Showing papers on "Urea cycle published in 2009"

Ammonia toxicity and its prevention in inherited defects of the urea cycle

Abstract: The urea cycle is the final pathway for removal of surplus nitrogen from the body, and the major route in humans for detoxification of ammonia. The full complement of enzymes is expressed only in liver. Inherited deficiencies of urea cycle enzymes lead to hyperammonaemia, which causes brain damage. Severe defects present with hyperammonaemic crises in neonates. Equally devastating episodes may occur in previously asymptomatic adults with mild defects, most often X-linked ornithine transcarbamylase (OTC) deficiency. Several mechanisms probably contribute to pathogenesis. Treatment aims to reduce plasma ammonia quickly, reduce production of waste nitrogen, dispose of waste nitrogen using alternative pathways to the urea cycle and replace arginine. These therapies have increased survival and probably improve the neurological outcome. Arginine, sodium benzoate, sodium phenylbutyrate and, less often, sodium phenylacetate are used. Long-term correction is achieved by liver transplantation. Gene therapy for OTC deficiency is effective in animals, and work is ongoing to improve persistence and safety.

Metabolic profiling of PPARα−/− mice reveals defects in carnitine and amino acid homeostasis that are partially reversed by oral carnitine supplementation

Abstract: Peroxisome proliferator-activated receptor-α (PPARα) is a master transcriptional regulator of β-oxidation and a prominent target of hypolipidemic drugs. To gain deeper insights into the systemic consequences of impaired fat catabolism, we used quantitative, mass spectrometry-based metabolic profiling to investigate the fed-to-fasted transition in PPARα+/+ and PPARα−/− mice. Compared to PPARα+/+ animals, acylcarnitine profiles of PPARα−/− mice revealed 2- to 4-fold accumulation of long-chain species in the plasma, whereas short-chain species were reduced by as much as 69% in plasma, liver, and skeletal muscle. These results reflect a metabolic bottleneck downstream of carnitine palmitoyltransferase-1, a mitochondrial enzyme that catalyzes the first step in β-oxidation. Organic and amino acid profiles of starved PPARα−/− mice suggested compromised citric acid cycle flux, enhanced urea cycle activity, and increased amino acid catabolism. PPARα−/− mice had 40–50% lower plasma and tissue levels of free carnitine, corresponding with diminished hepatic expression of genes involved in carnitine biosynthesis and transport. One week of oral carnitine supplementation conferred partial metabolic recovery in the PPARα−/− mice. In summary, comprehensive metabolic profiling revealed novel biomarkers of defective fat oxidation, while also highlighting the potential value of supplemental carnitine as a therapy and diagnostic tool for metabolic disorders.—Makowski, L., Noland, R. C., Koves, T. R., Xing, W., Ilkayeva, O. R., Muehlbauer, M. J., Stevens, R. D., Muoio, D. M. Metabolic profiling of PPARα−/− mice reveals defects in carnitine and amino acid homeostasis that are partially reversed by oral carnitine supplementation.

Urea cycle regulation by mitochondrial sirtuin, SIRT5

Abstract: Mammalian sirtuins have diverse roles in aging, metabolism and disease. Recently we reported a new function for SIRT5 in urea cycle regulation. Our study uncovered that SIRT5 localized to mitochondria matrix and deacetylates carbamoyl phosphate synthetase 1 (CPS1), an enzyme which is the first and rate-limiting step of urea cycle. Deacetylation of CPS1 by SIRT5 resulted in activation of CPS1 enzymatic activity. Indeed, SIRT5-deficient mice failed to up-regulate CPS1 activity and showed hyper ammonemia during fasting. Similar effects are also observed on high protein diet or calorie restriction. These data indicate SIRT5 also has an emerging role in the metabolic adaptation to fasting, high protein diet and calorie restriction.

Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease

Abstract: To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimer's disease (AD), we have developed a customized microarray containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT-PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC -389 G/A and -241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.

