ABSTRACT
Previous studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC, encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1. Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH, encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1, but not argGH, rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes (proC and rocA) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine. IMPORTANCE Staphylococcus aureus can cause infection in virtually any niche of the human host, suggesting that it has significant metabolic versatility. Indeed, bioinformatic analysis suggests that it has the biosynthetic capability to synthesize all 20 amino acids. Paradoxically, however, it is conditionally auxotrophic for several amino acids, including arginine. Studies in our laboratory are designed to assess the biological function of amino acid auxotrophy in this significant pathogen. This study reveals that the metabolic block repressing arginine biosynthesis in media lacking glucose is the transcriptional repression of ornithine carbamoyltransferase encoded by arcB1 within the native arginine deiminase operon in addition to limited intracellular pools of ornithine. Surprisingly, approximately 50% of S. aureus clinical isolates can grow in media lacking arginine, suggesting that mutations are selected in S. aureus that allow growth in particular niches of the human host.
Subject(s)
Ornithine Carbamoyltransferase , Staphylococcus aureus , Amino Acids/metabolism , Arginine/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Glucose/metabolism , Ornithine/metabolism , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase/metabolism , Staphylococcus aureus/enzymology , Staphylococcus aureus/geneticsABSTRACT
BACKGROUND: There is no published data comparing dietary management of urea cycle disorders (UCD) in different countries. METHODS: Cross-sectional data from 41 European Inherited Metabolic Disorder (IMD) centres (17 UK, 6 France, 5 Germany, 4 Belgium, 4 Portugal, 2 Netherlands, 1 Denmark, 1 Italy, 1 Sweden) was collected by questionnaire describing management of patients with UCD on prescribed protein restricted diets. RESULTS: Data for 464 patients: N-acetylglutamate synthase (NAGS) deficiency, n=10; carbamoyl phosphate synthetase (CPS1) deficiency, n=29; ornithine transcarbamoylase (OTC) deficiency, n=214; citrullinaemia, n=108; argininosuccinic aciduria (ASA), n=80; arginase deficiency, n=23 was reported. The majority of patients (70%; n=327) were aged 0-16y and 30% (n=137) >16y. Prescribed median protein intake/kg body weight decreased with age with little variation between disorders. The UK tended to give more total protein than other European countries particularly in infancy. Supplements of essential amino acids (EAA) were prescribed for 38% [n=174] of the patients overall, but were given more commonly in arginase deficiency (74%), CPS (48%) and citrullinaemia (46%). Patients in Germany (64%), Portugal (67%) and Sweden (100%) were the most frequent users of EAA. Only 18% [n=84] of patients were prescribed tube feeds, most commonly for CPS (41%); and 21% [n=97] were prescribed oral energy supplements. CONCLUSIONS: Dietary treatment for UCD varies significantly between different conditions, and between and within European IMD centres. Further studies examining the outcome of treatment compared with the type of dietary therapy and nutritional support received are required.
Subject(s)
Amino Acids, Essential/metabolism , Diet, Protein-Restricted , Urea Cycle Disorders, Inborn/diet therapy , Urea Cycle Disorders, Inborn/pathology , Adolescent , Adult , Amino-Acid N-Acetyltransferase/deficiency , Arginase/metabolism , Argininosuccinic Aciduria/diet therapy , Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/deficiency , Child , Child, Preschool , Citrullinemia/diet therapy , Europe , Humans , Infant , Infant, Newborn , Ornithine Carbamoyltransferase/metabolism , Surveys and Questionnaires , Treatment Outcome , Urea Cycle Disorders, Inborn/enzymologyABSTRACT
In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the hosts production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that citrulline especially should gain further attention in future treatment strategies.
