Effects of ornithine or arginine administration on serum amino acid levels

In healthy humans after overnight fasting, an oral administration of ornithine induced a new steady state: an accumulation of serum alanine and proline, a decrease in serum valine concentration, transient reductions in serum urea and urinary urea contents, and then an increased urea excretion. On the other hand, an oral administration of arginine resulted in an anabolic state: decreases in serum leucine and isoleucine concentrations, reductions in serum glucose and free fatty acid contents and a rapid increase in serum insulin level. It was assumed that the effect of ornithine administration may be exerted through an activation of hepatic System A transport and that of arginine is an insulin-mediated action.     

Accumulation of large neutral amino acids in the brain of sparse-fur mice at hyperammonemic state

Sparse-fur mice which are deficient in ornithine transcarbamylase, the second-step enzyme in the urea cycle, were examined for hyperammonemia and its relationship with encephalopathy. We compared amino acid concentrations in the serum and brain of spf mice with those of control mice. Unlike hepatic encephalopathy we could not find marked amino acid changes in the serum of spf mice besides low levels of citrulline and arginine. But in the brain of spf mice, glutamine was increased strikingly during hyperammonemia, and a concomitant accumulation of large neutral amino acids such as tyrosine, phenylalanine, methionine, and histidine was observed. The accumulation of these large neutral amino acids in the brain was not influenced by 24-hr fasting which caused increases in branched chain amino acids in the serum. From these results, we conclude that the accumulation of the large neutral amino acid in the brain of hyperammonemic state is caused by uptake of ammonia in the brain and the subsequent accumulation of glutamine, but is not influenced by a decreased ratio of branched chain amino acids to aromatic amino acids in the serum.     

Nickel deficiency disrupts metabolism of ureides, amino acids, and organic acids of young pecan foliage

The existence of nickel (Ni) deficiency is becoming increasingly apparent in crops, especially for ureide-transporting woody perennials, but its physiological role is poorly understood. We evaluated the concentrations of ureides, amino acids, and organic acids in photosynthetic foliar tissue from Ni-sufficient (Ni-S) versus Ni-deficient (Ni-D) pecan (Carya illinoinensis [Wangenh.] K. Koch). Foliage of Ni-D pecan seedlings exhibited metabolic disruption of nitrogen metabolism via ureide catabolism, amino acid metabolism, and ornithine cycle intermediates. Disruption of ureide catabolism in Ni-D foliage resulted in accumulation of xanthine, allantoic acid, ureidoglycolate, and citrulline, but total ureides, urea concentration, and urease activity were reduced. Disruption of amino acid metabolism in Ni-D foliage resulted in accumulation of glycine, valine, isoleucine, tyrosine, tryptophan, arginine, and total free amino acids, and lower concentrations of histidine and glutamic acid. Ni deficiency also disrupted the citric acid cycle, the second stage of respiration, where Ni-D foliage contained very low levels of citrate compared to Ni-S foliage. Disruption of carbon metabolism was also via accumulation of lactic and oxalic acids. The results indicate that mouse-ear, a key morphological symptom, is likely linked to the toxic accumulation of oxalic and lactic acids in the rapidly growing tips and margins of leaflets. Our results support the role of Ni as an essential plant nutrient element. The magnitude of metabolic disruption exhibited in Ni-D pecan is evidence of the existence of unidentified physiological roles for Ni in pecan.     

Accumulation of amino acids by the perfused rat liver in the presence of ethanol

1. The rate of gluconeogenesis from alanine in the perfused rat liver is affected by the presence of other metabolizable substances, especially fatty acids, ornithine and ethanol. Gluconeogenesis is accelerated by oleate and by ornithine. When both oleate and ornithine were present the acceleration was greater than expected on the basis of mere additive effects. 2. Much NH(3) and some urea were formed from alanine when no ornithine was added. With ornithine almost all the nitrogen released from alanine appeared as urea. 3. Lactate was a major product of alanine metabolism. Addition of oleate, and especially of oleate plus ornithine, decreased lactate formation. 4. Ethanol had no major effect on gluconeogenesis from alanine when this was the sole added precursor. Gluconeogenesis was strongly inhibited (87%) when oleate was also added, but ethanol greatly accelerated gluconeogenesis when ornithine was added together with alanine. 5. In the absence of ethanol the alanine carbon and alanine nitrogen removed were essentially recovered in the form of glucose, lactate, pyruvate, NH(3) and urea. 6. In the presence of ethanol the balance of both alanine carbon and alanine nitrogen showed substantial deficits. These deficits were largely accounted for by the formation of aspartate and glutamine, the formation of which was increased two- to three-fold. 7. When alanine was replaced by lactate plus NH(4)Cl, ethanol also caused a major accumulation of amino acids, especially of aspartate and alanine. 8. Earlier apparently discrepant results on the effects of ethanol on gluconeogenesis from alanine are explained by the fact that under well defined conditions ethanol can inhibit, or accelerate, or be without major effect on the rate of gluconeogenesis. 9. It is pointed out that in the synthesis of urea through the ornithine cycle half of the nitrogen must be supplied in the form of asparate and half in the form of carbamoyl phosphate. The accumulation of aspartate and other amino acids suggests that ethanol interferes with the control mechanisms which regulate the stoicheiometric formation of aspartate and carbamoyl phosphate.     

