Ammonium Metabolism in Humans

METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX

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Ammonium metabolism in humans

Maria M. Adeva a,⁎, Gema Souto b, Natalia Blanco c, Cristóbal Donapetry a a Hospital General Juan Cardona b Clinical Center of the National Institutes of Health c United Surgical Partners. La Coruña

ARTICLE INFO ABSTRACT

Article history: Free ammonium ions are produced and consumed during cell metabolism. Glutamine Received 23 March 2012 synthetase utilizes free ammonium ions to produce glutamine in the cytosol whereas Accepted 16 July 2012 glutaminase and glutamate dehydrogenase generate free ammonium ions in the mitochondria from glutamine and glutamate, respectively. Ammonia and bicarbonate are Keywords: condensed in the liver mitochondria to yield carbamoylphosphate initiating the urea cycle, Metabolic alkalosis the major mechanism of ammonium removal in humans. Healthy kidney produces Glutamate dehydrogenase ammonium which may be released into the systemic circulation or excreted into the Glutamine synthetase urine depending predominantly on acid–base status, so that metabolic acidosis increases Glutaminase urinary ammonium excretion while metabolic alkalosis induces the opposite effect. Brain Hyperammonemia and skeletal muscle neither remove nor produce ammonium in normal conditions, but they are able to seize ammonium during hyperammonemia, releasing glutamine. Ammonia in gas phase has been detected in exhaled breath and skin, denoting that these organs may participate in nitrogen elimination. Ammonium homeostasis is profoundly altered in liver failure resulting in hyperammonemia due to the deficient ammonium clearance by the diseased liver and to the development of portal collateral circulation that diverts portal blood with high ammonium content to the systemic blood stream. Although blood ammonium concentration is usually elevated in liver disease, a substantial role of ammonium causing hepatic encephalopathy has not been demonstrated in human clinical studies. Hyperammonemia is also produced in urea cycle disorders and other situations leading to either defective ammonium removal or overproduction of ammonium that overcomes liver clearance capacity. Most diseases resulting in hyperammonemia and cerebral edema are preceded by hyperventilation and respiratory alkalosis of unclear origin that may be caused by the intracellular acidosis occurring in these conditions. © 2012 Elsevier Inc. All rights reserved.

+ Abbreviations: NH3, ammonia; NH4, ammonium ions; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BCAT, branched-chain amino acids aminotransferase; KGA, kidney-type glutaminase; GAC, glutaminase C; GAM, glutaminase M; GDH1, glutamate dehydrogenase-1; GDH2, glutamate dehydrogenase-2; NAGS, N-acetylglutamate synthase; CPS1, carbamoylphosphate synthetase-1; OTC, ornithine transcarbamylase; ASL, argininosuccinate lyase; ORNT, mitochondrial ornithine transporter; HHH, hyperornithinemia, hyperammonemia and homocitrullinuria; IMP, inosine monophosphate; ALF, acute liver failure; UCD, urea cycle disorder; CPT1, carnitine palmitoyltransferase-1; CPT2, carnitine palmitoyltransferase-2; CACT, carnitine-acylcarnitine translocase; MCAD, medium-chain acyl-CoA dehydrogenase. ⁎ Corresponding author. Hospital General Juan Cardona, c/ Pardo Bazán s/n, 15406 Ferrol, La Coruña, Spain. Tel.: +34 664 527 257; fax: +34 981 17 81 59. E-mail address: [email protected] (M.M. Adeva).

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 2 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX

Molecular nitrogen (N2) present in the earth atmosphere has and 11 [8,14]. Glutamine synthetase expression and activity to be reduced to ammonia (NH3) by nitrogen-fixing bacteria have been detected in adult human skin, peripheral lympho- living independently in the soil or in the root of leguminous cytes, liver, brain (predominantly in astrocytes), and the plants before it may be utilized by humans. Ammonia gastrointestinal tract, where the activity is highest in the + dissolves in water to form ammonium ions (NH4) and this stomach, although the esophagus and both small and large form of reduced nitrogen is assimilated into amino acids and intestine have little synthesizing capacity [15,16]. In human other nitrogen-containing molecules. In aqueous solutions, skin, glutamine synthetase expression is predominantly ammonia is a base (any compound accepting hydrogen ions) associated with developing keratinocytes. It is present in all forming a conjugated pair with the ammonium ion, according layers of epidermis in young persons and primarily in the + ↔ + to the reversible reaction: NH3 +H NH4 stratum granulosum of elderly persons [17]. Dexamethasone The pKa of the reaction is 9.3, indicating that at this pH and the exposure to ammonium ions strongly induce the + value, the concentration of the ionized (NH4) and unionized activity of glutamine synthetase in spontaneously immortal- (NH3) forms is equal. When the pH of the solution is less than ized human keratinocytes [17]. Glutamine synthetase expres- 9.3, hydrogen ions are incorporated to ammonia to yield sion is also remarkably induced by glucocorticoids in human ammonium ions. Therefore, at physiological plasma and osteoblastic-like cells, while vitamin D inhibits basal and intracellular pH values, virtually only the protonated moiety glucocorticoid-stimulated glutamine synthetase activity by + (NH4) is present in aqueous solutions [1]. The concentration of affecting both the mRNA and protein levels of the enzyme [18]. ammonium in normal human plasma ranges between 11 and In astrocytes, the activity of the enzyme is suppressed by μ 50 mol/L and varies slightly in venous, arterial or capillary increasing the ADP concentration, which may be expected to blood. Free ammonium ions are continually produced and occur when the energy level of the cell is reduced [16]. consumed during cell metabolism in human body tissues. Glutamine synthetase is present at early gestational stages They arise during the breakdown of purine and pyrimidine in human fetuses and placenta [19,20].Humanskeletal derivatives, polyamines, and deamination of several amino muscle is capable of synthesizing glutamine, both during the acids, including glutamine, asparagine, serine, threonine, postprandial period [21,22] and the postabsorptive state [23]. glycine, histidine, lysine, proline, hydroxyproline, homocys- Skeletal muscle of healthy individuals also synthesizes teine, and cystathionine [2]. Some free ammonium ions may glutamine following an intravenous infusion of leucine [21] occasionally be supplied by urease-producing urea-splitting or a mixture of amino acids not containing glutamine [24]. organisms present in saliva [3], gastrointestinal tract [4,5], Congenital deficiency of glutamine synthetase has been urine [6], or other locations [7]. Free ammonium ions are rarely reported. A clinical picture with severe brain malforma- principally consumed to produce glutamine in the cytosol of tions, neonatal seizures, blistering skin lesions, multiorgan some human cells (principally skeletal muscle cells, hepato- failure, and frequent neonatal death has been associated with cytes, keratinocytes, gastrointestinal cells, lymphocytes, and homozygous mutations on the glutamine synthetase gene astrocytes) [8] and to generate carbamoylphosphate predom- [25,26]. Hyperammonemia has not been a consistent finding inantly inside liver mitochondria [9]. Ammonia in gas phase in the few patients reported with congenital glutamine has been detected in human skin [10] and exhaled air [11]. deficiency [25,26]. The activity of glutamine synthetase was The enzymes primarily involved in the metabolism of free markedly diminished with a concomitant reduction in the ammonium ions are the cytosolic enzyme glutamine synthe- amount of glutamine synthetase protein in the liver of two tase [8] and the mitochondrial enzymes glutaminase [12] and patients who had fatal hyperammonemia after orthotopic glutamate dehydrogenase [13]. In a reaction similar to the lung transplantation [27]. There may be a connection between glutaminase reaction, asparaginase yields free ammonium glutamine synthetase expression and some human tumors ions and aspartate from asparagine [2]. In addition, the associated with activating mutations in the gene encoding β- enzyme carbamoylphosphate synthetase-1 catalyzes the catenin, CTNNB1. In children, activating mutations in CTNNB1 condensation of bicarbonate and ammonia to form carba- occur in 80% of hepatoblastoma and 31% of nephroblastoma moylphosphate inside the mitochondrial matrix, starting the tumors. In hepatoblastoma with activated β-catenin, expres- urea cycle in the liver [9]. Aminotransferases (transaminases) sion of glutamine synthetase is detected in tumor areas with are both cytosolic and mitochondrial enzymes engaged in epithelial, but not with mesenchymal differentiation. Gluta- transferring amino groups between amino acid and keto acid mine synthetase expression was not observed in CTNNB1- pairs, without generating or consuming free ammonium ions. mutated nephroblastoma [28]. Important human transaminases include aspartate aminotransferase (AST), alanine aminotransferase (ALT) and branched-chain amino acids aminotransferases (BCAT) [2]. 2. Glutaminase

Glutaminase is a phosphate-activated mitochondrial matrix 1. Glutamine synthetase enzyme that catalyzes the hydrolysis of the amide group of glutamine to stoichiometric amounts of glutamate and free + Glutamine synthetase is a cytosolic enzyme that catalyzes the NH4 (Fig. 2). Two human genes with considerable degree of + synthesis of glutamine from glutamate and free NH4 in an sequence similarity, GLS1 and GLS2, encode several glutamin- ATP-dependent reaction (Fig. 1). The human gene encoding ase isoenzymes. Human glutaminase-1 gene (GLS1) is located glutamine synthetase has been mapped to chromosome 1q25. on chromosome 2q32 and it is thought to encode an initial Related genes of unclear significance lie on chromosomes 5, 9, mRNA transcript that undergoes tissue-specific alternative

- COO O = C – NH2

CH2 CH2

CH2 CH2 + NH4+ + + HC – NH3 HC – NH3

Glutamine COO- COO- synthetase Glutamate Glutamine Fig. 3 – Asparaginase reaction.

Fig. 1 – Glutamine synthetase reaction.