Subject(s)
Arginine/metabolism , Epithelial Cells/parasitology , Giardia lamblia/metabolism , Giardia lamblia/pathogenicity , Intestines/cytology , Caco-2 Cells , Cell Cycle/physiology , Cell Proliferation , Humans , Hydrolases/metabolism , Ornithine Carbamoyltransferase/metabolism , Polyamines/metabolismABSTRACT
Corynebacterium glutamicum shows a great potential for the production of the polyamide monomer putrescine (1,4-diaminobutane). Previously, we constructed the putrescine-producing strain PUT1 by deletion of argF, the gene for ornithine transcarbamoylase (OTC), and argR, encoding the L-arginine repressor, combined with heterologous expression of the Escherichia coli gene for L-ornithine decarboxylase SpeC. As a consequence of argF deletion, this strain requires supplementation of L-arginine and shows growth-decoupled putrescine production. To avoid costly supplementation with L-arginine and the strong feedback inhibition of the key enzyme N-acetylglutamate kinase (ArgB) by L-arginine, a plasmid addiction system for low-level argF expression was developed. By fine-tuning argF expression through modifications of the promoter, the translational start codon and/or the ribosome binding site, high productivity and titer could be obtained. OTC activity varied almost thousandfold between 960 and 1 mU mg⻹ resulting in putrescine yields on glucose from less than 0.001 up to 0.26 g g⻹, the highest yield in bacteria reported to date. The most promising strain, designated PUT21, was characterized comprehensively. PUT21 strain grew with a rate of 0.19 h⻹ in mineral salt medium without the need for L-arginine supplementation and produced putrescine with a yield of 0.16 g g⻹ glucose at a volumetric productivity of 0.57 g L⻹ h⻹ and a specific productivity of 0.042 g g⻹ h⻹. The carbon balance suggested that no major unidentified by-product was produced. Compared to the first-generation strain PUT1, the putrescine yield observed with PUT21 was increased by 60%. In fed-batch cultivation with C. glutamicum PUT21, a putrescine titer of 19 g L⻹ at a volumetric productivity of 0.55 g L⻹ h⻹ and a yield of 0.16 g g⻹ glucose could be achieved. Moreover, while plasmid segregation of the initial strain required antibiotic selection, plasmid segregation in C. glutamicum PUT21 was fully stable for more than 60 generations without antibiotic selection even in the presence of L-arginine. The ornithine decarboxylase gene speC was expressed from this argF addiction plasmid ensuring stable putrescine production by the engineered C. glutamicum strain.
Subject(s)
Corynebacterium glutamicum/enzymology , Genetic Engineering/methods , Ornithine Carbamoyltransferase/metabolism , Plasmids/genetics , Putrescine/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biotechnology , Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/metabolism , Culture Media , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Fermentation , Genes, Bacterial , Ornithine Carbamoyltransferase/geneticsABSTRACT
Cartilaginous fish comprise two subclasses, the Holocephali (chimaeras) and Elasmobranchii (sharks, skates and rays). Little is known about osmoregulatory mechanisms in holocephalan fishes except that they conduct urea-based osmoregulation, as in elasmobranchs. In the present study, we examined the ornithine urea cycle (OUC) enzymes that play a role in urea biosynthesis in the holocephalan elephant fish, Callorhinchus milii (cm). We obtained a single mRNA encoding carbamoyl phosphate synthetase III (cmCPSIII) and ornithine transcarbamylase (cmOTC), and two mRNAs encoding glutamine synthetases (cmGSs) and two arginases (cmARGs), respectively. The two cmGSs were structurally and functionally separated into two types: brain/liver/kidney-type cmGS1 and muscle-type cmGS2. Furthermore, two alternatively spliced transcripts with different sizes were found for cmgs1 gene. The longer transcript has a putative mitochondrial targeting signal (MTS) and was predominantly expressed in the liver and kidney. MTS was not found in the short form of cmGS1 and cmGS2. A high mRNA expression and enzyme activities were found in the liver and muscle. Furthermore, in various tissues examined, mRNA levels of all the enzymes except cmCPSIII were significantly increased after hatching. The data show that the liver is the important organ for urea biosynthesis in elephant fish, but, extrahepatic tissues such as the kidney and muscle may also contribute to the urea production. In addition to the role of the extrahepatic tissues and nitrogen metabolism, the molecular and functional characteristics of multiple isoforms of GSs and ARGs are discussed.
Subject(s)
Carbon-Nitrogen Ligases/metabolism , Fishes/physiology , Liver/enzymology , Ornithine Carbamoyltransferase/metabolism , Phylogeny , Urea/metabolism , Water-Electrolyte Balance/physiology , Alternative Splicing , Amino Acid Sequence , Animals , Arginase/genetics , Arginase/metabolism , Base Sequence , Bayes Theorem , Carbon-Nitrogen Ligases/genetics , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , Fishes/metabolism , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/metabolism , Kidney/metabolism , Models, Molecular , Molecular Sequence Data , Muscle, Skeletal/metabolism , Ornithine Carbamoyltransferase/genetics , Protein Isoforms/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Statistics, Nonparametric , VictoriaABSTRACT
Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No curative therapy exists except for liver transplantation which is limited by available donors and carries significant risk of mortality and morbidity. Adeno-associated virus 8 (AAV8) has been shown to be the most efficient vector for liver-directed gene transfer and is currently being evaluated in a clinical trial for treating hemophilia B. In this study, we generated a clinical candidate vector for a proposed OTC gene therapy trial in humans based on a self-complementary AAV8 vector expressing codon-optimized human OTC (hOTCco) under the control of a liver-specific promoter. Codon-optimization dramatically improved the efficacy of OTC gene therapy. Supraphysiological expression levels and activity of hOTC were achieved in adult spf(ash) mice following a single intravenous injection of hOTCco vector. Vector doses as low as 1×10(10) genome copies (GC) achieved robust and sustained correction of the OTCD biomarker orotic aciduria and clinical protection against an ammonia challenge. Functional expression of hOTC in 40% of liver areas was found in mice treated with a low vector dose of 1×10(9) GC. We suggest that the clinical candidate vector we have developed has the potential to achieve therapeutic effects in OTCD patients.
