Effect of caffeine on urea biosynthesis and some related processes, ketone bodies, ATP and liver amino acids.

An increase in urea synthesis has been found in rats administered large doses of caffeine. A parallel increase in urea biosynthesis was also found in hepatocytes isolated from caffeine-treated rats, which confirms a greater capacity for urea synthesis induced by caffeine. This increase appeared only after some days of caffeine treatment; during the first days there was no increase in serum urea levels or in in vitro studies of urea synthesis in isolated hepatocytes. However, no appreciable changes were found in either cytosolic or mitochondrial redox states, or in ATP levels in in vivo and in vitro studies. A parallelism was observed between the decreased amino acid levels in caffeine-treated rats and in isolated hepatocytes incubated with different concentrations of caffeine. Several possible mechanisms to explain these findings are considered in the discussion.

Orotic acid overproduction in experimental cirrhosis of rats.

Ammonia clearance, portal blood ammonia, and amino acid concentrations were studied during induction of cirrhosis by carbon tetrachloride in rats. Exposure to CCl4 vapors twice weekly for 7-16 weeks doubled orotic acid excretion. If exposure was discontinued for 7 days, the orotic acid excretion decreased despite the presence of cirrhosis proven histologically. Replacement of dietary casein with soybean protein eliminated the CCl4-induced orotic aciduria in growing rats but not in adults. Supplementation of casein with 1.5% arginine did not prevent CCl4-induced orotic aciduria. [14C]Orotate uptake into RNA and DNA of liver was not impaired. Perfusion of livers of cirrhotic animals with ammonia concentrations between 0.2 and 3.0 mM revealed no significant decreases in urea synthesis rates due to cirrhosis and no increase in the tendency to make orotic acid at a given ammonia concentration. However, ammonia uptake by cirrhotic livers was significantly reduced, resulting in higher ammonia concentrations in the effluent when there was moderate-to-severe cirrhosis. Portal blood samples taken from rats exposed to CCl4 had higher ammonia concentrations as cirrhosis worsened. The results lend support to the "intact hepatocyte" hypothesis of cirrhosis which attributes metabolic abnormalities to intrahepatic shunts.

A possible role of inorganic phosphate as a regulator of oxidative phosphorylation in combined urea synthesis and gluconeogenesis in perfused rat liver. A phosphorus magnetic resonance spectroscopy study.

Metabolic control of oxidative metabolism was studied in perfused rat liver by means of phosphorus magnetic resonance spectroscopy. Oxygen consumption, ATP, and Pi were measured with different rates of gluconeogenesis and urea synthesis by varying concentrations of the substrates in the perfusate. Five levels of oxygen consumption (VO2) were obtained: an average control value of 1.94 +/- 0.14 and 2.93 +/- 0.25, 3.29 +/- 0.46, 3.85 +/- 0.26, and 4.18 +/- 0.56 mumol/min/g liver (mean +/- S.D., n = 6). The corresponding ATP concentrations were 2.51 +/- 0.20, 2.39 +/- 0.08, 2.24 +/- 0.09, 2.13 +/- 0.12, and 1.91 +/- 0.13 mM. Pi increased stoichiometrically with the decrease in ATP. Free Pi (Pif) was calculated as NMR-visible Pi in control plus -delta ATP (1.94 mM + (-delta ATP]. The kinetic relationship of oxidative phosphorylation as a function of Pif followed a Michaelis-Menten type of equation: VO2 = 5.55/(1 + 0.24/[( Pif] - 1.81]. The observed Km value for Pi of 0.24 mM approximates the reported Km value in isolated mitochondria of 1 mM. The free Pi concentration of 1.94 mM is in the range of the Km value, while the free ADP concentration of 200 microM exceeds the Km value of 20 microM. Therefore, it is suggested that Pi play a major role in the regulation of mitochondrial oxidative phosphorylation in combined urea synthesis and gluconeogenesis.

Biosynthesis of hippurate, urea and pyrimidines in the fatty liver: studies with rats fed orotic acid or a diet deficient in choline and inositol, and with genetically obese (Zucker) rats.