- O = C – NH2 COO

CH2 CH2

CH2 CH2 + NH4+ + + Fig. 4 – Glutamate dehydrogenase reaction. HC – NH3 HC – NH3

Glutaminase COO- COO- activity, while interleukin-1, interleukin-6, tumor necrosis Glutamine Glutamate factor (TNF)-α, and interferon-γ have been shown to decrease the activity of the enzyme in human cells [32]. Glutaminase Fig. 2 – Glutaminase reaction. activity in human airway epithelial cells is enhanced in response to acidic challenge while it is inhibited by interferon-γ and TNF-α [11]. Glutaminase may have a role in human cancer. splicing generating three splice variants that are subsequently Human p53 gene exerts its tumor suppression action through translated into three human glutaminase isoenzymes: gluta- the transcriptional regulation of its target genes. Human minase-1 or kidney-type glutaminase (KGA), glutaminase C glutaminase-2 gene has been identified as a p53 target so that (GAC), and glutaminase M (GAM). The human GLS1 gene spans p53 activation induces expression of GLS2 mRNA and therefore 82 kb and is composed of 19 exons [12,29]. The full-length GLS2 may contribute to the role of p53 in tumor suppression by crystal structure of human GAC has been recently determined mediating some of its effects. GLS2 expression is lost or greatly [30]. Human glutaminase-2 gene (GLS2) is located on chromo- reduced in hepatocellular carcinomas and overexpression of some 12q13 and encodes glutaminase-2 or liver-type gluta- GLS2 reduces tumor cell colony formation [33,34]. On the other minase, a protein functionally similar to kidney-type hand, the oncogene Myc up-regulates glutaminase C expression glutaminase but a little smaller in size [31]. Glutaminase and promotes tumor cell proliferation in human B lymphoma expression and activity have been detected in numerous and prostate cancer cells [35] It appears that GLS1 and GLS2 may human tissues, including kidney, liver, brain (predominantly have different roles in tumorigenesis. The tumor suppressor in neurons), pancreas, airway epithelium, cardiac muscle, gene p53 induces the expression of GLS2 but not GLS1 in various skeletal muscle, small intestine, platelets, and fibroblasts. cells, whereas the oncogene Myc induces the expression of GLS1 Glutaminase-1 or kidney-type glutaminase is expressed but not GLS2 [33]. predominantly in kidney and brain [12,29]. Human airway epithelial cells also express mRNA for KGA and they produce ammonium ions stoichiometrically from glutamine [11]. 3. Asparaginase Glutaminase C is expressed principally in pancreas and cardiac muscle, but also appreciably in kidney, lung, and Analogous to the actions of glutaminase on glutamine, placenta [12,29]. Human airway epithelial cells express mRNA asparaginase catalyzes the conversion of asparagine into for GAC as well [11]. Glutaminase M is expressed in cardiac aspartate and a free ammonium ion (Fig. 3). Bacterial andskeletalmuscle[12]. Human tissue distribution of asparaginase has been used for decades to treat some glutaminase-2 or liver-type glutaminase is not well defined. lymphoproliferative disorders. It breaks down plasma aspar- While normal human hepatocytes express liver-type gluta- agine, interrupting the supply of this amino acid to cancer minase, hepatoma cells express the kidney-type isoform [29]. cells present in the blood, which lack asparagine synthetase In the human gastrointestinal tract, glutaminase specific and rely on blood-borne asparagine to survive. Asparaginase activity is highest in the small intestine, intermediate in the administration may be associated with severe side-effects, large intestine and lowest in the esophagus and stomach. including hyperglycemia, hypertriglyceridemia due to in- Therefore, both the small and large intestines have a high crease of endogenous synthesis of very low density lipopro- potential for glutamine hydrolysis, unlike the stomach and teins, pancreatitis, hepatotoxicity (diffuse liver steatosis and the esophagus [15]. Glucocorticoids enhance glutaminase very rarely acute fulminant hepatic failure), acquired