Subject(s)
Dependovirus/genetics , Genetic Therapy , Genetic Vectors , Ornithine Carbamoyltransferase Deficiency Disease/genetics , Ornithine Carbamoyltransferase Deficiency Disease/therapy , Ornithine Carbamoyltransferase/metabolism , Adult , Animals , Gene Expression , Humans , Liver/enzymology , Liver/pathology , Mice , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase Deficiency Disease/enzymology , Orotic Acid/urineABSTRACT
Human recombinant arginase I cobalt [HuArgI (Co)] coupled with polyethylene glycol 5000 [HuArgI (Co)-PEG5000] has shown potent in-vitro depletion of arginine from tissue culture medium. We now show that HuArgI (Co)-PEG5000 is toxic to almost all cancer cell lines and to some normal primary cells examined. In contrast, HuArgI (Co)-PEG5000 in combination with supplemental L-citrulline is selectively cytotoxic to a fraction of human cancer cell lines in tissue culture, including some melanomas, mesotheliomas, acute myeloid leukemias, hepatocellular carcinomas, pancreas adenocarcinomas, prostate adenocarcinomas, lung adenocarcinomas, osteosarcomas, and small cell lung carcinomas. Unfortunately, a subset of normal human tissues is also sensitive to HuArgI (Co)-PEG5000 with L-citrulline supplementation, including umbilical endothelial cells, bronchial epithelium, neurons, and renal epithelial cells. We further show that cell sensitivity is predicted by the level of cellular argininosuccinate synthetase protein expression measured by immunoblots. By comparing a 3-day and 7-day exposure to HuArgI (Co)-PEG5000 with supplemental L-citrulline, some tumor cells sensitive on short-term assay are resistant in the 7-day assay consistent with the induction of argininosuccinate synthetase expression. On the basis of these results, we hypothesize that HuArgI (Co)-PEG5000 in combination with L-citrulline supplementation may be an attractive therapeutic agent for some argininosuccinate synthetase-deficient tumors. These in-vitro findings stimulate further development of this molecule and may aid in the identification of tissue toxicities and better selection of patients who will potentially respond to this combination therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Arginase/pharmacology , Argininosuccinate Synthase/metabolism , Citrulline/pharmacology , Polyethylene Glycols/pharmacology , Arginine/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Culture Media , Drug Screening Assays, Antitumor , Epithelial Cells/drug effects , Humans , Male , Ornithine Carbamoyltransferase/metabolism , Protein Synthesis Inhibitors/pharmacology , Recombinant Proteins/pharmacologyABSTRACT
Ornithine transcarbamylase (OTC) deficiency, the most common urea cycle disorder, is associated with severe hyperammonemia accompanied by a high risk of neurological damage and death in patients presenting with the neonatal-onset form. Contemporary therapies, including liver transplantation, remain inadequate with considerable morbidity, justifying vigorous investigation of alternate therapies. Clinical evidence suggests that as little as 3% normal enzyme activity is sufficient to ameliorate the severe neonatal phenotype, making OTC deficiency an ideal model for the development of liver-targeted gene therapy. In this study, we investigated metabolic correction in neonatal and adult male OTC-deficient Spf(ash) mice following adeno-associated virus (AAV)2/8-mediated delivery of the murine OTC complementary DNA under the transcriptional control of a liver-specific promoter. Substantially supraphysiological levels of OTC enzymatic activity were readily achieved in both adult and neonatal mice following a single intraperitoneal (i.p.) injection, with metabolic correction in adults being robust and life-long. In the neonates, however, full metabolic correction was transient, although modest levels of OTC expression persisted into adulthood. Although not directly testable in Spf(ash) mice, these levels were theoretically sufficient to prevent hyperammonemia in a null phenotype. This loss of expression in the neonatal liver is the consequence of hepatocellular proliferation and presents an added challenge to human therapy.
Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , Ornithine Carbamoyltransferase Deficiency Disease/metabolism , Ornithine Carbamoyltransferase Deficiency Disease/therapy , Animals , Animals, Newborn , Blotting, Western , Cell Line , DNA, Complementary/genetics , Female , Humans , Immunohistochemistry , Liver/metabolism , Male , Mice , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase/metabolism , Ornithine Carbamoyltransferase/physiology , Ornithine Carbamoyltransferase Deficiency Disease/genetics , Ornithine Carbamoyltransferase Deficiency Disease/urine , Orotic Acid/urineABSTRACT
Ornithine transcarbamylase deficiency (OTCD) is an X-linked disorder of the urea cycle. It is often fatal in affected males. Treatment for affected individuals includes dietary protein restriction, activation of alternative pathways of nitrogen excretion and L-arginine supplementation. Depending on the amount of X chromosome inactivation skewing, females show variable clinical manifestations, and sometimes the need for treatment, including medications, is unclear. We conducted an n of 1 randomized controlled trial on an obligate OTC carrier. The treating physician and patient were blinded to treatment. Either placebo capsules or L-arginine capsules were given for weekly periods. Weekly efficacy indicators included plasma arginine and glutamine levels and a quality of life/mood assessment questionnaire scale. Clear evidence of benefit with L-arginine compared to placebo was shown. This is the first time an n of 1 randomized controlled trial has been reported for an X-linked metabolic condition. Despite some logistic hurdles, we have demonstrated that this method was an effective tool for determining the value of treatment. We propose that other rare metabolic conditions may be amenable to such trials, if the benefit of treatment is in doubt.
Subject(s)
Arginine/administration & dosage , Heterozygote , Ornithine Carbamoyltransferase Deficiency Disease/drug therapy , Ornithine Carbamoyltransferase/metabolism , Affect , Arginine/blood , Arginine/therapeutic use , Cross-Over Studies , Double-Blind Method , Female , Genetic Diseases, X-Linked/drug therapy , Glutamine/blood , Humans , Male , Middle Aged , Ornithine Carbamoyltransferase/genetics , Quality of Life , Surveys and Questionnaires , X Chromosome InactivationABSTRACT
The spf-ash mutation in mice results in reduced hepatic and intestinal ornithine transcarbamylase. However, a reduction in enzyme activity only translates in reduced ureagenesis and hyperammonemia when an unbalanced nitrogen load is imposed. Six-week-old wild-type control and spf-ash mutant male mice from different genetic backgrounds (B6 and ICR) were infused intravenously with [(13)C(18)O]urea, l-[(15)N(2)]arginine, l-[5,5 D(2)]ornithine, l-[6-(13)C, 4,4,5,5, D(4)]citrulline, and l-[ring-D(5)]phenylalanine to investigate the interaction between genetic background and spf-ash mutation on ureagenesis, arginine metabolism, and nitric oxide production. ICR(spf-ash) mice maintained ureagenesis (5.5 +/- 0.3 mmol.kg(-1).h(-1)) and developed mild hyperammonemia (145 +/- 19 micromol/l) when an unbalanced nitrogen load was imposed; however, B6(spf-ash) mice became hyperammonemic (671 +/- 15 micromol/l) due to compromised ureagenesis (3.4 +/- 0.1 mmol.kg(-1).h(-1)). Ornithine supplementation restored ureagenesis and mitigated hyperammonemia. A reduction in citrulline entry rate was observed due to the mutation in both genetic backgrounds (wild-type: 128, spf-ash: 60; SE 4.0 micromol.kg(-1).h(-1)). Arginine entry rate was only reduced in B6(spf-ash) mice (B6(spf-ash): 332, ICR(spf-ash): 453; SE 20.6 micromol.kg(-1).h(-1)). Genetic background and mutation had an effect on nitric oxide production (B6: 3.4, B6(spf-ash): 2.8, ICR: 9.0, ICR(spf-ash): 4.6, SE 0.7 micromol.kg(-1).h(-1)). Protein breakdown was the main source of arginine during the postabsorptive state and was higher in ICR(spf-ash) than in B6(spf-ash) mice (phenylalanine entry rate 479 and 327, respectively; SE 18 micromol.kg(-1).h(-1)). Our results highlight the importance of the interaction between mutation and genetic background on ureagenesis, arginine metabolism, and nitric oxide production. These observations help explain the wide phenotypic variation of ornithine transcarbamylase deficiency in the human population.