Valproate-Induced Hyperammonemic Encephalopathy and Normal Liver Functions: Possible Synergism With Topiramate

Abstract: A patient with valproate-induced hyperammonemic encephalopathy presented with altered mental status and hyperammonemia in the context of normal liver functions. Fortunately, altered mental status and elevated plasma ammonia level normalized 1 day after discontinuation of divalproex sodium (Depakote). The case analysis suggests a possible synergistic interaction of valproic acid and topiramate with respect to the emergence of hyperammonemic encephalopathy in the context of normal liver functions. Possible mechanisms of the encephalopathy and hyperammonemia are discussed. For example, valproate has diverse metabolic effects that include regulating levels of ammonia by altering activity of the urea cycle, whose first step uses HCO 3 - in the synthesis of carbamoylphosphate. Topiramate's inhibition of carbonic anhydrase activity may be the basis of its possible synergy with valproate by affecting levels of HCO 3 - .

Genetic variation in the urea cycle: a model resource for investigating key candidate genes for common diseases.

Abstract: The urea cycle is the primary means of nitrogen metabolism in humans and other ureotelic organisms. There are five key enzymes in the urea cycle: carbamoyl-phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). Additionally, a sixth enzyme, N-acetylglutamate synthase (NAGS), is critical for urea cycle function, providing CPS1 with its necessary cofactor. Deficiencies in any of these enzymes result in elevated blood ammonia concentrations, which can have detrimental effects, including central nervous system dysfunction, brain damage, coma, and death. Functional variants, which confer susceptibility for disease or dysfunction, have been described for enzymes within the cycle; however, a comprehensive screen of all the urea cycle enzymes has not been performed. We examined the exons and intron/exon boundaries of the five key urea cycle enzymes, NAGS, and two solute carrier transporter genes (SLC25A13 and SLC25A15) for sequence alterations using single-stranded conformational polymorphism (SSCP) analysis and high-resolution melt profiling. SSCP was performed on a set of DNA from 47 unrelated North American individuals with a mixture of ethnic backgrounds. High-resolution melt profiling was performed on a nonoverlapping DNA set of either 47 or 100 unrelated individuals with a mixture of backgrounds. We identified 33 unarchived polymorphisms in this screen that potentially play a role in the variation observed in urea cycle function. Screening all the genes in the pathway provides a catalog of variants that can be used in investigating candidate diseases.

The human and mouse SLC25A29 mitochondrial transporters rescue the deficient ornithine metabolism in fibroblasts of patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome.

Abstract: The hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a disorder of the urea cycle (UCD) and ornithine degradation pathway caused by mutations in the mitochondrial ornithine transporter (ORNT1). Unlike other UCDs, HHH syndrome is characterized by a less severe and variable phenotype that we believe may, in part, be due to genes with redundant function to ORNT1, such as the previously characterized ORNT2 gene. We reasoned that SLC25A29, a member of the same subfamily of mitochondrial carrier proteins as ORNT1 and ORNT2, might also have overlapping function with ORNT1. Here, we report that both the human and mouse SLC25A29, previously identified as mitochondrial carnitine/acyl-carnitine transporter-like, when overexpressed transiently also rescues the impaired ornithine transport in cultured HHH fibroblasts. Moreover, we observed that, in the mouse, the Slc25a29 message is more significantly expressed in the CNS and cultured astrocytes when compared with the liver and kidney. These results suggest a potential physiologic role for the SLC25A29 transporter in the oxidation of fatty acids, ornithine degradation pathway, and possibly the urea cycle. Our results show that SLC25A29 is the third human mitochondrial ornithine transporter, designated as ORNT3, which may contribute to the milder and variable phenotype seen in patients with HHH syndrome.

Evaluation of endogenous nitric oxide synthesis in congenital urea cycle enzyme defects