The activities of pathways for the biosynthesis of hippurate, urea and pyrimidines in hepatocytes isolated from lean livers were compared with those from three sources of fatty liver: a) the genetically obese Zucker rat, b) Sprague-Dawley rats fed a diet deficient in choline and inositol, and c) Sprague-Dawley rats fed a diet supplemented with orotic acid. The capacity for hippurate synthesis was not significantly affected by fat accumulation, but ureagenesis from saturating ammonia and ornithine was diminished about 50% in all models when fat content rose above 12% wet wt of liver. Pyrimidine biosynthesis under these conditions was similarly diminished with fat accumulation. Ureagenesis was inhibited by sodium benzoate in hepatocytes from lean livers, but not in hepatocytes from fatty livers. Other results suggest that higher rates of ureagenesis than could be achieved with the fatty liver are required in order to demonstrate inhibition by benzoate. Incorporation of [14C]NaHCO3 into orotate was also inhibited by sodium benzoate, but in hepatocytes from fatty as well as lean livers. The metabolic basis for impairment of ureagenesis and pyrimidine biosynthesis in the fatty liver requires further study. That the capacity for hippurate synthesis was not significantly affected suggests a pathway-specific mechanism.

Effects of the amount and quality of dietary protein on nitrogen metabolism and protein turnover of pigs.

1. The interrelation between protein accretion and whole-body protein turnover were studied by varying the quantity and quality of protein given to growing pigs. 2. Diets with 150 or 290 g lysine-deficient protein/kg were given in hourly meals, with or without lysine supplementation, to female pigs (mean weight 47 kg). 3. After the animals were adapted to the diets, a constant infusion of [14C]urea was given intra-arterially for 30 h, during the last 6 h of which an infusion of [4,5-3H]leucine was also infused at a constant rate. At the same time, yeast-protein labelled with 15N was given in the diet for 50 h. 4. The rate of urea synthesis was estimated from the specific radioactivity (SR) of plasma urea. The rate of leucine flux was estimated from the SR of plasma leucine. The irrevocable breakdown of leucine was estimated from the 3H-labelling of body water. Total N flux was estimated from the 15N-labelling of urinary urea. 5. Addition of lysine to the low-protein diet significantly increased N retention, with a substantial reduction in leucine breakdown, but there was no significant change in the flux of leucine or of total N. 6. Increasing the quantity of the unsupplemented protein also increased N retention significantly, with concomitant increases in leucine breakdown and in the fluxes of leucine and of total N. 7. It is concluded that a doubling of protein accretion brought about by the improvement of dietary protein quality is not necessarily associated with an increased rate of whole-body protein turnover.

Metabolic aberrations associated with arginine deficiency.

The significance of dietary arginine deficiency is often unrecognized since growth and nitrogen balance are generally positive. However, inadequate intakes of dietary arginine are typically associated with dramatic alterations in intermediary metabolism in mammals. Most of the symptoms that develop following arginine deprivation can largely be accounted for by a decreased efficiency of ammonia detoxification. However, species differences in the metabolic aberrations associated with arginine deficiency are clearly evident. Therefore, selected animals fed an arginine-deficient diet may serve as a useful model for the study of chronic hyperammonemia. In rats, mice, hamsters, guinea pigs and rabbits, the excretion of citric and orotic acid is a sensitive indicator of arginine availability. Increased orotic acid production is reduced or prevented by inclusion of the urea cycle intermediates arginine, citrulline or ornithine. However, growth in the rat is stimulated only when arginine or citrulline are included in the diet. Increased orotic biosynthesis is observed with increasing ammonia concentrations in rat, mouse and human liver and is reduced by in vitro arginine supplementation. The fatty infiltration of the rat fed an arginine-deficient diet is associated with changes in the ratio of purine to pyrimidine bases and is corrected by the dietary addition of adenine. The arginine-deficient rat should serve as a model for examining the dynamic interrelationship of the urea cycle with pyrimidine and purine biosynthesis.

Urea synthesis in the canine kidney.

