Showing papers on "Urea cycle published in 1975"

Hyperornithinemia, hyperammonemia, and homocitrullinuria associated with decreased carbamyl phosphate synthetase I activity.

Abstract: Extract: Six related subjects with severe feeding problems in infancy, hyperammonemia, hyperornithinenia, and homocitrullinuria are reported. The clinical picture includes episodic motor and mental impairment, seizures, intellectual deficits that vary from severe to very mild and, in two cases, a bleeding tendency early in life. The pedigree indicates autosomal recessive inheritance. In a liver biopsy in one patient decreased activity of carbamyl phosphate synthetase I (CPS I, EC. 2.7.2.5) was found. Enzymatic assays on peripheral leukocytes in all six subjects confirmed the liver results. Loading studies with ornithine and citrulline were consistent with a defect early in the urea cycle. Homocitrullinuria appeared to arise from excessive synthesis from lysine, but there was no impairment of the main lysine catabolic pathway. Large and bizarrely shaped hepatic mitochondria with curious periodic (350–400A) membranes were observed ultrastructurally. Speculation: Clinical entities in which liver CPS I deficiency has been reported represent a heterogeneous group. This heterogeneity may arise because the assays currently in use do not differentiate between individual steps in the complex synthetic reaction catalyzed by this enzyme. Associated abnormalities in ornithine transport into the hepatic mitochondria in some may also contribute to the varying expression of this group of disorders. It is conceivable that some unusual periodic structures observed just inside the inner mitochondrial membranes are related to altered ornithine mitochondrial entry. This disorder adds to the evidence from other inborn errors that there are one or more ways in which lysine metabolism and the urea cycle are specifically interrelated, but the details remain obscure.

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

Abstract: 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.

Abnormalities of Carbamyl Phosphate Synthetase and Ornithine Transcarbamylase in Liver of Patients with Reye's Syndrome

Abstract: Urea cycle function was evaluated in liver obtained from six patients with Reye's syndrome and from five control subjects. Reye's syndrome patients demonstrated normal activities for the extramitochondrial portion of the urea cycle, but showed marked abnormalities of the mitochondrial enzymes, i.e., carbamyl phosphate synthetase (CPS) and ornithine transcarbamylase (OTC) (Tables 2,3). CPS activity was reduced to less than 15% of control values in all four patients from whom tissues was obtained during the first 72 hr after the onset of encephalopathy. Two patents from whom tissue was not obtained until after 9 days of symptoms showed no reduction in CPS activity. The OTC activity was also reduced (3-67% of control values) in the four patients from whom tissue was obtained early in the illness. In addition, greater than 60% reduction in Vmax and Km for carbamyl phosphate was noted in all four patients in whom sample size permitted kinetic analysis, including both patients in whom CPS and OTC activity were not markedly reduced. The same kinetic abnormality as well as decreased CPS activity were experimentally produced in normal rate liver incubated in the presence of 1.0 mM 4-pentenoic acid, a short chain fatty acid and known hepatic mitochondrial toxin (Table 4).

Urinary metabolites characteristic of urea-cycle amino acid deficiency.

Abstract: Experiments with 45-75-g male rats gave conclusive evidence that the simultaneous absence from the diet of arginine, ornithine, or citrulline caused an immediate and persistent elevation of orotic and citric acids in the urine. The experiments also demonstrated that a deficiency of no other individual amino acid increased urinary citrate and orotate. Elevated urinary excretion of orotic and citric acid occurred independently of the form of nonessential nitrogen. Replacement of arginine isonitrogenously with ornithine or citrulline prevented the rise in urinary orotic acid, but had different effects on growth, urinary citrate, and urinary urea. These differences were probably due to differential uptake of arginine, ornithine, and citrulline by tissues; In the reported experiments employing L-amino acids as sources of dietary nitrogen, a deficiency of any amino acid indispensable for growth and nitrogen balance or a deficiency of arginine, ornithine, or citrulline retarded growth, increased urinary urea, and decreased urinary ammonia. It is concluded that the severe loss of orotic acid during urea-cycle amino acid deficiency arises from a decreased capacity of the urea cycle to detoxify ammonia, thereby causing increased shunting of intramitochondrial carbamyl phosphate into pyrimidine synthesis. The similarities in metabolism during arginine deficiency and ammonia intoxication are discussed. The evidence shows that urinary orotic acid may be a valuable measure of arginine nutrition in mammals.

