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Analysis of regulatory factors for urea synthesis by isolated perfused rat liver. I. Urea synthesis with ammonia and glutamine as nitrogen sources.

Urea synthesis was studied using the isolated liver perfusion with ammonium cholride and glutamine as nitrogen sources. The rate of urea formation increases with ammonium cholorde concentration up to 5mM, and the rate remained constant in the range between 5 and 20mM of ammonium chloride as the substrate. The concentration of ammonia in the medium to support the half-maximum velocity of urea formation was 0.7mM. The rate of urea formation was stimulated by the addition of 2.5mM ornithine, and the greater part of the ornithine which was taken up into the liver was accumulated as citrulline in the presence of ammonia. A considerable accelerating effect of N-acetylglutamate on the synthetic rate was observed, but a rather high concentration of N-acetylglutamate was required in order to obtain the maximum effect possibly, because its permeability into liver cells may be limited. A marked additive effect on the rate of urea formation was observed with the combined addition of ornithine and N-acetylglutamate. The metabolic conversion of glutamine nitrogen to urea in the perfused rat liver and the effect of several compounds which stimulated urea synthesis with ammonia were further examined. The process of conversion of glutamine nitrogen to urea might be composed of the following three steps. In the first lag phase, a small amount of glutamine was removed from the medium. In the second stage, the glutamine level decreased rapidly and ammonia was accumulated in the perfusate. The third stage was a period in which glutamine concentration remained at a constant low level, and the accumulated ammonia was rapidly conversed to urea. The rate of urea formation in this third stage was found to be much higher than that with ammonia as the substrate. The maximum rate of glutamine removal was obtained at pH 7.7 of the perfusate and at a concentration of 10mM glutamine. Urea formation with glutamine was also stimulated by the addition of ornithine, malate, or N-acetylglutamate, which had accelerating effects on the urea synthesis with ammonia. This stimulation was due to an effective conversion of ammonia to urea, but no change in the rate of removal glutamine was obtained.

Ammonium Chloride

The effects of sodium chloride, urea and mannitol on the permeability in vitro fo rat papillary collecting ducts to THO, 14C-urea and 22Na.

1. The diffusional permeabilities of collecting duct membranes to THO, 14C-urea and 22Na+ have been measured at different concentrations of urea, NaCl and mannitol. 2. In the absence of urea in perfusate and bath or in its presence in low concentrations, the diffusional permeability to urea was 2.0 (s.e.m. = 0.15, n = 58) micrometer s-1, compared with 0.87 (s.e.m. = 0.06, n = 29) microgram s-1 when 200 mmol/l urea was present. The permeability of the collecting ducts to THO or Na+ was not affected by the different urea concentrations. 3. High concentrations of sodium chloride increased the diffusional permeability of collecting ducts to water and urea but did not affect the diffusional permeability of the collecting duct to Na+. 4. Mannitol had effects similar to those of sodium chloride. 5. In all media tested there was an increase in THO and urea permeability when supramaximal amounts of antidiuretic hormone were added. The increases in the various media for each substance were similar, despite widely different starting permeabilities. 6. The results suggest that solutes and water move across collecting duct epithelium by several pathways that respond differently to various stimuli.

Animals

Urea amidolyase of Candida utilis. Characterization of the urea cleavage reactions.

Evidence is presented that the enzymes catalyzing the three reactions involved in urea cleavage in Candida utilis, biotin carboxylation, urea carboxylation, and allophanate hydrolysis occur as a complex of enzymes. The allophanate-hydrolyzing activity could not be separated from the urea-cleaving activity using common methods of protein purification. Further, urea cleavage and allophanate hydrolysis activities are induced coordinately in cells grown on various nitrogen sources. The reactions involved in urea cleavage can be distinguished from one another on the basis of their sensitivities to (a) heat, (b) pH, and (c) chemical inhibitors. Evidence is presented for the product of the first reaction in urea cleavage, biotin carboxylation. Production of carboxylated enzyme is ATP dependent and avidin sensitive. Carboxylated enzyme is not observed in the presence of 1 mM urea.

Acetamides

[Isobutylidene-di-urea as a new NPN source for ruminants. 2. Metabolism of 14C-15N-isobutylidene-di-urea in sheep].

2 male sheep (weighing 45 kg and 44 kg) were fitted with a ruminal fistula and a jugular vein catheter and received isobutylidendi-urea for a 42-day period of adjustment. The diet contained 25% starch, 23.8% glucose, 29.0% cellulose, 10.0% straw, 1.7% sunflower seed oil, 4.3% isobutylidendi-urea, 5.6% minerals and vitamins. Each animal received 60 g of isobutylidendi-urea in daily amounts of 1.4 kg of the ration-4.4% of the total dietary N came from the straw. At the begin of the trial each sheep received 30 g of 14C15N isobutylidendi-urea (C1-siobutyl labelling) administered as a suspension. The animals were then placedin respiration cages. The peak of specific 14C activity in the expired air (including ruminal gas) was observed 2 hrs after the beginning of the trial. 18--30 hrs after the beginning of the trial the highest level of 15N incorporation into the TCE (trichloroacetic acid) soluble fraction of the ruminal fluid was noted resulting from the reflow of urea via the rumeno-hepatic circulatory system in the rumen. A high concentration of 15N was shown to be present, for prolonged period, in the TCE soluble fraction of the ruminal fluid (up to the 30 hr of experiment). The 15N concentration in the blood plasma (TCE soluble portion) was found to increase reaching a peak value 23 hrs after administration of the isotope. The highest level of 14C activity in this fraction appeared 1 hr after isotope administration. The 15N incorporation into the protein fraction of blood plasma reached a constant high level between the 29th and 47th hr of experiment. The highest 15N concentrations in urine were noted after 1 day. 3.5% of the administered dose of 14C activity and 23% of the supplied amount of N were excreted in the urine. 20% of the total amount of 15N excreted in the urine could be detected as 14C isobutyl residues. An excess of between 0.05 and 0.17 atom% of the isotopes were found in muscular tissue and in different organs of the sheep when these were slaughtered on the 7th day of experiment (liver: 0.17%, kidneys: 0.14%, muscle: 0.05%, heart: 0.08%). The results obtained in the present trial clearly indicate that ruminants are able to utilize nitrogen from isobutyldi-urea.