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 4 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX antithrombin III deficiency, and thrombotic events [36]. L for men [42]. Alcoholics have higher serum glutamate Hyperammonemia has rarely been reported following aspar- dehydrogenase activity than normal individuals and the aginase use [37]. enzyme activity decreases rapidly after cessation of drinking [43,44]. Increased serum glutamate dehydrogenase activity in 3.1. Glutamate dehydrogenase alcoholics may be used as an index of the elapsed time from the last alcohol intake [43,44]. Deficiency of glutamate Glutamate dehydrogenase is an enzyme that localizes pre- dehydrogenase activity has been associated with a poorly dominantly to the mitochondrial matrix (and to a lesser defined neurological degenerative disorder with extrapyrami- extent to the rough endoplasmic reticulum) and catalyzes the dal features (atypical Parkinson's disease), cerebellar dysfunc- reversible oxidative deamination of glutamate to produce α- tion, peripheral neuropathy, and anterior horn cell signs, ketoglutarate and a free ammonium ion [13]. The oxidative suggesting that the phenotypic expression of the enzymatic deamination of glutamate uses NAD+ as cofactor and releases deficiency may be heterogeneous [45].Gainoffunction H+ and the reduced compound NADH, whereas NADPH and (activating) mutations on the GLUD1 gene increase GDH1 H+ are consumed in the synthetic reaction, releasing NADP+ activity by rendering this isoenzyme resistant to the allosteric (Fig. 4). Although reversible in the test tube, it is thought that in inhibition by GTP while retaining the stimulatory action of vivo the glutamate dehydrogenase reaction proceeds predom- ADP and L-leucine, resulting in a constitutive enzyme inantly toward the direction of the oxidative deamination of overactivity that leads to the hyperinsulinism hyperammo- glutamate, producing α-ketoglutarate and free ammonium nemia syndrome [38,39]. Affected patients manifest hyper- ions, probably due to the high concentration of glutamate and ammonemia and recurrent fasting or postprandial the low level of free ammonium ions usually present inside the hypoglycemia episodes related to enhanced insulin release mitochondria under baseline conditions. However, high con- by pancreatic β-cells thought to be due to an excess of centration of ammonium ions and α-ketoglutarate inside the intracellular ATP. In the liver, it is believed that overactivity of mitochondria might trigger biosynthesis of glutamate by GDH1 leads to hyperammonemia by increasing free ammoni- glutamate dehydrogenase [38,39]. um ions generation due to unrestrained oxidation of gluta- Two highly homologous human genes, GLUD1 and GLUD2, mate. Additionally, GDH1 overactivity might result in encode two glutamate dehydrogenase isoenzymes. GLUD1 is glutamate depletion, reducing N-acetylglutamate formation located to chromosome 10q and encodes the isoenzyme and consequently preventing urea cycle initiation [39]. glutamate dehydrogenase-1 (GDH1). GLUD1 is a housekeeping gene widely expressed in human tissues, including liver, 3.2. Aminotransferases (transaminases) kidney, pancreatic β-cells, brain, heart, intestine, spleen, skin, lymph nodes, leukocytes, fibroblasts, and placenta. GLUD2 is Aminotransferases catalyze the reversible transfer of α-amino an intronless gene mapped to Xq chromosome that encodes groups from α-amino acids to α-ketoacids without producing the glutamate dehydrogenase-2 isoenzyme (GDH2), being or consuming free ammonium ions. The reversible transport of predominantly expressed in retina, brain, and testicular tissue amino groups between alanine/pyruvate, aspartate/oxaloacetate, in humans [38]. In testicular tissue, GDH2 is highly expressed and branched-chain amino acids/branched-chain keto acids, in Sertoli cells and to some extent in Leydig cells, while takes place via coupled reactions with the partner pair gluta- spermatogonia and differentiated germ cells are negative for mate/α-ketoglutarate (Fig. 5). Alanine aminotransferase (ALT) this protein. In cerebral cortex, the expression of GDH2 is transports the α-amino group from alanine to α-ketoglutarate restricted to astrocytes, with neurons showing only faint with the generation of pyruvate and glutamate. Aspartate immunoreactivity. Human liver does not express endogenous aminotransferase (AST) catalyzes the transfer of α-amino groups GDH2 [40]. from aspartate to α-ketoglutarate with the formation of oxaloac- Glutamate dehydrogenase activity is regulated by allosteric etate and glutamate. The transfer of α-amino groups from the effectors. ADP and L-leucine are allosteric activators whereas branched-chain amino acids (leucine, isoleucine, and valine) to GTP, ATP, long-chain fatty acids (palmitoyl-CoA), estrogens, α-ketoglutarate, generating their cognate branched-chain keto epigallocatechin gallate (a polyphenol in green tea), and acids (α-ketoisocaproate, α-keto-β-methylglutarate, and α NADH act as allosteric inhibitors [38,39]. The two glutamate -ketoisovalerate, respectively) and glutamate, is performed by dehydrogenase isoenzymes differ in their allosteric regula- branched-chain amino acids aminotransferase (BCAT). Pyridox- tion. GDH1 activity is strongly inhibited by GTP while GDH2 is al phosphate, the functional form of vitamin B6, is a cofactor for resistant to the inhibitory effect of GTP, so that this isoenzyme aminotransferases reactions. is able to metabolize glutamate even when the housekeeping GDH1 protein is inhibited by sufficient amounts of GPT. In 3.3. Urea cycle enzymes addition, GDH2 depends on ADP for catalytic function. GDH2 maintains a very low baseline activity (less than 10% of its Human hepatocytes utilize two end-products of metabolism, capacity), its activation being dependent on rising ADP or L- ammonia and carbon dioxide (under the form of bicarbonate), leucine levels [38,39]. Estrogens inhibit to a greater extent the to generate some amino acids (citrulline, arginine, and GDH2 isoenzyme than the GDH1 isoform. The inhibition of the ornithine) through a chain of reactions referred to as ornithine two isoproteins by estrogens is inversely related to their state cycle, Krebs–Henseleit cycle or urea cycle (Fig. 6). Urea and of activation induced by ADP [41]. Serum glutamate dehydro- fumarate are produced while aspartate is consumed as a genase activity in healthy individuals is slightly gender- result of the cycle functioning. Activity of the mitochondrial dependent. The upper limit is 6.4 U/L for women and 11.0 U/ isoenzyme of carbonic anhydrase is thought to be required to

Alanine Aspartate BCAA (+α-ketoglutarate) (+α-ketoglutarate) (+α-ketoglutarate)

ALT AST BCAT

Glutamate Glutamate Glutamate (+pyruvate) (+oxaloacetate) (+BCKA)