Subject(s)
Mutation , Ornithine Carbamoyltransferase Deficiency Disease/metabolism , Ornithine Carbamoyltransferase/metabolism , Alanine/administration & dosage , Alanine/metabolism , Alanine/pharmacokinetics , Ammonia/blood , Ammonia/metabolism , Animals , Arginine/administration & dosage , Arginine/metabolism , Arginine/pharmacokinetics , Citrulline/blood , Citrulline/metabolism , Disease Models, Animal , Female , Glycine/administration & dosage , Glycine/metabolism , Glycine/pharmacokinetics , Injections, Intravenous , Intestinal Mucosa/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred ICR , Mice, Inbred Strains , Mice, Mutant Strains , Models, Biological , Nitric Oxide/metabolism , Ornithine/administration & dosage , Ornithine/metabolism , Ornithine/pharmacokinetics , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase Deficiency Disease/genetics , Phenotype , Phenylalanine/administration & dosage , Phenylalanine/metabolism , Phenylalanine/pharmacokinetics , Urea/blood , Urea/metabolismABSTRACT
The aim of the study was examining the effect of fluoride ions and caffeine administration on glucose and urea concentration in blood serum and the activity of protein metabolism enzymes and selected enzymes of the urea cycle in rat liver. The study was carried out using 18 male Sprague-Daowley rats (4.5 mo old). Rats were divided into three groups. Group I received distilled water ad libitum. Group II received 4.9 mg F-/kg body mass/d of sodium fluoride in the water, and group III received sodium fluoride (in the above-mentioned dose) and 3 mg/kg body mass/d of caffeine in the water. After 50 d, the rats were anesthetized with thiopental and fluoride ions, glucose, and urea concentration in blood serum were determined. Also determined were the activities of aspartate aminotransferase, alanine aminotransferase glutamate dehydrogenase, ornithine carbamoylotransferase and arginase in liver homogenates. Liver was taken for pathomorphological examinations. The applied doses of F- (4.9 mg/kg body mass/d) and F- + caffeine (4.9 mg F-/kg body mass/d + 3 mg caffeine/kg body mass/d) resulted in a statistically significant increase of fluoride ion concentration in blood serum, a slight increase of the glucose concentration, and no changes in the concentration of urea in blood serum. This might testify to the absence of kidney lesions for the applied concentrations of F-. No change in the functioning of hepatocytes was observed; however, slight disturbances have been noted in the functioning of the liver, connected with the activation of urea cycle, increase of arginase activity, and accumulation of F- in this organ. There was no observed significant influence of caffeine supplementation on the obtained results.
Subject(s)
Blood Glucose/analysis , Caffeine/pharmacology , Fluorides/blood , Liver/drug effects , Sodium Fluoride/pharmacology , Urea/blood , Alanine Transaminase/metabolism , Animals , Arginase/metabolism , Aspartate Aminotransferases/metabolism , Glutamate Dehydrogenase/metabolism , Liver/enzymology , Male , Ornithine Carbamoyltransferase/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
We showed that Otc(spf-ash) mice, a model of ornithine transcarbamylase deficiency, were able to sustain ureagenesis at the same rate as control mice, despite reduced enzyme activity, when a complete mixture of amino acids was provided. An unbalanced amino acid mixture, however, resulted in reduced ureagenesis and hyperammonemia. To study the effect of ornithine supplementation [316 micromol/(kg.h)] on urea and glutamine kinetics in conscious Otc(spf-ash) mice under a glycine-alanine load [6.06 mmol/(kg.h)], a multiple tracer infusion protocol ([(13)C(18)O]urea, [5-(15)N]glutamine, [2,3,3,4,4 D(5)]glutamine and [ring-D(5)] phenylalanine) was conducted. Ornithine supplementation increased ureagenesis [3.18 +/- 0.88 vs. 4.56 +/- 0.51 mmol/(kg.h), P < 0.001], reduced plasma ammonia concentration (1125 +/- 621 vs. 193 +/- 94 micromol/L, P < 0.001), and prevented acute hepatic enlargement (P < 0.006) in Otc(spf-ash) mice. Ornithine supplementation also increased [96 +/- 20 vs. 120 +/- 16 micromol/(kg.h), P < 0.001] the transfer of (15)N from glutamine to urea, to values observed in the control mice [123 +/- 17 micromol/(kg.h)]. De novo amido-N glutamine flux was higher [1.57 +/- 0.37 vs. 3.04 +/- 0.86 mmol/(kg.h); P < 0.001] in Otc(spf-ash) mice, but ornithine supplementation had no effect (P < 0.56). The flux of glutamine carbon skeleton was affected by both genotype (P < 0.0001) and by ornithine (P 0. 036). In conclusion, ornithine supplementation restored ureagenesis, mitigated hyperammonemia, prevented liver enlargement, and normalized the transfer of (15)N from glutamine to urea. These data strongly suggest that ornithine has the potential for the biochemical correction of OTCD in Otc(spf-ash) mice.