Possible interactions between the urea cycle and synthesis of pyrimidines and polyamines in regenerating liver.

Abstract: Ornithine levels rise progressively in the liver of partially hepatectomized rats, probably as a consequence of the increased flow of metabolites through the urea cycle. Ammonia and urea concentrations in the blood and liber of partially hepatectomized animals are not significantly different from those of sham-operated rats. However, in regenerating livers, the ability to remove ammonia from the blood is close to its maximal limit. Ammonia overload leads to the production of large amounts of orotic acid and causes a marked elevation of hepatic ornithine decarboxylase activity. Among the pyrimidine precursors dihydroorotic acid injections increase the activity of the enzyme while orotic acid is without effect. A peak of labeled material that corresponds to dihydroorotic acid was identified by partition chromatography of acid-soluble extracts of livers of partially hepatectomized rats previously given injections of [14-C2 bicarbonate. The labeling of dihydroorotic acid from [14-C] bicarbibate is increased in the liver of rats given injections of ornithine. Despite the difficulties involved in studies of ornithine decarbozylase activity in vivo, our results suggest that mutual interactions between urea, pyrimidine, and polyamine synthesis take place during liver regeneration.

Hyperammonemia due to a urea cycle enzyme deficiency in two dogs.

Abstract: Hyperammonemia in 2 dogs was associated with a deficiency of arginosuccinate synthetase, one of the enzymes of the urea cycle. The usual criteria for evaluation of hepatic function were normal.

Citrullinemia: enzymatic evidence for genetic heterogeneity.

Abstract: Extract: The specific activity of argininosuccinate synthetase (micromoles of 14CO2 per milligram of protein per hour) was 0.00104 and 0.00087 in fibroblasts derived from two patients with citrullinemia, and was undetectable in both fibroblasts and cultured lymphocytes from a third patient. In five obligate heterozygotes the specific activity in fibroblasts was 0.012 0.029 and in nine control subjects was 0.058 ± 0.014 (0.030–0.076). In both control and patient cells, the maximum activity was obtained at pH 8.5 and there was no inhibition of normal argininosuccinate synthetase by any of the mutant cells. Kinetic studies were consistent with decreased binding of citrulline and/or aspartate in fibroblasts from all three patients. The Km for citrulline was 4.2 × 10−3 and >2.0 × 10−2 M for the enzyme from patients 1 and 2, respectively (normal 1.1 × 10−4 M). Corresponding Km values for aspartate were 1.8 × 10−2 and >6.5 × 10−3 M, respectively (normal 3.4 × 10−5 M). Hill constants derived from the citrulline substrate curves from patients 1 and 2 were 1.01 and 1.50 (normal 0.91). Corresponding constants obtained from aspartate binding curves were 1.06 and 1.35, respectively (normal 0.97). The activity of homoargininosuccinate synthetase was undetectable in fibroblasts from all three patients and a control subject. Speculation: Heterogeneity of the primary enzyme defect will become a frequent observation in many inborn errors of metabolism. In disorders of the urea cycle, alternative and as yet unknown pathways of urea formation may be discovered.

Reye syndrome. A correlated electron-microscopic, viral, and biochemical observation.

Abstract: Liver biopsy specimens of two patients with Reye syndrome were examined for ultrastructural features, viral isolation, and urea-cycle enzyme activity. Concurrent presence of herpes-like virus and myxovirus/paramyxovirus was demonstrated by electron microscopy, and viral infections were confirmed by isolation or serologic tests. A concomitant hepatic ornithine transcarbamoylase deficiency was also noted. The pathogenesis in these instances seems to consist of an initial synergistic insult on the liver by mixed types of viruses and subsequent breakdown of urea cycle, Krebs cycle, and possible other hepatic functions. An exodus of glycogen granules into the hepatic spaces of Disse and sinusoids suggests that the viruses have injured the plasma membranes as well as the mitochondria of hepatocytes. Since Kapila et al reported similar disorders five years before Reye et al, the name of Kapila-Reye disease is suggested.