The ability of the kidney to synthesize urea is of interest and some controversy. We tested the hypothesis that low plasma urea concentrations would stimulate renal urea synthesis, thereby enhancing urinary concentrating ability. We defined a ratio of specific activity of plasma/specific activity of urine (SA ratio) greater than 1.0 as indicative of de novo intrarenal urea synthesis. We studied dogs on 3 dietary regimens: (1) commercial chow ad libitum; (2) restricted rations of the same chow; (3) protein-free diet ad libitum with vitamin/mineral supplement. A significant inverse relationship between plasma urea concentration and SA ratio was seen only in the ration-restricted group, despite the fact that plasma urea concentrations in this group were highest of the three groups. In addition, dogs in this group were more likely to demonstrate renal urea synthesis than dogs in the other groups. Thus, the dog kidney is capable of de novo urea synthesis, but a reduced plasma urea concentration does not necessarily stimulate this metabolic activity. Other factors in addition to the plasma urea concentration appear to be involved in the regulation of renal urea synthesis.

Application of commercial enzymes to measure the activity of the arginine pathway-urea cycle in intact cells.

The activity of the complete arginine pathway-urea cycle was assessed in intact plant cells by employing the commercial enzymes arginase (EC 3.5.3.1) and urease (EC 3.5.1.5) to determine the amount of NaH14CO3 incorporated into [guanido-14C]arginine and/or into [14C]urea during a 3-h labeling period. Recovery of [guanido-14C]arginine was linear from 5 to 1000 nmol/g tissue and averaged 80 +/- 5% (mean +/- SE, N = 3). The procedure is reliable, inexpensive, well suited to the simultaneous analysis of numerous samples, and significantly more sensitive than existing methods. The method is ideally suited for assessing the activity of the complete arginine biosynthetic pathway in intact cells. In addition, the method has the distinct advantage of providing simultaneous measurement of the amount of NaH14CO3 accumulating in arginine relative to the amount accumulating as urea. Evidence is presented demonstrating that both the activity of the arginine pathway and the relative amounts of [guanido-14C]arginine and [14C]urea synthesized from NaH14CO3 were influenced by changes in the level of ornithine, NH+4, or phosphorus available to plant tissues.

Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice.

L-Carnitine protects mice against acute ammonia intoxication. The effect of L-carnitine on in vivo incorporation of [14C] bicarbonate into urea has been investigated in mice given large doses of ammonium acetate. The hepatic content of N-acetylglutamate has been measured. Following ammonia administration the animals given L-carnitine have much higher production of urea than the unprotected mice. The marked protective effect of L-carnitine on ammonium acetate-induced hyperammonemia and on the increase in urea synthesis is not due primarily to activation of N-acetylglutamate synthetase.

Species comparison of the influence of ammonia on orotic acid and urea biosynthesis in liver.

Although orotic aciduria occurs in both male and female rats fed an arginine-deficient diet, only hepatocytes from male rats exhibited an enhanced rate of orotate biosynthesis to increasing ammonium compared to controls. Inhibition of incorporation [14C]NaHCO3 into urea by norvaline in the rat and mouse was accompanied by a 20 and 25% increase in orotate biosynthesis, respectively. Injection of ammonium chloride (2 mmol/kg) or consumption of a diet devoid of arginine resulted in an increased urinary orotate excretion in the rat. Injection of a similar quantity of ammonium chloride in mice did not result in an orotic aciduria. The influence of ammonia and arginine on the biosynthesis of orotic acid and urea in isolated liver slices from various mammals was also examined. In rat, mouse and pig liver, increasing quantities of ammonia stimulated the incorporation of [14C]NaHCO3 into orotic acid. In porcine, bovine and ovine liver the incorporation of [14C]NaHCO3 into orotic acid was low and was minimally effected by supplemental ammonia. Addition of arginine to the incubation medium diminished the incorporation of [14C]NaHCO3 into orotic acid in the liver of all species examined except the cow and sheep. This decrease was accompanied by a stimulation of urea biosynthesis in the rat, but a depression in the pig. Addition of ammonium (5.0 mM) markedly increased the incorporation of [14C]NaHCO3 into urea in all mammalian liver slices examined. These studies show that the liver of various mammals can respond to increasing ammonia by increasing orotic acid biosynthesis. However, species differences in the response to ammonia are evident.

