Urea Synthesis In Liver Research Articles

Glutamine-dependent urea synthesis in elasmobranch fishes

Marine elasmobranchs (sharks, skates, and rays) synthesize and retain in their tissues high concentrations of urea (up to 0.4 M) and certain amines, such as trimethylamine oxide (up to 0.2 M), for the purpose of osmoregulation. Although the pathway of urea synthesis in ureosmotic elasmobranchs is similar to the classical urea cycle in ureotelic species, there are several properties that are unique. Indeed, this might be anticipated, since the primary function of urea synthesis in elasmobranchs is osmoregulation, rather than ammonia detoxification. The existence in different species of a major biosynthetic pathway resulting in the same end product, but which serves quite different physiological purposes in each species, can provide an opportunity for comparative investigations that contribute to our understanding of the regulatory properties and evolutionary development of the pathway. The purpose of this commentary is to consider the unique properties of urea synthesis in elasmobranchs and to comment on the comparative relationships to teleost fishes and mammalian species; most studies discussed here have been carried out with spiny dogfish shark, Squalus acanthias, a representative elasmobranch. Like ureotelic species, hepatic urea synthesis in ureosmotic elasmobranchs requires both mitochondrial and cytosolic enzymes, but there are notable differences (Fig. 1). The most significant difference is that the initial step of ammonia assimilation for urea synthesis in hepatic mitochondria of elasmobranchs is the formation of glutamine, which is subsequently utilized as the substrate for carbamoyl-phosphate synthesis (Anderson and Casey, J. Biol. Chem., 259, 456-462, 1984). This is due to the presence of high levels of glutamine synthetase (GSase) and a unique acetylglutamateand glutamine-dependent carbamoyl-phosphate synthetase (CPSase 111), both localized exclusively in the mitochondrial matrix (Casey and Anderson, J. Biol. Chem., 257,8449-8453, 1982, and Comp. Biochem. Physiol., 82B, 307-315,1985). GSase is a cytosolic enzyme in liver of mammalian species and in some, but not all, teleost fishes. In mammalian and amphibian species the first step of ammonia assimilation for urea synthesis in liver is direct conversion of ammonia to carbamoyl phosphate catalyzed by a corresponding mitochondrial acetylglutamateand ammoniadependent CPSase I (which cannot utilize glutamine as a substrate). The properties of elasmobranch mitochondrial CPSase I11 are similar to the mammalian mitochondrial CPSase I, except for the fact that glutamine rather than ammonia serves as the nitrogen-donating substrate (Anderson, J. Biol. Chem., 256, 12 228 12 238, 1981).

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish)

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation.

HEPATIC UREA SYNTHESIS AND SITE AND RATE OF UREA REMOVAL FROM BLOOD OF BEEF STEERS FED ALFALFA HAY OR A HIGH CONCENTRATE DIET

Five Hereford × Angus steers, 240 ± 6 kg liveweight with chronic indwelling catheters in various sites in the splanchnic bed were used to evaluate effects of dietary energy source and nitrogen (N) level on urea synthesis and site and rate of urea removal from blood. Alfalfa hay (162 g N d−1) or a high concentrate diet (95 g N d−1) were fed at similar metabolizable energy intake (54.8 MJ d−1). Compared with the alfalfa, the high concentrate, low N diet decreased (P < 0.05) blood concentrations of urea N, hepatic urea synthesis, urinary urea N excretion, transfer of urea N to poststomach tissues, and salivary transfer of urea N. Similarly, the high concentrate diet decreased net absorption of ammonia N and increased net absorption of glucose from portal-drained viscera decreased hepatic removal of ammonia N and α-amino N, and increased release of α-amino N by splanchnic tissues (P < 0.01). Blood flow (dilution of para-aminohippurate) through the liver and gut was not affected (P > 0.10) by diet. A greater (P < 0.05) portion of urea synthesized in the liver was transferred to the gut when the high concentrate diet was fed. Site of total urea transfer to the gut (from blood plus saliva) shifted from 37% of total transfer into poststomach (alfalfa) to 75% into stomach (high concentrate diet). Increased intake of readily fermentable carbohydrate at the expense of N and fiber increased the percentage of liver urea synthesis recycled to the gut and focused that recycling into the stomach (rumen) in these steers. Key words: Bovine, energy, nitrogen, urea flux, gut, liver

Kinetics of urea synthesis and alanine uptake by perfused rat livers.