Abstract: Nitric oxide (NO) is synthesized from arginine and O(2) by nitric oxide synthase (NOS). Citrulline, which is formed as a by-product of the NOS reaction, can be recycled to arginine by the 2 enzymes acting in the urea cycle: argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Although the complete urea cycle is expressed only in the liver, ASS and ASL are expressed in other organs including the kidney and vascular endothelium. To examine possible alterations of the NO pathway in urea cycle defects, we measured plasma concentrations of arginine and citrulline and serum concentrations of nitrite/nitrate (NOx(-), stable NO metabolites) and asymmetric dimethylarginine (ADMA, an endogenous NOS inhibitor) in patients with congenital urea cycle disorders of 3 types: ornithine transcarbamylase (OTC) deficiency, ASS deficiency, and ASL deficiency. All were receiving oral arginine replacement at the time of this study. The same parameters were also measured in healthy subjects, who participated as controls. The OTC-deficient patients had significantly high NOx(-) and nonsignificantly high ADMA concentrations. Their NOx(-) was significantly positively correlated with arginine. The ASS-deficient patients had significantly low NOx(-) and significantly high ADMA concentrations. The ASL-deficient patients had normal NOx(-) and nonsignificantly high ADMA concentrations. In ASS-deficient and ASL-deficient patients, the NOx(-) was significantly inversely correlated with citrulline. These results suggest that NO synthesis is enhanced in OTC-deficient patients while receiving arginine but that NO synthesis remains low in ASS-deficient patients despite receiving arginine. They also suggest that endogenous NO synthesis is negatively affected by citrulline and ADMA in ASS-deficient and ASL-deficient patients. Although the molecular mechanisms remain poorly understood, we infer that the NO pathway might play a role in the pathophysiology related to congenital urea cycle disorders.

Quantitative comparison of diversity and conformity in nitrogen recycling of ruminants.

Abstract: Domestic ruminant animals are reared in diverse production systems, ranging from extensive systems under semi-arid and tropical conditions with poor feed resources to intensive systems in temperate and cold areas with high quality feed. Nitrogen (N) recycling between the body and gut of ruminants plays a key role in the adaptation to such diverse nutritional conditions. Ammonia and microbial protein produced in the gut and urea synthesized in the liver are major players in N-recycling transactions. In this review, we focus on the physiological factors affecting urea production and recycling. Sheep and buffalo probably have higher abilities to reabsorb urea from the kidney compared with cattle. This affects the degree of urea-N recycling between the body and gut at both low and high N intakes. The synthesis and gut entry of urea also differs between cattle bred for either dairy or beef production. Lactating dairy cows show a higher gut entry of urea compared with growing cattle. The synthesis and recycling of urea dramatically increases after weaning, so that the functional development of the rumen exerts an essential role in N transactions. Furthermore, high ambient temperature increases urea production but reduces urea gut entry. An increase in total urea flux, caused by the return to the ornithine cycle from the gut entry, is considered to serve as a labile N pool in the whole body to permit metabolic plasticity under a variety of physiological, environmental and nutritional conditions.

Arginase isoenzymes in human cirrhotic liver.

Abstract: Cirrhosis leads to an inability of the liver to perform its biochemical functions. It can also lead to hepatocellular carcinoma in which, as we showed lately, arginase isoenzyme pattern changes. The present work presents our results on arginase isoenzymes and their possible role in liver cirrhosis. The study was performed on tissues obtained during liver transplantation from 60 patients with liver cirrhosis, and on samples of histologically normal liver (control) from 40 patients with benign or colorectal cancer liver metastases removed during surgery, 6-7 cm from the tumor border. Arginase isoenzymes AI (so-called liver-type arginase) and AII (called extrahepatic arginase) were identified by Western blotting and isolated by ion-exchange chromatography. Their expression on mRNA level was studied by RT-PCR. A significant decrease in arginase activity, dependent of the liver clinical stage, was observed in cirrhotic tissue. Arginase AI activity and its mRNA level were significantly decreased in cirrhotic liver, whereas the activity and expression of arginase AII were concurrently raised, as compared to normal liver. Since arginase AI is a key enzyme of the urea cycle, whereas arginase AII most probably takes part in the biosynthesis of ornithine and polyamines, the defective ammonia inactivation and increased collagen biosynthesis observed in cirrhotic liver may be related to the changes in arginase AI and AII levels, respectively.

1H NMR spectroscopic study of blood serum for the assessment of liver function in liver transplant patients.