Uptake and excretion of amino acids and utilization of glucose by Schistosoma japonicum eggs

The utilization of amino acids and glucose in the external nutrients and the excretion of nitrogenous compounds by Schistosoma japonicum eggs were investigated with the eggs cultured in a chemically defined medium (MEMSE-J). Of the 15 amino acids in MEMSE-J, arginine and glutamine markedly decreased in concentration during cultivation of S. japonicum eggs. The nitrogenous excretory products of developing eggs were demonstrated to be at least four amino acids (alanine, proline, glutamic acid and ornithine), urea and ammonia. Glucose was consumed at an estimated rate of 32 ng/living egg/day during the period of egg growth and differentiation. When 14C-labelled glucose was included in the culture medium, the radioactivity was incorporated into three amino acids (alanine, proline and glutamic acid), which were excreted by S. Japonicum eggs. The results were discussed with reference to the possible role in stimulating fibrosis in the granuloma of schistosomiasis.     

Inhibition of arginine synthesis by urea: A mechanism for arginine deficiency in renal failure which leads to increased hydroxyl radical generation

We have reported that (1) the synthesis of GSA, a uremic toxin, increases depending on the urea concentration and (2) GSA is formed from argininosuccinic acid (ASA) and the hydroxyl radical or SIN-1 which generates superoxide and NO simultaneously. However, an excess of NO, which also serves as a scavenger of the hydroxyl radical, inhibited GSA synthesis. We also reported that arginine, citrulline or ammonia plus ornithine, all of which increase arginine, inhibit GSA synthesis even in the presence of urea. To elucidate the mechanism for increased GSA synthesis by urea, we investigated the effect of urea on ASA and arginine, the immediate precursor of NO.Isolated rat hepatocytes were incubated in 6 ml of Krebs-Henseleit bicarbonate buffer containing 3% bovine serum albumin, 10 mM sodium lactate, 10 mM ammonium chloride and with or without 36 mM of urea and 0.5 or 5 mM ornithine at 37°C for 20 min. In vivo experiments, 4 ml/100 g body weight of 1.7 M urea or 1.7 M NaCl were injected intra-peritoneally into 5 male Wistar rats. Two hours after the intra-peritoneal injection of urea or 1.7 M NaCl, blood, liver and kidney were obtained by the freeze cramp method and amino acids were determined by an amino acid analyzer (JEOL:JCL-300).ASA in isolated hepatocytes was not detected with or without 36 mM (200 mgN/dl) urea, but the arginine level decreased from 36 to 33 nmol/g wet cells with urea. Ornithine which inhibits GSA synthesis, increased ASA markedly in a dose dependent manner and increased arginine. At 2 h after the urea injection the rat serum arginine level decreased by 42% (n = 5), and ornithine and citrulline levels increased significantly. Urea injection increased the ASA level in liver from 36–51 nmol/g liver but this was not statistically significant.We propose that urea inhibits arginine synthesis in hepatocytes, where the arginine level is extremely low to begin with, which decreases NO production which, in turn, increases hydroxyl radical generation from superoxide and NO. This may, also, be an explanation for the reported increase in oxygen stress in renal failure.     

Regulation of sinefungin biosynthesis by the wild-type strain and mutants of Streptomyces incarnatus.

Sinefungin, an antifungal and antiparasitic antibiotic, is produced efficiently from ammonium citrate by prototrophic strains of Streptomyces incarnatus. The regulation of the biosynthesis of this nucleoside, composed of adenosine and ornithine, was studied by using auxotrophic mutants and a resting-cell system. Mutants blocked in arginine synthesis were not able to produce sinefungin. A uridine-negative mutant produced sinefungin in the presence of ATP, but this production was strongly inhibited when amino acids of the urea cycle were added. The same mutant produced sinefungin from aspartic acid, and this production was enhanced by ornithine. Our results show that the ornithine part of the molecule originates from arginine, liberated by either anabolic or catabolic processes.     

Regulation of sinefungin biosynthesis by the wild-type strain and mutants of Streptomyces incarnatus

Sinefungin, an antifungal and antiparasitic antibiotic, is produced efficiently from ammonium citrate by prototrophic strains of Streptomyces incarnatus. The regulation of the biosynthesis of this nucleoside, composed of adenosine and ornithine, was studied by using auxotrophic mutants and a resting-cell system. Mutants blocked in arginine synthesis were not able to produce sinefungin. A uridine-negative mutant produced sinefungin in the presence of ATP, but this production was strongly inhibited when amino acids of the urea cycle were added. The same mutant produced sinefungin from aspartic acid, and this production was enhanced by ornithine. Our results show that the ornithine part of the molecule originates from arginine, liberated by either anabolic or catabolic processes.     