Fig. 5 – Aminotransferases reaction. ALT: alanine aminotransferase; AST: aspartate aminotransferase; BCAA: branched-chain amino acids; BCAT: branched-chain amino acids aminotransferase; BCKA: branched-chain keto acids.

from glutamate and acetyl-CoA by N-acetylglutamate synthase, activating the enzyme carbamoylphosphate synthetase-1 to initiate the urea cycle. Briefly, bicarbonate and ammonia are condensed in the mitochondrial matrix of hepatocytes to produce carbamoylphosphate by carbamoylphosphate synthetase-1. Carbamoylphosphate is combined with ornithine entering from the cytoplasm by ornithine transcarbamylase to produce citrulline, which exits the mitochondria. In the cytosol, the condensation of citrulline and aspartate by argininosuccinate synthetase-1 yields argininosuccinate, which is transformed into arginine by argininosuccinate lyase, liberating the carbon skeleton of aspartate in the form of fumarate. Arginine is split by arginase-1 producing ornithine and urea. Ornithine travels from the cytosol to the mitochondrial matrix via the ornithine transporter to complete the cycle. Several unarchived polymorphisms have been identified that may be implicated in the inter-individual variation observed in urea cycle function [9].

3.3.1. N-acetylglutamate synthase The formation of N-acetylglutamate from glutamate and acetyl-CoA inside the mitochondrial matrix is catalyzed by the enzyme N-acetylglutamate synthase (NAGS). N-acetylglutamate is an obligatory allosteric activator of carbamoylphosphate synthetase-1, being therefore required to initiate the urea cycle. The human NAGS gene is located on chromosome 17q21.31, being expressed in adult liver and small intestine as Fig. 6 – Urea cycle. NAGS: N-acetylglutamate synthase; CPS1: well as in fetal liver. The intestinal transcript is smaller in carbamoylphosphate synthetase-1; ORNT: mitochondrial size than the liver transcript. The NAGS gene is not expressed ornithine transporter; OTC: ornithine transcarbamylase; in adult brain, colon, heart, kidney, lung, muscle, placenta, ASL: argininosuccinate lyase; ASS1: argininosuccinate spleen, stomach or testis [47,48]. The activity of human NAGS synthetase-1. NAGS, CPS1, and OTC reactions take place in is augmented by the presence of arginine [49] and inhibited the mitochondrial matrix. ASS1, ASL, and arginase-1 by xanthine and uric acid [50]. Deficiency of NAGS activity reactions occur in the cytosol. may be congenital or secondary to poorly defined causes, such as primary carnitine deficiency [47,51]. Inherited NAGS deficiency is an autosomal recessive disorder whose phenotypic expression is similar to carbamoylphosphate supply bicarbonate from carbon dioxide, as urea synthesis in synthetase-1 deficiency. Neonatal presentation with respi- healthy humans is inhibited by acetazolamide [46]. Amino ratory alkalosis, hyperammonemia and coma usually occurs groups derived from various amino acids are funneled by when the disease-producing mutations abolish NAGS hepatic aminotransferases to glutamate, which is deaminated enzymatic activity whereas patients with partial NAGS in the mitochondria by glutamate dehydrogenase, producing deficiency may present later in life with less severe free ammonium ions and α-ketoglutarate. Some amino acids manifestations including recurrent vomiting and neurobe- and other nitrogen-containing compounds do not participate havioral changes [52]. N-carbamylglutamate is a functional in transamination reactions, being directly deaminated to analog of N-acetylglutamate that activates carbamoylpho- generate free ammonium ions. When the rate of amino acid sphate synthetase-1 and restores urea cycle function in catabolism increases, N-acetylglutamate is also synthesized patients with NAGS deficiency [52]. The NAGS inhibition by