Abstract: Background and Aims: Plausible reasons for the failure of liver graft in liver transplantation are explored. 1 H-NMR spectroscopy of serum is employed for assessment of liver graft function. Differences in concentrations of specific metabolites between patients with successful and unsuccessful liver grafts following transplantation were used as possible markers to assess the graft quality. Methods: Blood samples from the patients undergoing liver transplantation were obtained preoperatively, immediately after transplant followed by every 24 hrs of post-transplantation until patients were discharged or expired. 1 H-NMR spectroscopic studies of serum were performed at each time point and concentrations of various metabolites measured. Conventional biological tests were also performed at each time point. Results: Elevation of concentrations of the nine metabolites (lactate, alanine, lysine, glutamine, methionine, asparagine, tyrosine, histidine and phenylalanine) in nonsurvivors using NMR was attributed to the graft dysfunction. The information on the graft dysfunction using conventional biological tests was obtained much later. However, elevation in aminotransferases and bilirubin levels was indicated after about one week and 3 days respectively in non-survivors. Hepatic failure causes alteration in the concentrations of amino acids due to impairment of amino acid metabolism and urea cycle. 1 H-NMR spectroscopy provides the information of all the metabolites in a single step without involving any chemical pretreatment implying better accuracy since each step involved can introduce its own experimental error. Conclusion: Distinct metabolic profile in non-survivors compared to survivors following transplantation promises potential of 1 H-NMR studies in the assessment of liver graft function.

First example of hepatocyte transplantation to alleviate ornithine transcarbamylase deficiency, monitored by NMR-based metabonomics.

Abstract: We demonstrate the effective use of NMR spectroscopic profiles of urine and plasma from the first successful use of hepatocyte transplantation as a bridge to auxiliary partial orthotopic liver transplantation in a child antenatally diagnosed with severe ornithine transcarbamylase deficiency. In this single-patient study, NMR profiles indicated that the disrupted urea cycle could be normalized by hepatocyte cell infusion and this was confirmed using orthogonal partial least-squares-based chemometrics. However, despite dietary manipulations and adminstration of ammonia scavengers, the desired reduction in plasma ammonia was not consistently achieved between sessions of hepatocyte transplantation due to episodes of sepsis. A subsequent liver transplant corrected the metabolic abnormalities. The use of metabolic profiling has been shown to be a promising method for evaluating the efficacy of cell infusions and has demonstrated the capability for the early detection of response to therapy in real time, an approach that may be of use in wider clinical settings.

Inborn Errors of Metabolism: Part 2: Specific Disorders

Abstract: 1. Paul A. Levy, MD* 1. *Assistant Professor of Pediatrics and Pathology, Children's Hospital at Montefiore, Bronx, NY The following article is included online only as a second part of the article “Inborn Errors of Metabolism: Part 1.” There is no one prototypical disorder of amino acid metabolism; each disorder has its own unique collection of symptoms. Four well-described amino acid disorders have been chosen as examples of this group. Phenylketonuria, a disorder of phenylalanine metabolism, leads to intellectual disability if untreated. Maple syrup urine disease involves an enzyme common to the degradation of the branched-chain amino acids (leucine, isoleucine, and valine). Although there are five subtypes of maple syrup urine disease, the classic form has a neonatal onset and generally progresses from poor feeding to coma and death if not treated. Tyrosinemia also has multiple subtypes. Hepatorenal tyrosinemia (type I) may present with liver failure (elevated transaminase concentrations, hyperbilirubinemia, coagulopathy, ascites, and gastrointestinal bleeding) as well as kidney involvement (tubular dysfunction) and peripheral nerve involvement (painful crises, weakness or paralysis). Type II tyrosinemia is an oculocutaneous form of the disease that has corneal lesions and skin findings. Homocystinuria, most commonly caused by cystathionine beta-synthase deficiency, presents with ocular (ectopia lentis), skeletal (marfanoid features such as dolichostenomelia and arachnodactyly), vascular (thromboembolic), and central nervous system (intellectual disability, stroke, and seizures) abnormalities. When an amino acid disorder is suspected, measurement of plasma amino acids generally is sufficient to make the diagnosis. Assessment of urine amino acids can be helpful for homocystinuria and some abnormalities of amino acid transport (cystinuria, dicarboxylic amino aciduria) that affect the kidneys and for detecting generalized amino aciduria found with some kidney disease and mitochondrial disorders. The degradation of amino acids results in their deamination, generating ammonia as the waste nitrogen. The urea cycle removes the excess ammonia by generating urea, which is eliminated in the urine. Six disorders of the urea cycle are known. …

A novel mutation in ARG1 gene is responsible for arginase deficiency in an Asian family.