Animal Feed

Urea biosynthesis I. The urea cycle and relationships to the citric acid cycle.

The urea cycle consist of five enzymatically controlled steps that are catalyzed by carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase, and arginase, respectively. The complete cycle is present in physiological meaningful levels in the liver of terrestrial vertebrates, and in man represents the sole mechanism for ammonia disposal. The formation of carbamyl phosphate and the synthesis of argininosuccinate are potential limiting steps in urea biosynthesis but substrate and not enzymes levels are rate-limiting under physiological conditions. In the adult, urea cycle enzymes change as a unit, and are largely influenced by dietary protein content. The urea cycle is closely linked to the citric acid cycle deriving one of its nitrogens through transamination of oxalacetate to form asparate and returns fumarate to that cycle. The biosynthesis of urea demands the expenditure of energy but less than 20% of the energy derived from metabolism of gluconeogenic amino acids is required for ureogenesis. Embryological development of the urea cycle in the tadpole and in mammalian fetal liver therefore permits use of amino acids as new sources of energy to meet oxidative demands for continuing growth.

Ammonia

Studies of renal urea cycle enzymes. I. Renal concentrating ability and urea cycle enzymes in the rat during protein deprivation.

Activities of renal urea enzymes were studied in normally fed (21% dietary protein) rats and rats deprived of protein (6% dietary protein) for 3 weeks. Protein deprivation resulted in growth retardation and defective urine concentrating ability. Compared to rats on an optimal diet containing 21% of protein, the protein starved animals had decreased concentrations of protein and urea in serum, reduced urinary excretion of urea and decreased levels of all five urea cycle enzyme activities in the liver. In the kidney, however, protein malnutrition resulted in a significant increase in arginase specific activity from 11.5 +/- 1.1 to 16.3 +/- 1.5 (M +/- SD) whereas the other urea cycle enzymes remained unchanged. It is postulated that this increase in renal arginase might be an early compensatory mechanism to preserve a net synthesis of urea in a situation involving arginine deficiency, thereby preserving an intact hypertonic gradient in the renal medulla.

Animals

[Contribution to the problem of microbially induced urea transformation in soil. I. On the ability of urea utilization by soil micro-organisms (author's transl)].

The present publication is the first of a series on the enzymatic urea tranformation in soil. With about 2,000 pure cultures of micro-organisms it was possible to prove the very good urea utilization by the soil micro-organisms (bacteria, actinomycetes, and fungi). Above all the fungi showed an excellent utilization of urea, while bacteria and actinomycetes were somewhat poorer. Contrary to this is the urease activity of these organisms, and that is the reason why fungi in soil may be regarded as short-time accumulators for urea nitrogen and must not be suppressed by inhibitors.

Actinomycetales

The mechanism of passage of endogenous urea through the rumen wall and the role of ureolytic epithelial bacteria in the urea flux.

1. The rumen urea concentration in gnotobiotic lambs lacking ureolytic bacteria was equal to that of blood. 2. Bacterial urease (EC 3.5.1.5) activity in sheep fed by intraruminal and intra-abomasal infusion was inversely related to rumen ammonia concentration. 3. A model is proposed for the facilitation and control of urea flux by wall-found ureolytic bacteria.

Ammonia

Determination of ammonia and urea in urine and of urea in blood by use of an ammonia-selective electrode.

I propose a single, quick method for measuring ammonia in urine and urea in plasma and urine. An ammonia-selective electrodie is used, set up on a microcell, which allows use of small sample volumes. Ammonia is measured directly after partial conversion of ammonium ions to NH3. Urea is measured after its hydrolysis by urease. With urine, the two procedures can be carried out successively in the same cell and on the same sample without changing the procedural conditions. Linear electrode response and accuracy have been checked for concentrations in the expected (normal) range.

Ammonia

Investigations of the antiviral activity of some nitroso-urea derivatives. Inhibitory action of the IOB-252 nitroso-urea derivative on vaccinia virus incell cultures.

The nitroso-urea derivative IOB-252 was administrered in monkey kidney cell cultures in a concentration of 40 mug/ml 24 hours before inoculation of vaccinia virus and maintained afterwards in a concentration of 25 mug/ml. The drug inhibited vaccinia virus multiplication, hemagglutinin synthesis and late cytopathic effect, but did not prevent early cytopathic lesions. IOB-252 inhibited the synthesis of interferon initiated by a viral inductor and blocked the antiviral effect of an exogeneous interferon. The mechanism of action of the drug is discussed.

Animals