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 6 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX xanthine and uric acid is reversed by supplementation with disease characterized by tissue accumulation of argininosuc- N-carbamylglutamate [50]. cinate and excessive excretion of this compound in urine. Similarly to other urea cycle disorders except arginase-1 3.3.2. Carbamoylphosphate synthetase-1 deficiency, ASL deficiency leads to reduced arginine synthesis. The enzyme carbamoylphosphate synthetase-1 (CPS1) cata- Patients with ASL deficiency share the acute clinical phenotype lyzes the condensation of bicarbonate and ammonia to form of hyperammonemia, encephalopathy, and respiratory alka- carbamoylphosphate in the mitochondrial matrix, being depen- losis common to other urea cycle defects, but they also display dent on N-acetylglutamate for activity. The carbamoylphos- chronic neurological symptoms that seem to be inevitable in phate synthetase reaction takes place at the expense of two spite of careful treatment of hyperammonemia, believed to be molecules of ATP and is essentially irreversible. The human caused by a combination of tissue specific deficiency of gene encoding CPS1 has been mapped to chromosome 2 [53] arginine and/or elevation of argininosuccinate [64,65]. and a compilation of 222 molecular changes has been reported [54]. A gene coding a novel isoform of CPS1 that shows high 3.3.6. Arginase-1 expression in human testicular tissue has been cloned [55]. Arginase is the enzyme that catalyzes the hydrolysis of arginine CPS1 has been shown to be among the most antigenically to ornithine and urea. In humans, two isoenzymes of arginase dominant proteins in human liver mitochondria [56].Immuno- have been demonstrated. Arginase-1 is a cytosolic protein, histochemical and Western blot analyses reveal strong and while arginase-2 is located to the mitochondrial matrix [66]. diffuse CPS1 expression within normal human small intestine Arginase-1 protein has been found in human liver, erythrocytes, mucosa and small-intestinal adenomas while protein expres- granulocytes, kidney, brain, and gastrointestinal tract, whereas sion is lost in adenocarcinomas [57]. Congenital CPS1 deficiency arginase-2 is detected in kidney, brain, gastrointestinal tract, is an autosomal recessive disorder that usually presents during and fibroblasts. Therefore, arginase-1 is the only isoenzyme the neonatal period with severe hyperammonemia and coma, found in liver and red blood cells while kidney, brain, and but a delayed onset form has also been observed [53].Secondary gastrointestinal tract express the two arginase isoproteins low CPS1 activity in liver has been reported in a patient with [67–69]. The human gene encoding arginase-1 has been mapped hyperinsulinism hyperammonemia syndrome [58]. to chromosome 6q23 [70]. Mutations on this gene cause deficiency of arginase-1 activity and congenital argininemia, a 3.3.3. Ornithine transcarbamylase rare autosomal recessive disorder that shows phenotypic Ornithine transcarbamylase (OTC) catalyzes the synthesis of variability, similarly to other urea cycle disorders. Most patients citrulline from carbamoylphosphate and ornithine that enters experience progressive mental impairment and neurologic the mitochondria from the cytosol. The human gene encoding manifestations such as spastic tetraplegia during childhood. OTC is located on Xp21.1 [59,60]. OTC deficiency is an X-linked Neonatal hyperammonemia is uncommon but metabolic crisis disease with phenotypic heterogeneity that usually presents may occur later in life [71–73]. with neonatal hyperammonemia, respiratory alkalosis and cerebral edema, although it also may be diagnosed in 3.3.7. Mitochondrial ornithine carrier (ORNT) adulthood or discovered in asymptomatic patients with only In humans, the transport of ornithine from the cytosol to the biochemical abnormalities. Episodes are usually triggered by mitochondrial matrix across the mitochondrial membrane is catabolism, such as infections, and pregnancy is an additional carried out by the mitochondrial ornithine transporter risk [59,60]. (ORNT). Mutations in the human mitochondrial ornithine carrier-1 abolish its transport activity and cause the hyper- 3.3.4. Argininosuccinate synthetase-1 ornithinemia, hyperammonemia and homocitrullinuria The enzyme argininosuccinate synthetase-1 (ASS1) combines (HHH) syndrome [74,75]. citrulline and aspartate in the cytosol of hepatocytes to generate argininosuccinate. Citrulline and aspartate leave 3.3.8. Citrin (aspartate–glutamate carrier) the mitochondrial matrix and enter the cytoplasm through The aspartate export from the mitochondrial matrix to the the ornithine transporter and the aspartate–glutamate carrier cytosol in the liver is performed by citrin, an aspartate– (citrin), respectively. The gene encoding ASS1 is located on glutamate carrier that catalyzes a 1:1 exchange of aspartate chromosome 9q34.1 and mutations producing ASS1 deficien- for glutamate and a proton, being therefore electrogenic. The cy have been recently compiled [61]. Congenital deficiency of human gene encoding citrin (SLC25A13) has been localized to ASS1 activity causes type 1 citrullinemia, an autosomal chromosome 7q21.3. Mutations on the SLC25A13 gene produce recessive disorder with phenotypic variability that ranges defective citrin activity reducing aspartate export from the from severely affected patients with neonatal hyperammo- mitochondria to the cytosol and leading to type-2 citrulline- nemia to asymptomatic adult individuals with biochemical mia, an adult-onset autosomal recessive disorder in which manifestations of the disease [61,62]. aspartate is not available to generate argininosuccinate in the liver [62]. Most patients with type 2 citrullinemia may exhibit 3.3.5. Argininosuccinate lyase hepatic steatosis which is not accompanied by obesity or the Argininosuccinate lyase (ASL) is a cytosolic enzyme that metabolic syndrome [76]. Type 2 citrullinemia has been catalyzes the breakdown of argininosuccinate to arginine and associated with high incidence of hepatocellular carcinoma fumarate. The human gene that encodes argininosuccinate [77,78]. Citrin protein is reduced in lymphocytes isolated from lyase is positioned on chromosome 7 [63]. ASL deficiency peripheral blood in patients with citrin deficiency, suggesting results in argininosuccinic aciduria, an autosomal recessive an alternative diagnostic method for this disorder [79].

