value of 0.6 mM giving a value of one-third maximum. Thus, while it is clear that alterations in N-acetylglutamate content of mitochondria can regulate the ability of these mitochondria to synthesize citrulline, the milieu of the organelle may alter the affinity of carbamyl phosphate synthetase I for its activator, N-acetylglutamate. An alternative explanation is that a good portion of the total mitochondrial N-acetylglutamate may be bound to carbamyl phosphate synthetase 1, which may be as high as 1 mM in the matrix (35). If this were the case, the measured amount of activator yielding one-half maximal activation is not a measure of the activation constant.
N-Acetylglutamate Synthetase-An alternative mechanism to rapidly augment N-acetylglutamate content, and hence carbamyl phosphate synthetase I activity, could be rapid changes in activity of the enzyme N-acetylglutamate synthetase (EC 2.3.1.1). Levels of this enzyme before and after amino acid loads are documented in Table I. No change in activity occurred when N-acetylglutamate was maximal either with dose or with time. Thus, alterations in mitochondrial N-acetylglutamate do not appear to be secondary to alterations in N-acetylglutamate synthetase on a short term basis. Indeed, no change in activity was found even up to 3 h after administration of 1.5 g/kg of amino acids. The activity of this enzyme, when measured 3 days after alterations in the protein content of the diet, has been found to change in concert with N-acetylglutamate content (16). This finding suggests that Nacetylglutamate synthetase may affect N-acetylglutamate content on a long term basis, in contradistinction to the regulation described here.
It is of interest to estimate whether the activity of Nacetylglutamate synthetase is sufficient to increase N-acetylglutamate levels as rapidly as was seen in this experiment. With an amino acid load of 1.5 g/kg, liver N-acetylglutamate content increased in 5 min by 75 nmol/g wet weight. Assuming 60 mg of mitochondrial protein/g of liver, this would require N-acetylglutamate synthesis at a rate of 0.25 m o l min-l mg-l protein. This compares with a maximal in vitro rate obtained in these experiments of 0.34 nmol min" mg" a t a temperature of 25°C.
Urea Cycle Amino Acids in the Liver-In rats injected with a complete amino acid mixture, hepatic levels of both ornithine and arginine, measured 15 min after injection, rose linearly with dose of amino acids (Figs. 9 and lo), reflecting the rapid conversion of arginine to ornithine via arginase (EC 3.5.3.1) and glycine transamidinase (EC 2.6.2.1). The increment in ornithine was twice as great as the increment in arginine, indicating that at least two-thirds of the arginine carbon skeletons present in the liver at this time had been converted to ornithine. (The fraction present as an increment in citrulline, as noted below, was smaller; the amount present as argininosuccinate was too low to be determined.) These inferences are based on the assumption that de novo synthesis of ornithine or arginine, or both, via ornithine transaminase (EC 2.6.1.13), was negligible. This assumption is borne out by the results of experiments in which arginine was omitted from the injected mixture. Under these conditions, liver arginine and ornithine remained nearly constant with increasing dose of amino acids, except that there was a slight upward trend in arginine at the largest doses. These results contrast with the effects of a high protein intake in rats, recently reported by Saheki et al. (36). Within 15 h of the change in diet, liver ornithine increased substantially in their experiments. Thus, de novo ornithine synthesis may play a role in accelerating urea synthesis within 15 h of a change in dietary protein intake. But the data reported in the present studies suggest that neither arginine nor ornithine plays more than a minor role in the acute response to a protein load.
When the time course of hepatic levels of these two amino acids following a dose of 1.5 g/kg was examined (Figs. 11 and  12), confiiatory findings were seen. Arginine rose about 4fold a t 15 min and was back to control values at 60 min. Ornithine rose from 210 to 510 nmol/g, a 2.5-fold increase, and peaked later, returning to control values at a slower rate. With an arginine-free mixture, no change in hepatic arginine content occurred. Hepatic ornithine content apparently increased at 15 and 30 min, but only one-third as much as when compared to the same dose of the complete mixture, and returned to control values at 45 min. This increase was inconsistent, since, as noted above, ornithine was not increased at 15 min following a dose of 0.5, 1, or 2 g/kg (Fig. 10).
The conversion of an exogenous load of arginine to ornithine in the liver is rapid and apparently complete. In addition, the disposal of the resulting ornithine (presumably by ornithine aminotransferase) is also rapid. It is noteworthy that the fraction of the increment in amino acids (at 15 min) made up of the sum of the molar concentrations of arginine, ornithine, and citrulline (4.3% average) was only slightly greater than the mole fraction of arginine in the injected mixture (2.9%). The difference may reflect the fact that liver/plasma ratio of ornithine in these rats was greater than unity (data not shown).