Eight livers of 200 g rats were isolated and perfused in a single pass system with a semi-synthetic medium to which alanine was added to concentrations from 0.5 to 15 mmol/l. In each liver, 4-5 sets of urea synthesis rate and alanine uptake rate at different alanine concentrations were measured. The urea synthesis rate in relation to the alanine concentration was compatible with substrate inhibition kinetics. The kinetic constants were (mean +/- SD): Vmax = 10.34 +/- 3.41 mumol urea-N/(min X 100 g b.w.), Km = 1.56 +/- 0.67 mmol/l, and Ki = 5.35 +/- 2.44 mmol/l. The alanine uptake rate followed Michaelis-Menten kinetics with the constants (mean +/- SD) Vmax = 7.51 +/- 1.68 mumol/(min X 100 g b.w.) and Km = 2.14 +/- 1.04 mmol/l. The constants were assessed by non-linear iterative regression analysis. Urea synthesis exceeded alanine uptake at alanine concentrations below 2 mmol/l, and was smaller at higher concentrations. In two experiments, alanine metabolites were measured. The glucose production rate in relation to the alanine concentration suggested substrate inhibition. At high alanine concentrations, ammonia, lactate and pyruvate were released by the livers. The results indicate that whole liver urea synthesis and gluconeogenesis is inhibited by high blood alanine concentrations, in contrast to alanine uptake.

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

1. 1. The submitochondrial localization of the four mitochondrial enzymes associated with urea synthesis in liver of Squalus acanthias (spiny dogfish), a representative elasmobranch, was determined. 2. 2. Glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, ornithine carbamoyltransferase, glutamine synthetase, and arginase were all localized within the matrix of liver mitochondria. 3. 3. The subcellular and submitochondrial localization and activities of several related enzymes involved in nitrogen metabolism and gluconeogenesis in liver of spiny dogfish are also reported. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase were localized in the mitochondrial matrix. 4. 4. 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.

The glutamine analog acivicin as antipyrimidine. Studies on the interrelationship between pyrimidine and urea synthesis in liver

The inhibition of cytosolic carbamoyl-phosphate synthetase II by acivicin was used to study the role of the cytosolic carbamoyl phosphate pool as the exclusive substrate source for de novo pyrimidine synthesis rat hetacoytes. De novo pyrimidine synthesis was stimulated: 1. by uridine triphosphate deficiency (incubation with d-galactosamine) leading to a stimulation of cytosolic carbamoyl phosphate synthesis, and 2. by accumulation and efflux of mitochondrial carbamoyl phosphate (incubation with ammonium ions and l-norvaline). The stimulated orotate formation from cytosolic carbamoyl phosphate in UTP depleted cells was completely blocked by acivicin. It was not influenced by an inhibition of mitochondrial carbamoyl phosphate synthesis mediated by 4-pentenoate, since mitochondrial carbamoyl phosphate did not partipate in cytosolic pyrimidine synthesis even in the presence of ammonium ion concentrations maintaining physiological rates of urea synthesis. An excess of ammonium ion sled to an artificial accumulation and efflux of mitochondrial carbamoyl phosphate, which could be avoided by 4-pentenoate. The non-regulated stimulation of pyrimidine synthesis from surplus mitochondrial carbamoyl phosphate was not inhibited by acivicin. Utilization of mitochondrial carbamoyl phosphate for de novo pyridimidine synthesis presumably does not occur under physiological conditions because mitochondrial CP efflux depends on the accumulation of this metabolite in the mitochondria under experimental or pathological circumstances. Acivicin inhibition of CPS II thus cannot be bypassed by mitochondrial CP. It is suitable as inhibitor of the physiological de novo pyrimidine synthesis.

Regulation of the rate of urea synthesis in liver by extracellular pH. A major factor in pH homeostasis in mammals.

The views that catabolism of protein leads to net production of acid and that urinary excretion of ammonium ion represents an equimolar excretion of proton are not compatible with basic chemical relationships (Atkinson, D.E., and Camien, M.N. (1982) Curr. Top. Cell. Regul. 21, 261-302). Metabolism of protein produces significant amounts of base (bicarbonate), which is disposed of in the synthesis of urea. In perfused rat liver and in isolated rat hepatocytes, the rate of urea synthesis increases with increase in pH but is not affected by change in the concentration of bicarbonate when pH is held constant. An increase in the concentration of ammonium ion in the suspending medium causes an increase in the rate of urea synthesis by hepatocytes when lactate is the energy source, as previously reported by others, but causes a decrease in the rate of urea synthesis during incubation with glucose or with no added energy source. The rate of urea synthesis decreases when glucose is added to lactate medium. All of these observations are consistent with the view that disposal of bicarbonate is a major function of urea synthesis, and that regulation of the rate of ureagenesis is an important factor in the maintenance of pH homeostasis.