Roles of arginine and canavanine in the synthesis and repression of ornithine transcarbamylase by Escherichia coli

Conditions were found under which the processes of repression and derepression of ornithine transcarbamylase were separated from the process of enzyme synthesis. After 10 min of arginine deprivation followed by the addition of 2 to 200 mug of l-arginine per ml, a number of strains of Escherichia coli exhibited a significant burst of ornithine transcarbamylase synthesis which lasted 3 to 4 min before the onset of repression. The rapid increase of enzyme activity was shown to require protein synthesis, and was not due to a slow uptake of arginine or induction of an arginine-inducible ornithine transcarbamylase. The capacity of E. coli to synthesize the burst of ornithine transcarbamylase reached a maximum after 10 min of arginine deprivation and then remained constant. The observed increase in enzyme synthesis may reflect the level of unstable messenger ribonucleic acid (RNA) for ornithine transcarbamylase present in the cell at the time protein synthesis was reinitiated. After the addition of arginine in the absence of protein synthesis, the burst of ornithine transcarbamylase decayed with a half-life of about 3 min. The data implied that arginine prevents synthesis of new messenger RNA that can translate this enzyme. Repression of ornithine transcarbamylase by l-canavanine (100 to 200 mug/ml) was observed, and no active enzyme was formed in the presence of this analogue. The action of canavanine as a repressor was distinguished from the inhibitory effect of this compound on protein synthesis.     

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

Basic amino acids and inorganic polyphosphates in Neurospora crassa: independent regulation of vacuolar pools

At least 78%, and perhaps all, of inorganic polyphosphate is shown to be contained within the vesicles (vacuoles) of Neurospora crassa, where over 97% of the soluble arginine, lysine, and ornithine pools are known to accumulate. Furthermore, synthetic polyphosphate can concentrate arginine up to 400-fold from dilute (0.01 mM) solutions in equilibrium dialysis. For these reasons and because the molar ratio of basic amino acids and polyphosphate phosphorus is approximately 1, we tested the hypothesis that there was an obligate physiological relationship between them. Experiments in which nitrogen starvation and arginine excess were imposed upon cells showed that polyphosphate content was insensitive to changes in the basic amino acid content. Experiments involving phosphate starvation and restoration showed that basic amino acid content was almost wholly independent of polyphosphate pools. Moreover, the normal high degree of compartmentation of arginine in vesicles was maintained despite polyphosphate depletion, and arginine was still exchanged across the vesicular membrane. We conclude that N. crassa, like yeasts, can regulate polyphosphates and basic amino acids independently, and that the accumulation of basic amino acids in vesicles may depend upon an energy-requiring mechanism in addition to the demonstrated charge interaction with polyphosphate.     

Effect of hyperammonemia on brain amino acids in young and adult ferrets

Summary Effects of arginine deficiency and hyperammonemia on the brain concentrations of amino acids and urea cycle enzyme activities in young and adult ferrets were investigated. Only young ferrets developed hyperammonemia and encephalopathy immediately after consuming the arginine-free diet. Brain ornithine and citrulline concentrations in young ferrets fed arginine containing diet were significantly lower than those in adult ferrets. Compared to rats and other animals, young and adult ferrets had lower concentrations of brain glutamic acid and glutamine. Unlike in other species, brain glutamine was not elevated in young, hyperammonemic ferrets. Brain arginase and glutamate dehydrogenase activities were significantly increased in young ferrets fed arginine-free diet. Young ferrets provide a useful animal model for investigating the neurotoxicity of acute hyperammonemia.Abbreviations ACD Arginine-containing diet - AFD Arginine-free diet This work was presented, in part, at the annual meeting of the Midwest Society for Pediatric Research, Chicago, IL, 1991.     

Comparison of the inhibitory effects of diverse amino acids and amino acid analogs on 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity in isolated epidermal cells