At a dose of 1.5 g/kg of amino acids, liver citrulline was only slightly increased and no trend with time could be discerned. With increasing dosage, however, the increment was substantially greater in animals that received the complete mixture than in those receiving the arginine-free mixture (Fig. 13). These findings are of interest because of the possibility that argininosuccinate synthetase (EC 6.3.4.5) might become ratelimiting for urea synthesis. If this were the case, a progressive increase in citrulline levels might be detected in supramaximal loads. However, it is known that ornithine inhibits arginase (37) and that argininosuccinate synthetase is inhibited by both arginine and argininosuccinate (38). These two effects alone could result in a compensatory elevation in steady state citrulline levels without necessarily reflecting any change in the rate of urea synthesis.
Aspartate and Glutamate-As one of the nitrogen donors for urea, aspartate is likely to vary in conjunction with rapid changes in ureagenesis. As shown in Fig. 14 . 9 (left). Liver arginine as a function of the injected dose of amino acids, measured 15 min after injection. Symbols and vertical bars as in Fig. 1.   FIG. 10 (right). Liver ornithine, shown as in Fig. 9. Nearly the same results are seen as in Fig. 9.   FIG. 11 (left). Time course of liver arginine following the injection of 1.5 g/kg of amino acids. Symbols and vertical bars as in Fig. 1.   FIG. 12 (right). Time course of liver ornithine plotted as in Fig. 11. A transient increase in liver ornithine is seen even when arginine is omitted from the injected mixture. fell to control values by 45 min. Subsequently, values below control were seen. Clearly, amino groups derived from the injected mixture were transaminated with oxalacetate to form aspartate, presumably intramitochondrially, and thence incorporated into argininosuccinate extramitochondrially. Other possible sources of hepatic aspartate are circulating asparagine or aspartate itself. The approximately linear dose dependence of the increase in aspartate is shown in Fig. 15.
There appears to be scarcely any difference between the results seen with complete and arginine-free mixtures. Thus, the availability of ornithine had little influence on the disposal of aspartate under these conditions. The time course (Fig. 16) and dose dependence (Fig. 17) of glutamate levels were quite similar to those of aspartate, although the percentage increase was smaller. Again, the results were essentially the same with or without arginine in the injected mixture. Since glutamate is believed to be the direct source of ammonia for carbamyl phosphate synthesis as well as the source of amino groups for aspartate synthesis (39), these changes presumably reflect the driving force for ureagenesis.
An additional effect of the rise in glutamate may have been stimulation of the synthesis of N-acetylglutamate. As noted above, this compound rose within a few minutes after injection of amino acids in a dose-dependent manner. This effect occurred independently of the presence or absence of arginine in the injected mixture, and thus could not be attributed to stimulation of N-acetylglutamate synthetase by this mechanism. The K , of N-acetylglutamate synthetase for glutamate is reported to be 3.0 mM (28). Hence, the changes in hepatic glutamate seen here, if they reflect changes in mitochondrial glutamate concentration, could well account for the observed increases in N-acetylglutamate simply by a substrate effect. Thus, glutamate may facilitate its own disposal into urea, not only by direct effects on substrate availability, but also by modulating N-acetylglutamate synthesis and flux through carbamyl phosphate synthetase. The increase in hepatic glutamate comprised the same fraction of the increase in hepatic amino acid nitrogen as glutamate comprised of the injected mixture (Fig. 17). In all probability, this is a coincidence, since measured glutamate was almost certainly synthesized intrahepetically. The permeability of the hepatocyte membrane to this amino acid is low (40). FIG. 13 (left). Liver citrulline measured 15 min after the inmixture. Aspartate levels fall below control values at 1 h and then jection of varying doses of amino acids. Symbols and vertical return to control. bars as in Fig. 1.   FIG. 15 (right). Liver aspartate measured 15 min after injec- FIG. 14 (center). Liver aspartate at intervals after the injec-tion of varying doses of amino acids. Symbols and vertical bars tion of 1.5 g/kg of amino acids. Symbols as in Fig. 1. A rapid as in  . 16 (lefl). Liver glutamate at intervals after the injection of 1.5 g/ kg of amino acids. Symbols and vertical bars as in Fig. 1.   FIG. 17 (right). Liver glutamate, measured 15 min after the intraperitoneal injection of varying doses of amino acids. Symbols and vertical bars as in Fig. 1.