Abstract: Argininemia is a rare autosomal recessive metabolic disorder caused by a deficiency in the arginase enzyme, which is the final enzyme in the urea cycle and responsible for the hydrolysis of arginine to urea and ornithine. The disease becomes symptomatic during childhood and is characterized by progressive spastic quadriplegia, progressive mental impairment, growth retardation, and periodic episodes of hyperammonemia. At least 19 distinct mutations in the ARG1 gene have been identified indicating the molecular heterogeneity of this condition. We report a homozygous novel mutation (c.93 delG) in the ARG1 gene from 3 affected children of a Pakistani family living in the United Arab Emirates. The mutation is expected to lead to a frame shift after the thirtieth residue and a stop codon at residue 44 (p.T30fsX14). Therefore, this mutation is expected to result in complete loss-of-function of the arginase enzyme and therefore is the mostly likely cause of argininemia in this family.

Hepatic amino acid transport primary to the urea cycle in regulation of biologic neutrality.

Abstract: A biologic determination appears to be made as to whether the nitrogenous portion of food amino acids reaches the kidney for excretion as ammonium ion, or whether it is sent to the liver to form urea. It now becomes likely that this determination occurs primarily by hydrogen ion inhibition of at least one transport system by which the liver receives amino acids, and not by regulation applied directly to the liver-ornithine cycle.

Hyperammonemia from a Urea Cycle Disorder Presenting in Adulthood

Abstract: OBJECTIVE: The aim of this report is to describe a patient with late presentation of carbamyl phosphate syn- thetase I (CPS-I, EC 6.3.4.16) deficiency, a rare urea cycle deficiency, and to facilitate recognition and treatment of pa- tients presenting with encephalopathy and hyperammonemia in a critical care setting. DESIGN: Case Report. SETTING: Intensive care unit of Saint Mary's Hospital, Mayo Clinic, Rochester, Minnesota. PATIENT: A 65-year-old woman ad- mitted with progressive encephalopathy. INTERVENTIONS: Intubation and mechanical ventilation, protein-restricted parenteral nutrition, intravenous arginine, hemodiafiltration, and intravenous antibiotic therapy. MEASUREMENTS AND MAIN RESULTS: Serum ammonia and glutamine levels were elevated, but other laboratory and imaging investigations were unremarkable. Despite the above interventions, her mental status deteriorated. She developed ventilator associated pneumonia, which worsened despite antibiotic treatment. The family decided to withdraw care and the patient expired on hospital day 10. A postmortem enzyme assay on fresh-frozen liver tissue showed severely diminished CPS-I activity. CONCLUSIONS: To our knowledge, this is the oldest reported age at presentation of CPS-I deficiency. Urea cycle disor- ders should be part of the differential diagnosis of hyperammonemia regardless of age as early treatment may ameliorate mortality and morbidity in these patients.

Arginine metabolism in a small animal model of sepsis and after hemihepatectomy

Abstract: Asymmetric dimethylarginine (ADMA) is an inhibitor of the arginine–NO pathway. ADMA accumulates when degradation in the liver by dimethylarginine dimethylaminohydrolase is impaired. In theory, plasma citrulline, formed when arginine is converted by NO synthase, and when ADMA is metabolized, would be lowered and ornithine, formed by the degradation of arginine in the urea cycle, would be potentially elevated when ADMA accumulates as in sepsis and in liver failure [1].

Consenso para o tratamento nutricional das Doenças do Ciclo da Ureia

Abstract: The urea cycle disorders result from several enzy- matic deficiencies on the nitrogen excretion metabolic path- way. All the diseases have autosomal recessive inheritance with the exception of ornithine transcarbamoylase deficiency, which is X-linked. The clinical presentation may develop in the neonatal period, during infancy, or even during adoles- cence or adulthood. The main biochemical markers of urea cycle disorders are the hyperammonaemia and the abnormal blood and urinary concentrations of amino acids and organic acids. The nutritional treatment is based on a low natural pro- tein diet, supplemented with an essential amino acid mixture. The protein ingestion must be rigorously established accord- ing to the metabolic control, anthropometric evaluation and pharmacological therapy used for the alternative pathways for nitrogen excretion. Monitoring the nutritional status will be crucial in the management of these diseases in order to better characterize the nutritional needs, preventing states of protein insufficiency which may affect growth and development.