At a concentration of 1.25 mM, 14 amino acids were capable of inhibiting the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in isolated epidermal cells. The greatest percentages of inhibition of TPA-induced epidermal ornithine decarboxylase activity were as follows: cysteine, 98%; tryptophan, 74%; methionine, 64%; phenylalanine, 51%; glycine, 44%; asparagine, 43%; glutamic acid, 42%; leucine, 40%; and arginine, 39%. These amino acid treatments did not alter the time- and concentration-response curves for induction of ornithine decarboxylase activity by TPA. Moreover, there was no difference between the rates at which [3H]arginine, [3H]leucine, [3H]phenylalanine, [3H]methionine, [3H]tryptophan and [14C]cysteine were taken up by freshly isolated epidermal cells or incorporated into epidermal proteins. Arginine, phenylalanine and methionine inhibited the induction of ornithine decarboxylase activity by the tumor promoter to degrees comparable to those elicited by their analogs canavanine and homoarginine, beta-2-thienyl-DL-alanine, and ethionine, respectively. These amino acids and amino acid analogs did not alter the overall rate of protein synthesis. In contrast, both the amino acids and their analogs increased the rates of proteolysis in isolated epidermal cells, an effect which correlated well with the abilities of these different compounds to inhibit TPA-induced ornithine decarboxylase activity. Moreover, both methionine and phenylalanine decreased the half-life and increased the rate of heat denaturation of the TPA-induced enzyme, a result identical to that obtained after treatment with the analogs ethionine and beta-2-thienyl-DL-alanine, respectively. Taken together, these results suggest that millimolar concentrations of exogenous amino acids might induce the synthesis of abnormal proteins and nonfunctional enzymes. Therefore, it is speculated that the uptake of unbalanced amounts of amino acids into the epidermal target cells might alter the stability and the ultrastructure of the TPA-stimulated enzyme just as the amino acid analogs do.     

Changes in free and bound amino acids in experimental silicosis.

The amino acid composition of lung, serum and liver in silicotic rats was studied in order to assess the availability of precursors in lung for fibrogenesis. It was observed that the pool of ornithine, arginine, alanine, leucine, valine, glutamic acid, lysine, proline and glycine underwent marked alterations. Free arginine, proline and leucine were only detectable in silicotic lung, while free glycine, glutamic acid and glutamine pools decreased significantly in liver. Changes in amino acid metabolism as a result of silicosis are discussed.     

Morphogenesis and free amino acid composition of the ascomycete Sphaerostilbe repens as influenced by nitrogen and calcium

Abstract Sphaerostilbe repens utilizes nitrate and ammonium as nitrogen sources. Differentiation of mycelium into rhizomorphs and coremia was reduced in the presence of nitrate and completely inhibited in the absence of calcium. The most abundant free amino acids were, in decreasing order: alanine, glutamine, glutatomic acid, serine, aspartic acid, γ-aminobutyric acid, arginine and threonine. These compounds represented 90% of the total amino acid pool.
The free amino acid composition did not vary with cultural conditions although concentrations of individual amino acids differed. In ammonium-grown cells, γ-aminobutyric acid increased in concentration and glutamate, aspartate and alanine decreased. Calcium-deficient media reduced amino acid concentrations, especially of arginine and ornithine. Amino acid contents increased during the growth period and were higher in rhizomorphs than in vegetative mycelia.     

Lactation during hibernation in wild black bears: effects on plasma amino acids and nitrogen metabolites.

This study examined the seasonal and reproductive influences on individual plasma amino acid concentrations and nitrogen metabolites in a black bear population (Ontario, Canada). During hibernation, 11 of 23 plasma amino acids were significantly higher (13%-108%) in lactating than in nonlactating females, without an alteration in plasma total protein or total essential or nonessential amino acid levels. The greatest changes were observed in glutamine, arginine, and glycine levels. Plasma urea, urea/creatinine, and ammonia levels were significantly lower in hibernating compared with active female bears, but lactation had no effect on these parameters. Taken together these results show that lactation during hibernation is an additional metabolic challenge that results in increased mobilization of individual plasma amino acids and no accumulation of nitrogen end products, underlining the remarkable efficiency of amino acid and urea recycling in denning female black bears.     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Incorporation of dl-[2-14C]ornithine and dl-[5-14C]arginine in milk constituents by the isolated lactating sheep udder

1. Lactating mammary glands of sheep were perfused for several hours in the presence of dl-[2-(14)C]ornithine or dl-[5-(14)C]arginine and received adequate quantities of acetate, glucose and amino acids. 2. In the [(14)C]ornithine experiment 1.4% of the casein and 1% of the expired carbon dioxide came from added ornithine; 96% of the total radioactivity in casein was recovered in proline; 13% of the proline of casein originated from plasma ornithine. 3. In this experiment the results of chemical degradation of proline of casein as well as relative specific activities in the isolated products are consistent with the view that ornithine is metabolized, by way of glutamic gamma-semialdehyde, to proline or glutamic acid. 4. In the [(14)C]arginine experiments 3% of the casein and 1% of the expired carbon dioxide came from arginine; 84% of the arginine and 9% of the proline of casein originated from plasma arginine. 5. In these experiments the relative specific activities of arginine, ornithine and proline in plasma are in agreement with the view that arginine is metabolized by way of ornithine to proline. The conversion of arginine into ornithine is probably catalysed by arginase, so that arginase in mammary tissue may be involved in the process of milk synthesis.