Diamino acid transport into granulocytes and liver slices of patients with lysinuric protein intolerance.

Abstract: Extract: The amounts of 14C-labeled lysine, arginine, and ornithine taken up by isolated granulocytes of 11 patients with lysinuric protein intolerance (LPI) and of 16 control subjects were measured. Transport against a concentration gradient was evident in all. The initial 5-min uptake was measured in a series of substrate concentrations varying from 0.025—4.0 mM. One transport system was seemingly present for all three amino acids. The rates of transport into the granulocytes in the patients and control subjects were comparable. The Vmax, in millimoles × kilogram of cell water−1 × 5 min−1, ranged only from 0.30—0.38 in the patients and from 0.32—0.34 in the control subjects. The Km values were 0.06 aqd 0.06 mM for lysine, 0.05 and 0.06 for arginine, and 0.10 and 0.09 mM for ornithine in the patients and control subjects, respectively. The transport of the nonmetabolizable diamino acid analog, homoarginine, into liver slices of three patients and five control subjects was also studied. There was no clear difference in the time course of uptake at 0.05 mM homoarginine concentration in the medium. In kinetic studies of two patients and three control subjects, at least two transport systems were evident. The “large capacity, low affinity” system had Vmax of 0.43 and 3.08 mmol × kg cell water−1 × 15 min−1 and Km of 0.18 and 2.49 mM in the patients and control subjects, respectively. The “small capacity, high affinity” system had Vmax of 0.07 and 0.14 mmol × kg cell water−1 × 15 min−1 and Km of 0.03 and 0.05 mM, respectively. The Vmax of the large capacity, low affinity system in the patients was only [fraction 1 over 7] of that in the control subjects, showing clearly impaired diamino acid uptake by the liver cells in LPI. Speculation: In LPI diamino acids show depressed plasma concentrations, probably because of their impaired intestinal absorption and increased renal loss. If my findings on the kinetically abnormal diamino acid transport in liver slices of the patients are valid in vivo, the intestinal, renal, and hepatic defects must result in ornithine deficiency in the liver cells. As ornithine is the molecule on which urea is formed in the urea cycle, this deficiency will incapacitate the urea cycle and result in hyperammonemia. This mechanism is consistent with the basic chemical characteristics of LPI, the hyperdibasic aminoaciduria, and hyperammonemia after amino nitrogen loading. The defect of hepatic diamino acid transport is suggested to distinguish LPI from other diseases with low plasma diamino acid concentrations, the classic cystinuria, and the nonhyperammonemic hyperdibasic aminoacidurias.

Leukocyte urea cycle enzymes in hyperammonemia.

Abstract: All enzymes of the urea cycle are demonstrable in circulating leukocytes. They show the same relative activities as those in liver except for argininosuccinate synthetase + lyase (combined) which seems to be disproportionately active. To see whether leukocytes reflect liver activity, blood from patients with three hepatic urea cycle disorders was tested. In each case, the leukocytes showed the same enzyme deficiency as was apparent from a liver biopsy (Table 4). Leukocyte assays appear to be reliable indicators of the enzyme lesions in inherited urea cycle enzyme defects and therefore may obviate the need for liver biopsy.

Hepatic albumin and urea synthesis: The mathematical modelling of the dynamics of [14C]carbonate-derived guanidine-labelled arginine in the isolated perfused rat liver.