I I
The role that compartmentation plays in altering the tissue concentrations of all these metabolites cannot be assessed. At least in the isolated mitochondria (19), there is no gradient of ornithine across the mitochondrial membrane, so that its tissue concentration is likely to approximate its intramitochondrial concentration. But the compartmentation of other measured amino acids between cell and extracellular fluid and between cytosol and mitochondrion under ihe conditions of these experiments is conjectural.
Other Amino Acids in the Liver-The time course and dose dependence of hepatic amino acid levels are shown in Tables I1 to V. Although a full interpretation of these results cannot be given in our present state of knowledge, they may provide a useful basis for further reference, in that they define the response of hepatic amino acid levels, both in time and in varying dose, to complete amino acid loads.
Dose 01 ammo acldr. 9, kg Alanine rose more than any other amino acid, reaching a peak of 6.86 pmol/g after 1.5 g/kg of a complete mixture, and reaching an earlier peak following an arginine-free load. With increasing dosage, alanine rose significantly more steeply following the arginine-free mixture, suggesting some limitation in disposal of load, although this limitation was not apparent in levels of blood a-amino nitrogen.
Glutamine, by contrast, showed no significant changes with time or dose except a transient increase immediately after injection. This is surprising, because blood glutamine rises in hyperammonemic states, suggesting that it serves as a storage site for excess amino groups (41). However, Lund (42) has shown that net glutamine synthesis fails to occur in isolated perfused liver despite high tissue levels of ammonia and glutamate. The present findings suggest that the injected glutamine has been rapidly metabolized, presumably intra-

Changes with time in lzuer amino acids in rats given an arginine-free mixture of amino acids intraperitoneally at a dose of 1.5 g/kg body weight
Min after Injection  hepatically, and furthermore, that its hepatic concentration is subject to close control. The importance of glutamine as a precursor of urea in the isolated perfused liver has been pointed out by Lund and Watford (43). The alternate explanation, that glutamine was not extracted by the liver, seems unlikely.

TABLE V Liver amino acids in rats given an arginine-free mixture of amino acids intraperitoneally at varying doses in grams per kg body weight, measured 15 min after injection
The results seen with other amino acids can best be summarized by comparing increments in hepatic concentrations, calculated as fractions of the increments in total hepatic amino acid nitrogen, with the fractions that each amino acid com-prised of the injected dose of nitrogen, as shown in Fig. 18. Four amino acids are enriched significantly in the liver relative to their proportions in the injected mixture: aspartate, serine, alanine, and tyrosine, indicating net intrahepatic synthesis. The increases in alanine and aspartate are not surprising, and doubtless reflect the synthesis of these amino acids by transamination from some of the injected amino acids or from glutamate (43). Tyrosine synthesis presumably reflects the action of phenylalanine hydroxylase (EC 1.99.1.2); indeed, phenylalanine was more depleted in the liver relative to its proportion in the dose than any other amino acid. The rise in serine, which comprised only 1% of the dose, was unexpected. It may be related in part to the relative depletion of glycine.
The increments in hepatic concentrations of essential amino acids, expressed as fractions of the total increase in hepatic a-amino nitrogen, were equal to or less than the fractions they comprised of the injected mixture. These results could reflect varying rates of equilibrium with hepatic cells, varying intracellular/extracellular accumulation ratios, or varying degrees of intrahepatic as well as extrahepatic metabolism, or both.
An additional difficulty in interpretation of these changes in hepatic amino acids is that the freeze-clamped liver also includes amino acids present in the extracellular space. At present, there is no generally accepted method for estimating the extracellular space of the liver, and values obtained with differing markers may vary more than 2-fold (44). Total liver a-amino nitrogen, in micromoles per g wet weight, was several times higher than blood a-amino nitrogen, in micromoles per ml , 15 min after the injection of every dose of amino acids.
Consequently, the fraction of the hepatic content present in the extracellular space was presumably small.

DISCUSSION
These results establish that mitochondrial carbamyl phosphate synthetase I is rapidly activated following loads of amino acids varying from 0.5 to 3.0 g/kg. With doses of 3 g/kg or higher, carbamyl phosphate synthetase I activity is maximally activated and reached the same level as is seen in disrupted mitochondria assayed with optimal concentrations of N-acetylglutamate. Since urea production could not be measured in these experiments, it is not certain that these changes in carbamyl phosphate synthetase I activity were accompanied by parallel changes in the rate of urea synthesis.
However, the minor increase in blood ammonia concentration induced by amino acid loads of less than 3 g/kg, compared with the sharp increase seen following larger doses (Fig. 3), is compatible with this inference. The simultaneous changes in liver N-acetylglutamate content (Fig. 7) strongly suggest that this substance was responsible for the activation of carbamyl phosphate synthetase I observed, although other possibilities, such as alterations in mitochondrial free calcium content (45), cannot be excluded from having an effect.