Potentiation by piridoxilate of the synthesis of hippurate from benzoate in isolated rat hepatocytes. An approach to the determination of new pathways of nitrogen excretion in inborn errors of urea synthesis.

The synthesis of hippurate from benzoate as compared to ureagenesis has been investigated in isolated rat hepatocytes. Half-maximal synthesis of hippurate was observed at 0.3 mmol/l benzoate. In the presence of 1 mmol/l benzoate, hippurate synthesis proceeded linearly with time at a rate of 40 +/- 10 mumol X h-1 X g-1 dry weight. This provided less than 10% of nitrogen epuration supported by concomitant urea synthesis (350 +/- 82 mumol X h-1 X g-1 dry weight). The incorporation of benzoate to hippurate was markedly limited by the availability of glycine. Half-maximal hippurate synthesis was observed at 2 mmol/l glycine. In the absence of glycine, piridoxilate, a glyoxylate derivative, markedly potentiated hippurate synthesis. Half-maximal stimulation was observed at 10 mmol/l piridoxilate. In the presence of 10 mmol/l piridoxilate, hippurate synthesis (420 +/- 35 mumol X h-1 X g-1 dry weight) provided more than 50% of nitrogen epuration supported by urea synthesis. It is concluded that supplementation with nitrogen-free analogues of glycine such as piridoxilate are required to potentiate hippurate synthesis in an attempt to replace ureagenesis as an alternative pathway of waste nitrogen excretion in inborn errors of urea synthesis.

Cellular and subcellular organization of pathways of ammonia assimilation and ureide synthesis in nodules of cowpea (Vigna unguiculata L. Walp.).

Fractionation of cell organelles of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp) by discontinuous and continuous sucrose density centrifugation indicated that starch-containing plastids possessed the complete pathway for purine nucleotide synthesis together with significant activities of some other enzymes associated with the provision of substrates in purine synthesis; triosephosphate isomerase (EC 5.3.1.1), NADH-glutamate synthase (EC 2.6.1.53), aspartate aminotransferase (EC 2.6.1.1), phosphoglycerate oxidoreductase (EC 1.1.1.95), and methylene tetrahydrofolate oxidoreductase (EC 1.5.1.5). Enzymes of purine oxidation, xanthine oxidoreductase (EC 1.2.3.2), and urate oxidase (EC 1.7.3.3) were recovered in the soluble fraction; glutamine synthetase (EC 6.3.1.2) occurred in bacteroids and in the cytosol. Intact, infected (bacteroid-containing) and uninfected cells were prepared by enzymatic maceration of the central zone of the nodule and partially separated by centrifugation on discontinuous sucrose gradients. Glutamine synthetase was largely restricted to infected cells whereas plastid enzymes, de novo purine synthesis, and urate oxidase were present in both cell types. Although the levels of all enzymes assayed were higher in infected cells, both cell types possessed the necessary enzyme complement for ureide formation. A model for the cellular and subcellular organization of nitrogen metabolism and the transport of nitrogenous solutes in cowpea nodules is proposed.

The influence of excess lysine on urea cycle operation and pyrimidine biosynthesis.

Rats fed a purified L-amino acid diet with 0.72, 1.50, 2.9 or 4.3% lysine excreted 117, 124, 237 and 628 micrograms/day orotic acid, respectively. Dietary arginine supplementation prevented the orotic aciduria induced by excess dietary lysine. Increased orotic acid excretion was accompanied by a significant depression in urinary urea in rats fed a diet containing 4.3% lysine compared to those fed a diet containing 0.72% lysine. As measured by incorporation of [14C]HCO3, lysine addition to liver slices or isolated hepatocytes resulted in a progressive increase in the rate of orotic acid biosynthesis. The minimum quantity of lysine tested that significantly increased the rate of orotic acid biosynthesis was 0.5 mM or 1 mM for studies with slices and hepatocytes, respectively. Ammonia at concentrations of 0, 0.75, or 5.0 mM NH4Cl linearly increased orotate and urea synthesis. Inhibition of urea biosynthesis resulting from lysine supplementation coincided with an increase in pyrimidine biosynthesis. Addition of 1 mM arginine to the liver incubation media prevented the increased rate of orotic acid biosynthesis caused by lysine. Arginine addition may overcome an approximate 90% competitive inhibition of arginase by excess lysine.