Subject(s)
Amino Acids/metabolism , Glutamine/metabolism , Hyperammonemia/drug therapy , Ornithine Carbamoyltransferase Deficiency Disease , Ornithine/therapeutic use , Urea/metabolism , Amino Acids/blood , Animals , Mice , Ornithine/administration & dosage , Ornithine Carbamoyltransferase/metabolism , Urea/bloodABSTRACT
The role of nitric oxide (NO) in maintaining homeostasis and regulating organ function during hemorrhagic shock is complex. The inducible NO synthase (iNOS) has been hypothesized to play a critical role in the pathophysiologic consequences of severe hemorrhage. Heat shock protein (HSP) expression is increased by hemorrhage and is a marker of the magnitude of ischemic injury in the liver. HSP induction is protective against injury in animal models of inflammation and is regulated by NO in hepatocytes. To clarify the role of iNOS in hepatic injury and its relationship to HSP expression in hemorrhagic shock, NOS was inhibited with L-N-6-(1-iminoethyl) lysine (L-NIL), which is reported to be a selective inhibitor of the inducible NOS isoform. Doses of 50 microg/kg or 150 microg/kg were infused over 1 h at the end of compensated shock. Plasma ornithine carbamoyltransferase (OCT), a specific marker of liver injury, was significantly reduced after hemorrhage with low-dose L-NIL (7.1+/-1.5 IU/L) compared to saline-treated control rats (13.0+/-1.5 IU/L, P < 0.005), while high-dose L-NIL significantly increased OCT release (35.9+/-7.2 IU/L, P< 0.05 versus shock alone) despite a greater MAP after resuscitation. HSP expression (HSP-72 and HSP-32) after hemorrhage was increased by L-NIL treatment at the highest dose. We conclude that excessive NO production from iNOS contributes to shock-induced hepatic injury. Our data suggest HSP expression may reflect the degree of ischemic injury after hemorrhage.
Subject(s)
Gene Expression Regulation , Heat-Shock Proteins/genetics , Liver/metabolism , Liver/pathology , Lysine/analogs & derivatives , Nitric Oxide Synthase/antagonists & inhibitors , Shock, Hemorrhagic/genetics , Shock, Hemorrhagic/pathology , Tyrosine/analogs & derivatives , Animals , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Hemodynamics/drug effects , Liver/drug effects , Liver/enzymology , Lysine/pharmacology , Male , Nitric Oxide Synthase Type II , Ornithine Carbamoyltransferase/metabolism , Rats , Rats, Sprague-Dawley , Shock, Hemorrhagic/enzymology , Time Factors , Tyrosine/metabolismABSTRACT
Influence of alimentary zinc deficiency on nitrogen elimination and activities of urea cycle enzymes This study was conducted to investigate whether the hyperammonaemia shown in earlier zinc-deficiency experiments was the result of disturbed enzyme activities of the urea cycle. For this study 36 male Sprague-Dawley rats with an average body weight of 85 g were divided into three experimental groups of 12 animals each. Group 1 received the semisynthetic zinc-deficient diet (AIN-93G; 1.2 mg Zn/kg DM) ad libitum over 33 experimental days. Group 2 received the zinc-sulphate-supplemented control diet (60 mg Zn/kg DM) ad libitum and group 3 received the same diet matched to the feed intake of the zinc-deficient rats. Alimentary zinc deficiency reduced the zinc concentration and the activity of the alkaline phosphatase in serum by 75 and 67%, respectively. The activity of the glutamate dehydrogenase and the concentrations of ammonia and urea in the serum of the zinc-deficient rats showed no significant differences compared with pair-fed control rats. On the other hand the hepatic activity of the mitochondrial localized glutamate dehydrogenase of the zinc-deficient rats was significantly increased and the carbamoylphosphate synthetase and ornithine carbamoyltransferase were reduced about half in comparison with both control groups. The activities of the cytosolic liver enzymes such as argininosuccinate synthetase, argininosuccinase and arginase were again significantly increased in zinc-deficient rats compared with both control groups. The increased hepatic activity of the glutamate dehydrogenase possibly led to an enhanced NH(3) elimination in addition to urea synthesis. The typical reduction of feed intake in consequence of zinc deficiency is therefore not the cause of hyperammonaemia due to disturbed urea synthesis, as has been hypothesized in earlier studies.