Liver Arginase Activity and Urea Metabolism in Beef Heifers Fed Urea Diets and Abomasally Infused with Arginine and(or) Ammonium Acetate

Eight growing crossbred beef heifers, surgically fitted with indwelling abomasal cannulas, were used in a 4 x 4 Latin square design and examined for the effects of continuous abomasal infusion of arginine and(or) ammonium acetate on liver arginase activity and urea and ammonia metabolism. Heifers were fed complete diets containing 1.2% urea as the only source of supplemental N. Infusion treatments were: (1) control (C): 2 liters water/24 h; (2) arginine (Arg): 24.00 g arginine in 2 liters water/24 h; (3) ammonium acetate (NH4Ac): 116.34 g ammonium acetate in 2 liters water/24 h; (4) arginine plus ammonium acetate (Arg + NH4Ac): 24.00 g arginine . HCl plus 116.34 g ammonium acetate in 2 liters water/24 h. Infusions solutions contained 0, 1, 1 and 2% crude protein equivalent, respectively, above the level of crude protein provided by the diet. NH4Ac and Arg + NH4Ac increased (P less than .05) apparent digestibility of N over that observed with treatment C. Arg tended to increase N retention over that observed with C, while both NH4Ac and Arg + NH4Ac resulted in lower (P less than .05) N retention than did C and Arg. Mean liver arginase activities (mumol . mg protein-1 . h-1) were 166.9, 137.5, 226.3 and 194.5 for heifers infused with C, Arg, NH4Ac and Arg + NH4Ac, respectively. NH4Ac and Arg + NH4Ac resulted in higher (P less than .05) arginase activity than did C or Arg. Arg infusion increased plasma urea N (PUN) over that observed with C, while PUN was higher (P less than .05) with NH4Ac or Arg + NH4Ac than with C or Arg. NH4Ac infusion increased (P less than .05) plasma ammonia-N (PAN) over that observed with Arg or Arg + NH4Ac, while PAN levels were the same with C, Arg and Arg + NH4Ac. Both Arg and NH4Ac infusion resulted in higher (P less than .05) rumen ammonia levels than C, while heifers given Arg + NH4Ac had rumen ammonia levels similar to those of controls. Mean daily urea excretion was 13.16, 16.02, 28.92 and 32.44 g/24 h for heifers infused with C, Arg, NH4Ac and Arg + NH4Ac, respectively. The major difference in excretion of urea was attributable to NH4Ac and not Arg. Daily urea excretion decreased between 1530 and 2330 h and increased between 2330 and 0730 h. Arginine infusion appeared to have a protective effect against ammonia infusion because PUN increased and PAN decreased in heifers infused with arginine. The reduction (P less than .05) in plasma amino acids thought to be limiting for protein synthesis in ruminants in conjunction with increased (P less than .05) N retention and the trend toward decreased arginase activity observed in heifers infused with Arg suggests that the effects of arginine on ammonia metabolism are not wholly mediated by liver urea synthesis.

Synthesis of urea after stimulation with amino acids: relation to liver function.

Hepatic urea synthesis is the organism's main channel for the disposal of nitrogen and it may be an 'essential' liver function. In six control subjects and five patients with cirrhosis of the liver urea synthesis was studied during continuous infusion for six to 24 hours of about 3 mmol alpha-amino nitrogen/h X kg body weight. The urea synthesis rate was calculated in intervals of two hours as urinary excretion with correction for accumulation in the total body water and for hydrolysis of urea in the gut. The peripheral venous plasma alpha-amino nitrogen concentration increased from 3 to about 14 mmol/l and the urea nitrogen synthesis rate from 25 to about 215 mmol/h. In all cases the urea synthesis rate rose linearly with the alpha-amino concentration throughout the examined range. The slope of this linear relationship is an expression of the hepatic conversion of alpha-amino nitrogen to urea nitrogen ('functional hepatic nitrogen clearance'). The functional hepatic nitrogen clearance was 22.4 l/h in control subjects and 13.7 1/h (P < 0.025) in the patients with cirrhosis. It was correlated with quantitative measures of the liver function (the galactose elimination capacity, r = 0.84, and the clearance of antipyrine, 4 = 0.80). These observations, while confirming the abundant capacity of the urea synthesis system, imply that a given urea synthesis rate requires a higher alpha-amino level in patients with reduced liver function.

EFFECT OF HYDROCORTISONE ON AMMONIA INTOXICATION IN THE ADRENALECTOMIZED RAT

Adrenalectomy induces a hypersensitivity in the rat to ammonia intoxication. Daily injection of hydrocortisone hydrochloride to adrenalectomized rats restored normal sensitivity to ammonia intoxication, with concomitant restoration of liver urea-synthesizing capacity to the normal value. When injected with a large dose of ammonium acetate, hydrocortisone-treated adrenalectomized rats were able to reduce the plasma ammonia concentration much more rapidly than the adrenalectomized control rats. However, neither the increase in liver urea synthesis nor the more rapid decrease in the plasma ammonia concentration were sufficient to explain the protective aciton of hydrocortisone against ammonia intoxication.