Rapid augmentation of N-acetylglutamate synthetase activity has been excluded as a possible explanation of the increase in N-acetylglutamate content. Furthermore, nearly the same results were obtained whether or not arginine was a component of the injected mixture of amino acids. Liver arginine levels rose minimally following the injection of arginine-free mixtures, but rose about 14-fold when arginine was included. Even allowing for uncertainties as to compartmentation within the liver, it is inconceivable that the increment in mitochondrial arginine content was the same in these two groups of experiments.
Ornithine has recently been reported to stimulate carbamyl phosphate synthetase I about 15 to 20% (35), and a transient increase in liver ornithine was seen following a dose of 1.5 g/ kg of amino acids lacking arginine (Fig. 12). Furthermore, no change in hepatic ornithine with increasing amino acid dosage was seen when arginine was omitted, in contrast with an increase up to &fold when arginine was included (Fig. 10). Since carbamyl phosphate synthetase I activation did not differ whether or not arginine was injected (Figs. 4 and 5), and this enzyme was assayed in the presence of high concentrations of ornithine, it is clear that ornithine derived from the injected arginine cannot have been responsible for the activation of carbamyl phosphate synthetase I observed in intact mitochondria. Nevertheless, it could have played a role in facilitating the ureagenic response, either by its minor stimulatory effect on carbamyl phosphate synthetase I (which would not have been revealed by these experiments) or by some other mechanism. The small but consistent difference in blood ammonia concentration between those animals injected with the arginine-free mixture (Fig. 3) is likely to be a reflection of an effect of ornithine.
Ornithine must have an effect on the carbamyl phosphate synthetase I reaction, since it not only augments ureagenesis from amino acid precursors in isolated perfused liver but also reduces ammonia accumulation (46). Kuchel et al. (47) have suggested that decomposition of carbamyl phosphate, either spontaneously or through the action of cytosolic acyl phosphate phosphohydrolase (EC 3.6.1.7) could result in regeneration of ammonia when carbamyl phosphate concentration is high and ornithine concentration is low. The stimulatory effect of added ornithine on ureagenesis seen in liver slices (48), isolated perfused liver (49,50) or in isolated hepatocytes (51,52) may well have different explanations, perhaps because tissue levels of ornithine are depleted in such systems.
Whatever the explanation of the effect of ornithine on ureagenesis, it is clear that the rapid activation of carbamyl phosphate synthetase I as measured in these experiments cannot be attributed to an increase in hepatic ornithine, and also cannot have been caused by a stimulatory effect of arginine on N-acetylglutamate synthetase. A more likely explanation is the increase in hepatic glutamate levels (Figs. 16 and 1 3 , thereby augmenting N-acetylglutamate by a substrate effect. These changes in glutamate occurred rapidly and to the same extent whether or not arginine was included in the injected mixture. Although hepatic glutamate rose no more than its mole fraction of the injected dose (9%), it is likely, in view of the low permeability of cells to this amino acid, that the injected glutamate largely bypassed the liver and that the measured glutamate was synthesized intrahepatically. In any case, it is clear that the increment in hepatic glutamate was sufficiently rapid to account for the increase in N-acetylglutamate levels, while at the same time, stimulating the production of both ammonia and aspartate, the immediate precursors of the nitrogen atoms incorporated into urea. Thus, it appears that hepatic glutamate may play a central role in regulating ureagenesis, not only serving as a precursor of both nitrogen atoms incorporated into urea but also facilitating its own disposal by means of carbamyl phosphate synthetase I activation. This latter mechanism evidently exhibits saturation at doses of amino acids of 3 g/kg and above. At these larger doses, another enzyme in the cycle may have become rate-limiting, such as argininosuccinic acid synthetase. This could explain the increase in hepatic citrulline levels seen even when arginine was omitted (Fig. 13), but only at higher doses of amino acids. Saturation of this enzyme could explain the fact that blood ammonia concentration increased more, proportionately, than the injected dose of amino acids at doses of 3 g/kg and greater (Fig. 3).
Thus, the picture that emerges from these studies is that of an autoregulatory control of ureagenesis seen only at moderate doses of amino acids, and mediated by glutamate effects on N-acetylglutamate synthesis. At larger doses of amino acids, this regulatory mechanism is no longer effective, and hyperammonemia supervenes.
Although these inferences are consistent with the data, confiiatory evidence is needed. In particular, study of amino acid tolerance in animals in which this regulatory mechanism has been incapacitated experimentally would be of interest.

Short Term Regulation
of Ureagenesis