Subject(s)
Alkaline Phosphatase/metabolism , Glutamate Dehydrogenase/metabolism , Nitrogen/metabolism , Urea/blood , Zinc/deficiency , Ammonia/blood , Animals , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Liver/enzymology , Male , Mitochondria/enzymology , Ornithine Carbamoyltransferase/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The aim of this investigation was to determine if the hyperammonaemia shown in previous zinc-deficiency experiments was the result of disturbed enzyme activities for urea synthesis caused by zinc deficiency per se or was a secondary effect of the reduced feed intake accompanying energy and protein deficiency. For this, 24 male Sprague-Dawley rats with an average body weight of 109 g were divided into two groups of 12 animals each. Both groups were force fed by intragastric tube four times daily over 11 experimental days. Group 1 received a zinc-deficient diet (1.3 mg Zn/kg diet) in a total amount of 11.6 g/day/animal. Group 2 received the zinc sulphate-supplemented control diet (25 mg Zn/kg diet) in the same amount. This technique made it possible to supply even the zinc-deficient rats with sufficient nutrients over the whole experimental period in the same manner as for the control rats, at the same time and with the same dietary amounts. At the end of the experiment, the serum zinc concentration and the alkaline phosphatase activity were significantly reduced in the zinc-deficient rats by 59 and 37%, respectively, in comparison with control animals. This showed a severe alimentary zinc-deficiency status of the animals. The concentrations of ammonia and urea, as well as the activity of glutamate dehydrogenase in serum, were not influenced by the zinc-deficient nutrition within the experimental time. Likewise, the mitochondrial activities of glutamate dehydrogenase and carbamoylphosphate synthetase in the liver were not affected by the alimentary zinc concentration. On the contrary, the activities of ornithine carbamoyltransferase and cytosolic liver enzymes argininosuccinate synthetase, argininosuccinase and arginase were significantly increased in comparison with control rats. In the case of a sufficient supply of nutrients, alimentary zinc deficiency did not cause hyperammonaemia owing to disturbed urea synthesis, as previously hypothesized.
Subject(s)
Ammonia/blood , Zinc Sulfate/administration & dosage , Zinc/deficiency , Alkaline Phosphatase/metabolism , Animals , Arginase/metabolism , Argininosuccinate Lyase/metabolism , Argininosuccinate Synthase/metabolism , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Disease Models, Animal , Enteral Nutrition/veterinary , Glutamate Dehydrogenase/metabolism , Intubation, Gastrointestinal/veterinary , Liver/enzymology , Male , Ornithine Carbamoyltransferase/metabolism , Rats , Rats, Sprague-Dawley , Urea/blood , Zinc/bloodABSTRACT
Kidney bean (Phaseolus vulgaris L.) ornithine carbamoyltransferase (OCT; EC 2.1.3.3) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonoacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 8540-fold-purified OCT exhibited a specific activity of 526 micromoles citrulline per minute per milligram of protein at 35 degrees C and pH 8.0. The enzyme represents approximately 0.01% of the total soluble protein in the leaf. The molecular mass of the native enzyme was approximately 109 kDa as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single band of molecular mass 36 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at a single isoelectric point of 6.6 when subjected to denaturing isoelectric focusing. These results suggest that the enzyme is a trimer of identical subunits. Among the tested amino acids, L-cysteine and S-carbamoyl-L-cysteine were the most effective inhibitors of the enzyme. The OCT of kidney bean showed a very low activity towards canaline. The OCTs of canavanine-deficient plants have very low canaline-dependent activities, but the OCTs of canavanine-containing plants showed high canaline-dependent activities. It was assumed that the substrate specificity of this enzyme determines the canavanine synthetic activity of the urea cycle.