Abstract: A mathematical model was constructed to define the dynamics of incorporation of radioactivity into urea carbon and the guanidine carbon of arginine in plasma albumin after the rapid intraportal-venous administration of Na214CO3 in the isolated perfused rat liver. 2. The model was formulated in terms of compartmental analysis and additional experiments were designed to provide further information on subsystem dynamics and to discriminate between alternative model structures. 3. Evidence for the rapid-time-constant of labelling of intracellular arginine was provided by precursor-product analysis of precursor [14C]carboante and product [14C]urea in the perfusate. 4. Compartmental analysis of the dynamics of newly synthesized urea was based on the fate of exogenous [13C]urea, endogenous [14C]urea and the accumulation of [12C]urea in perfusate water, confirming the early completion of urea carbon labelling, the absence of continuing synthesis of labelled urea, and the presence of a small intrahepatic urea-delay pool. 5. Analysis of the perfusate dynamics of endogenously synthesized and exogenously administered [6-14C]arginine indicated that although the capacity for extrahepatic formation of [14C]-urea exists, little or no arginine formed within the intrahepatic urea cycle was transported out of the liver. However, the presence of a rapidly turning-over intrahepatic arginine pool was confirmed. 6. On the basis of these subsystem analyses it was possible to offer feasible estimations for the parameters of the mathematical model. However, it was not possible to stimulate the form and magnitude of the dynamics of newly synthesized labelled urea and albumin which were simultaneously observed after administration of [14C]carbonate on the basis of a preliminary model which postulated that both products were derived from a single hepatic pool of [16-14C]arginine. On the other hand these observed dynamics could be satisfied to a two-compartment arginine model, which also provided an explanation for discrepancies observed between albumin synthesis measured radioisotopically and immunologically. This was based on a relative overestimation of [14C]urea specific radioactivity resulting from the rapid dynamics of [14C]carbonate and the [14C]urea subsystem relative to the labelled albumin subsystem. The effects of arginine compartmentalization could be minimized in the model by minor slowing of the rate of [14C]carbonate turnover or by constant infusion of [14C]carbonate, both of which permitted valid determination of albumin-synthesis rates.

Studies on the Growth Effects of the Canaline-Urea Cycle Amino Acids with Lemna minor L.

Abstract: The aquatic microphyte, Lemna minor L., was utilized to assess the relative toxicity and general growth effects of canavanine, canaline, ureidohomoserine (UHS), and canavaninosuccinate (CSA). These amino acids are constituents of the canaline-urea cycle and structural analogues of the ornithine-urea cycle amino acids. Comparative growth studies with L. minor revealed that the canaline-urea cycle amino acids are potent antimetabolites. With the exception of CSA, they are extremely toxic at a concentration of 5 μm. Over a concentration range of 1 to 4 μm, canavanine is the most growth-inhibiting of the canaline-urea cycle amino acids. At or above 5 μm, canavanine and canaline possess comparable toxicity. UHS is less growth-inhibiting than canavanine or canaline, and CSA is the least toxic of the canaline-urea cycle intermediates.

Absence of ureogenic pathways in liver of the hagfish Bdellostoma cirrhatum

Abstract: 1. 1. Of the five ornithine-urea cycle enzymes, only arginase was found in liver of the hagfish Bdellostoma cirrhatum. None of the purine pathway enzymes, uricase, allantoinase or allantoicase was detected. 2. 2. Only low or trace amounts of urea were detected in blood serum of fasted animals and urea excretion was very low. In contrast, the rate of ammonia excretion was high. 3. 3. However, as confirmation of an earlier finding with Myxine, Bdellostoma appears to have an active secretory system for urea, being able to concentrate injected urea in urine of the mesonephric duct.

Studies on nitrogen catabolism in Panagrellus redivivus, Goodey, 1945 (Nematoda: Cephalobidae).

Abstract: 1. 1. The activities of ornithine-urea cycle, amino acid and purine deaminating and purine degradating enzymes were measured in whole nematode homogenates, and radiocarbon incorporation from urea-cycle intermediates into urea determined. 2. 2. Evidence for a functional urea cycle of low activity was obtained; purine degradation was shown to be insignificant. 3. 3. The significance of these studies is discussed in relation to the nitrogenous end-products excreted by Panagrellus redivivus.

Studies on urea cycle enzymes in rat liver during acute uraemia.