Recently, the analysis of the urine metabolome based on gas and the quantity of ammonium released into the systemic chromatography/mass spectrometry has been reported to be a circulation approximates the amount of ammonium excreted reliable diagnostic tool for citrin deficiency, readily differenti- in the urine. The release of ammonium into the renal vein ating this disease from other causes of hyperammonemia [80]. represents a major source of the normal ammonium concentration in blood [81,85–89]. The total amount of ammonium produced by the kidney and its partition into the renal vein or 4. Ammonium metabolism in healthy humans the urine may be modified in response to acid–base balance and potassium status and kidney function. In healthy in- Liver, portal-drained viscera (including stomach, small and dividuals, ammonium chloride-induced acidosis is associated large intestines, spleen, and pancreas), kidney, skeletal with an increase in the total kidney ammonium production muscle, brain, lung, skin, and red blood cells are involved in and a significant rise in the urinary excretion of ammonium. ammonium homeostasis in the human body. In contrast, metabolic alkalosis is associated with a marked reduction in urinary ammonium excretion and a rise in the 4.1. Liver ammonium released into the kidney venous blood [87,89,90]. In healthy humans, potassium depletion enhances kidney The role of the liver in maintaining normal ammonium ammoniagenesis and urinary ammonium excretion (likely metabolism is crucial. In healthy humans, the ammonium associated with a decrease in urinary potassium excretion). concentration measured in the portal vein has been found Conversely, the administration of potassium supplements higher than the ammonium concentration in the hepatic vein, decreases urinary ammonium excretion [87,89]. In patients indicating that ammonium is used by the liver, which with chronic kidney failure, total renal ammonium produc- substantially reduces the high ammonium content present tion is decreased in relation to the reduction of functioning in portal blood [81]. The source of the elevated ammonium renal mass and consequently both urinary ammonium and concentration in the portal vein has not been entirely ammonium added to the renal vein are reduced. However, elucidated. Ammonium release associated with glutamine the rate of ammonium production per unit of glomerular extraction has been observed by the small and large intestines filtration rate is fourfold greater in patients with chronic in humans, being slightly more pronounced in the jejunum kidney failure than in healthy controls [88]. Glutamine is a than in ileum and colon [81,82]. Pancreas metabolism might major contributor to kidney ammonium production both contribute ammonium to the portal vein via the glutamate under normal acid–base conditions and metabolic acidosis, dehydrogenase reaction while spleen contribution to portal but other amino acids such as glutamate, glycine, and proline, ammonium level has not been explored. Urea-splitting may also contribute [87,88]. bacteria living in the mouth and the gastrointestinal tract (including Helicobacter pylori) may generate ammonium from 4.3. Skeletal muscle urea. Additionally, ammonium in a concentration ranging from 45 to 240 μmol/L (average 110 μmol/L) has been detected In healthy individuals under basal conditions, there is no in the hepatic bile in healthy humans undergoing a cholecys- significant net uptake or release of ammonium ions by resting tectomy for gallstones [83]. The fate of the ammonium present muscle [91–94], but normal skeletal muscle releases ammoni- in the bile is unclear, but it may be released into the intestinal um during exercise and healthy individuals show an increase of lumen being subsequently transported by the portal vein to ammonium concentration in the vein draining the exercising the liver. Human hepatocytes use ammonia and bicarbonate muscles. In some studies, simultaneous elevation of ammoni- to form carbamoylphosphate in the mitochondria, initiating um in either arterial blood or venous blood draining the the urea cycle reactions. In individuals with normal liver contralateral resting limb has not been observed [95,96].The function, hepatocytes seem to have a remarkable functional precise origin and fate of the ammonium released by the active reserve, and urea synthesis per gram of liver tissue increases muscles are unclear [1]. During heavy exercise, ATP is rapidly rapidly following major hepatectomy, so that liver resections consumed, rendering ADP. Two molecules of ADP are combined of up to 70%–80% are generally well tolerated and arterial in the adenylate kinase or myokinase reaction, re-synthesizing ammonium concentration remains unchanged. In contrast, ATP with AMP as a by-product. The AMP is broken down to much smaller liver resections precipitate liver failure in inosine monophosphate (IMP) and an ammonium ion by the patients with underlying liver disease [84]. Normal human enzyme AMP deaminase or myoadenylate deaminase. Free liver also shows glutamine synthetase and glutaminase ammonium ions may then be released into the venous blood of activity, being therefore capable of glutamine synthesis and the exercising limb. It is believed that the IMP may be converted hydrolysis [12,15,16,29,46]. into adenylosuccinate which in turn is transformed again into AMP, completing the proposed purine nucleotide cycle func- 4.2. Kidney tioning in human skeletal muscle. Aspartate is consumed by the cycle, while fumarate is produced [97,98].Duringhyper- Human kidney also plays a fundamental role in ammonium ammonemia, skeletal muscle may become a major ammoni- homeostasis. Normal kidney cells produce free ammonium um-removing organ, particularly due to its large mass. The ions that are either excreted into the urine or released into the intravenous administration of ammonium salts increases renal vein. In healthy individuals under normal acid–base peripheral uptake of ammonium in normal persons, although balance conditions, total kidney ammonium production is considerable individual variation is observed and once reached approximately half-divided between urine and venous blood a threshold plasma ammonium level, additional rise in arterial