Subject(s)
Canavanine/metabolism , Fabaceae/enzymology , Ornithine Carbamoyltransferase/isolation & purification , Ornithine Carbamoyltransferase/metabolism , Plants, Medicinal , Animals , Carbamyl Phosphate/metabolism , Hydrogen-Ion Concentration , Mice , Molecular Weight , Ornithine/metabolism , Plant Leaves/enzymology , Rabbits , Substrate SpecificityABSTRACT
Glutamine synthetase and carbamoylphosphate synthetase I expression was examined immunohistochemically in livers of spf-ash homozygous and hemizygous mice, in which one of the urea cycle enzymes (ornithine carbamoyltransferase) is deficient and hyperammonemic disorders are obvious. In the mutant adult mouse liver, only hepatocytes lining central veins expressed glutamine synthetase. In contrast, other hepatocytes expressed carbamoylphosphate synthetase I but not glutamine synthetase. This complementary expression pattern is similar to that seen in wild-type mouse liver. In the liver of mutant young mice, which showed severe retarded growth and abnormal hair and skin development, the developmental expression pattern of both enzymes was also similar to that of the corresponding wild-type liver. However, suppression of carbamoylphosphate synthetase I expression in the pericentral hepatocytes occurred later in the mutant than in wild-type liver. These results show that high plasma concentrations of ammonium ions, which are one of the substrates for both the enzymes, do not change their complementary expression. Instead they support the idea that factor(s) associated with central veins rather than humoral factors direct pericentral hepatocytes to express glutamine synthetase and to suppress carbamoylphosphate synthetase I expression.
Subject(s)
Carbamoyl-Phosphate Synthase (Ammonia)/biosynthesis , Glutamate-Ammonia Ligase/biosynthesis , Liver/enzymology , Ornithine Carbamoyltransferase Deficiency Disease , Ammonia/blood , Animals , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Female , Glutamate-Ammonia Ligase/metabolism , Immunohistochemistry , Male , Mice , Mice, Inbred C3H , Ornithine Carbamoyltransferase/metabolismABSTRACT
Biotin deficiency is well known as a cause of hyperammonemia, but there has been no report on the effect of biotin deficiency on hepatic ureagenesis. In this study, we examined the changes in the activities and gene expression of urea cycle enzymes using rats fed raw egg white as a model of biotin deficiency. All rats were made biotin-deficient by feeding them an avidin-containing diet for 6 wk. The rats were divided into two groups at the beginning of this experiment: biotin-deficient rats (BD rats) and biotin-supplemented rats (BS rats) which were treated with biotin once a day at a dose of 1 mg per rat intraperitoneally. The plasma ammonia concentration of the BD rats (92.8 +/- 12 mumol/L) was significantly higher than that of BS rats (63.9 +/- 16 mumol/L, P < 0.05). The activities of ornithine transcarbamylase (OTC) was significantly lower in the liver of the BD (110.2 +/- 5.5) rats than in the BS rats (154 +/- 3.8 U/mg protein, P < 0.01). Activities of the other urea cycle enzymes were not significantly different in the two groups. OTC gene expression in the liver of BD rats was 40% lower than in BS rats (P < 0.05). These data suggest that biotin deficiency decreases OTC activity and the amount of OTC mRNA.
Subject(s)
Biotin/deficiency , Liver/enzymology , Ornithine Carbamoyltransferase/metabolism , RNA, Messenger/analysis , Amino Acids/blood , Ammonia/blood , Animals , Biotin/blood , Biotin/pharmacology , Blotting, Northern , Body Weight/physiology , Gene Expression Regulation, Enzymologic , Liver/metabolism , Male , Ornithine Carbamoyltransferase/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Urea/metabolismABSTRACT
The aim of the study was to examine the effects of oral zinc supplementation on liver ornithine transcarbamylase activity (OTC), a key enzyme in the urea cycle, in cirrhotic rats. OTC was studied in two groups of rats treated with carbon tetrachloride (CCl4): the first received zinc in the drinking water during the induction of cirrhosis; the second was the control group. Cirrhotic rats which received zinc supplementation showed an increase in liver OTC activity, positively correlated with serum and hepatic zinc content. The results suggest that zinc dietary supplementation may modify hepatic OTC activity and, therefore, plasma ammonia levels in cirrhotic rats.
Subject(s)
Liver Cirrhosis, Experimental/metabolism , Liver/enzymology , Ornithine Carbamoyltransferase/metabolism , Zinc/pharmacology , Ammonia/blood , Animals , Carbon Tetrachloride , Disease Models, Animal , Liver/drug effects , Liver/metabolism , Liver Cirrhosis, Experimental/blood , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/drug therapy , Male , Rats , Rats, Wistar , Zinc/blood , Zinc/metabolismABSTRACT
In crude protein extracts of aseptically grown plantlets of Datura innoxia ornithine carbamoyltransferase was detected. The ornithine carbamoyltransferase of plant extracts was inhibited by phaseolotoxin. The level of this inhibition was dependent on the concentration of the toxin applied. Subsequent accumulation of ornithine in plant vegetative parts was observed. Phaseolotoxin influenced both the vitability and alkaloid accumulation of treated plants.