Abstract: . Activities of urea cycle enzymes were measured in the liver of starved rats 12 and 48 h after bilateral nephrectomy. Control experiments (sham-operated, starved rats) revealed that the activities of only two enzymes of the cycle are altered in the uraemic state: argininosuccinic acid synthetase (EC 6. 3. 4. 5.), which is considered to be rate limiting for urea production and carbamyl phosphate synthetase (EC 2. 7. 2. 5.). Alterations in ornithine concentration of the liver, a possible cause of an increased urea production rate, could not be detected previously (21). Our present results do not support the concept that a decrease of the activity of ornithine-6-amino transferase (EC 2. 6. 1. 13), leading to an increase in the ornithine content of the liver is responsible for the accelerated urea production rate in the liver of acute uraemic rats.

Ornithine transcarbamylase deficiency in reye's syndrome

Abstract: Hyperammonemia is observed regulary in patients with Reye's Syndrome /RS/, and may be a crucial determinant of the encephalopathy in this often lethal condition. Since ammonia detoxification depends on an intact urea cycle and since patients with inherited defects of a single urea cycle enzyme also demonstrate hyperammonemia, we sought to define the mechanism of hyperammonemia in RS by examining activities of the first two urea cycle enzymes, carbamyl phosphate synthetase I /CPS I/ and ornithine transcarbamylase /OTC/ in liver homogenates from 4 RS patients. OTC activity was reduced to 10–15 % of control mean in 2 autopsy specimens and to 40–42 % of control in 2 biopsy samples. In 2 livers Km's for the substrates, ornithine /Orn/ and carbamyl phosphate /Cp/, were normal and the Vmax's were much reduced; in a third, the Km's for Orn and Cp were increased 10 fold and 5 fold, respectively, but the Vmax's were normal. Significantly CPS I activity was normal in all 4 livers and activity of a third urea cycle enzyme, arginase, was normal in the one RS liver assayed. These data suggest that specific deficiency of OTC activity may be responsible for the hyperammonemia in RS and that more then one kind of kinetic disturbance may underlie the OTC impairment.

Quantitative aspect of the endogenous urea cycle in goats.

Abstract: The turnover of plasma urea was studied in goats fed rations with differentprotein contents by using "C- and "N-urea as tracers. The biological half-life time ofurea determined with "N-urea was always longer than that determined with "C-urea, reflecting the difference in the metabolic pathway between the urea hydrolysates, ammoniaand carbon dioxide. It was roughly estimated from the turnover studies that more thana half the quantity of urea synthesized might be transferred to the alimentary canal, including the rumen, where it underwent dissociation. With a decrease in the proteincontent of the ration, the percentage of transfer and dissociation increased. The "C-ureadischarged into the urine was less than 57% of the administered dose. It decreased to3.9% in goats fed rations with a low protein content.The rate of urea turnover determined with "N-urea was nearly consistent with the rateof urea discharge into the urine in goats fed higher protein rations. In goats fed lowerprotein rations, on the other hand, the rate of urea discharge decreased and its consistencywith the rate of turnover determined with "N-urea was upset. This was caused byassimilation of urea-nitrogen in rumen microorganisms, as was convinced by studying theincorporation of urea-"N into the fraction of rumen microorganisms. It was assumedthat in goats fed lower protein rations the bulk of urea synthesized might have enteredthe endogenous recycling system within the body and assimilated by rumen micro-organusms.

The effect of electrical stimulation of the hypothalamus and other brain areas on urea cycle enzymatic activities in the liver in rabbits--experimental studies on brain and liver interrelationship--.

Abstract: Changes in urea cycle enzymatic activities in the liver induced by intracerebral electrical stimulation in rabbits with chronically implanted electrodes in the hypothalamus, the thalamus, and the limbic area were studied in comparison with these activities in non-stimulated controls. In stimulating the ventromedial nucleus of the hypothalamus, the two-hour sessions were followed by significantly reduced activity in arginase and the arginine synthetase system in the liver, but the six-hour stimulation sessions were followed by normal range activity in these enzymes. In stimulating the lateral hypothalamic area, the two-hour sessions were followed by only slightly reduced activity in the arginine synthetase system in the liver. Stimulation of the center median nucleus of the thalamus, the medial area of the amygdala and the dorsal hippocampus produced no significant changes in urea cycle enzymatic activities in the liver. The possibility that ammonia metabolism in the liver may be modified through some functional linkage between the brain and the liver was suggested.