Treatment of inborn errors of urea synthesis: activation of alternative pathways of waste nitrogen synthesis and excretion.

Children with inborn errors of urea synthesis accumulate ammonium and other nitrogenous precursors of urea, leading to episodic coma and a high mortality rate. We used alternative pathways for the excretion of waste nitrogen as substitutes for the defective ureagenic pathways in 26 infants. These pathways involve synthesis and excretion of hippurate after sodium benzoate administration, and of citrulline and argininosuccinate after arginine supplementation. The children were treated for seven to 62 months; 22 survived. The mean plasma level of ammonium ( +/- S.E.) was 36 +/- 2 mumol per liter, and that of benzoate was 1.5 +/- 1.0 mg per deciliter. Alternative pathways accounted for between 28 and 59 per cent of the total "effective" excretion of waste nitrogen. Nineteen infants had normal height, weight, and head circumference, and 13 had normal intellectual development. Activation of alternative pathways of waste nitrogen excretion can prolong survival and improve clinical outcome in children with inborn errors of urea synthesis.

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 mitochondria isolated from livers removed 15 min after injection of amino acids, with N-acetylglutamate at its endogenous levels, rose up to 5-fold with increasing doses up 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 N-acetylglutamate was not attributable to activation by arginine of N-acetylglutamate synthetase. These results indicate that moderate loads of amino acids activate unreagenesis 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.

[Effect of arginine ketoglutarate on the detoxifying capacity of the liver in cirrhosis of the liver (author's transl)]. / Wirkung von Argininketoglutarat auf die Entgiftungsleistung der Leber bei Leberzirrhose

According to the present studies it is shown that arginine ketoglutarate in sufficiently high dosage (3x3 g daily by mouth) produces a significant lowering of plasma ammonia and free serum phenols with a high-protein diet in patients with liver cirrhosis, compared to a previous day without this substance. The ammonia reduction can be explained by a significant increase in urea synthesis, measured by the urea nitrogen content of the 24-hour urine. The simultaneous lowering of pathologically raised serum levels of free phenols can be explained by an improved oxidative decomposition of these substances. No stimulation of insulin secretion worthy of note occurred after oral administration of 9 g arginine ketoglutarate.

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.

Control of arginine utilization in Neurospora.

The response of Neurospora to changes in the availibility of exogenous arginine was investigated. Upon addition of arginine to the growth medium, catabolism is initiated within minutes. This occurs prior to expansion of the arginine pool or augmentation of catabolic enzyme levels. (Basal levels are approximately 25% of those found during growth in arginine-supplemented medium.) Catabolism of arginine is independent of protein synthesis, indicating that the catabolic enzymes are active but that arginine is not available for catabolism unless present in the medium. Upon exhaustion of the supply of exogenous arginine, catabolism ceases abruptly, despite an expanded arginine pool and induced levels of the catabolic enzymes. The arginine pool supports protein synthesis until the cells regain their normal capacity for endogenous arginine synthesis. These observations, combined with the known small level of induction of arginine catabolic enzymes, non-repressibility of most biosynthetic enzymes, and vesicular localization of the bulk of the arginine pool, suggest that compartmentation plays a significant role in controlling arginine metabolism in Neurospora.

Compartmentation and control of arginine metabolism in Neurospora.

The fate of [14-C]arginine derived from the medium or from biosynthesis has been examined in Neurospora growing in arginine-supplemented medium. In both cases the label enters the cytosol, where it is used efficiently for both protein synthesis and catabolism before mixing with the majority of the endogenous [12C]arginine pool. Both metabolic processes appear to use the same cytosolic arginine pool. It is calculated that the nonorganellar cytoplasm contains approximately 20% of the intracellular arginine pool when the cells are growing in arginine-supplemented medium. The results suggest that compartmentation of arginine is a significant factor in controlling arginine metabolism in Neurospora. The significance of these results for studies of amino acid metabolism in other eukaryotic systems is discussed.