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 8 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX ammonium concentration may not be accompanied by a of normal [46,106]. Similarly to healthy humans, acetazol- significant increase in peripheral uptake [93]. amide inhibits urea synthesis in cirrhotic liver slices by about 50%, suggesting that hepatic urea synthesis depends on the 4.4. Brain activity of mitochondrial carbonic anhydrase [46]. Glutamine synthesis is also diminished in cirrhotic livers, compared In healthy humans, ammonium cerebral arterio-venous with normal controls. Conversely, the flux through hepatic difference is negligible, indicating that there is neither a glutaminase is increased in cirrhosis 4- to 6-fold [46]. significant net uptake nor release of ammonium by the brain Compared to healthy individuals in whom ingestion of [91,92]. However, the rate of cerebral ammonium utilization amino acids does not induce hyperammonemia, blood increases as a linear function of the arterial ammonium level ammonium concentration in patients with hepatic cirrhosis, and net uptake of ammonium ions by the brain is observed in particularly those with a transjugular intrahepatic portosys- healthy humans during hyperammonemia [99,100]. temic shunt, is transiently elevated following oral administration of some amino acids, such as glutamine, glycine, 4.5. Lung serine, and threonine, indicating a deficient handling of ammonium in liver disease [102,107]. Human airway epithelial cells display glutaminase activity Similarly to healthy individuals, the kidney releases and produce ammonium ions from glutamine. Ammonia has ammonium into the renal venous circulation in patients been detected in gas phase in the exhaled breath and alveolar with liver disease and this delivery represents a major source air of healthy humans [11]. of the ammonium concentration in blood [100,103,108]. The kidney is also largely responsible for the hyperammonemia 4.6. Skin induced by administration of acetazolamide, chlorothiazide and mercurial diuretics to patients with liver disease. The The skin could be an important organ to dispose of ammoni- intravenous administration of acetazolamide or chlorothia- um ions, as ammonia is present in gas emanated from the zide results in a significant elevation in the ammonium skin surface of healthy persons and patients with liver released into the renal vein and a reduction in the ammonium disease; its concentration being correlated with blood ammo- excretion in the urine that correlates with a rise in urine pH nium concentration [10,17]. [108,109]. Administration of mercurial diuretics to cirrhotic patients also produces an elevation in arterial ammonium 4.7. Red blood cells concentration associated with an increase in the amount of ammonium released into the renal venous blood while Human Rh proteins expressed in erythroid cells and epithelial extremities, liver, and brain do not contribute ammonium to tissues have been defined as ammonium transporters, the circulation under these conditions. [110]. In addition, although their precise localization, carrier mechanisms, and voluntary hyperventilation is associated with hyperammone- clinical significance are yet to be clarified [101]. mia of unclear origin in patients with liver disease [86,111].It has been recently shown that the urinary ammonium excretion increases during the anhepatic phase of a liver 5. Ammonium metabolism in liver disease transplantation and after reperfusion of the organ [112]. Skeletal muscle may contribute to ammonium clearance in Ammonium homeostasis and the interorgan trafficking of patients with liver disease and hyperammonemia. Unlike ammonium are altered in hepatic disease. Similarly to healthy healthy individuals, peripheral arterio-venous ammonium persons, the portal-drained viscera produce ammonium in concentration is slightly positive in patients with liver disease, patients with cirrhosis and the ammonium production is indicating that the skeletal muscle takes up ammonium in related to glutamine uptake [102,103]. Hepatocellular dysfunc- these patients [91,94,100,103,113]. Further, the uptake of tion results in impaired clearance of ammonium by the liver. ammonium by skeletal muscle rises with increasing levels of In addition, patients with liver disease may develop portal blood ammonium [100]. However, the capacity of skeletal collateral veins that divert portal blood with high ammonium muscle to extract ammonium from the systemic circulation is content to the systemic circulation. Both the incomplete reduced in cirrhotic patients with gross muscle wasting [94].In clearance of ammonium and the development of collateral patients with hepatic insufficiency, the peripheral arterio- portal circulation contribute to hyperammonemia generally venous difference for glutamine is threefold greater than that present in liver failure. The role of the portal collateral for alanine, in contrast to healthy humans, in whom the circulation is suggested by studies showing that patients release of alanine exceeds that of glutamine, suggesting that a with portal vein collateral circulation or portacaval anasto- major fraction of ammonium taken up by muscle is released moses exhibit an increase in peripheral blood ammonium as glutamine in patients with liver disease [94]. In cirrhotic concentration exceeding the concomitant rise in ammonium patients, muscular exercise produces greater ammonium values in hepatic vein blood following ingestion of ammoni- release by skeletal muscle than in normal individuals, um chloride [104,105]. Diminished ammonium clearance by resulting in larger increase in the venous ammonium the cirrhotic liver is suggested by the high ammonium concentration of blood draining the exercising forearm [95,96]. concentration present in the hepatic vein in patients with Unlike healthy individuals, patients with hepatic disease liver disease [103]. Urea synthesis capacity is reduced in exhibit a positive cerebral arterio-venous difference in am- cirrhotic patients, the maximal rate ranging from 10% to 90% monium concentration, indicating ammonium uptake by the

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX 9 brain that shows a correlation to the level of ammonium irrigant agent during transurethral resection of the prostate, entering the brain in these patients [91,92,114]. amino acid total parenteral nutrition, and administration of drugs such as asparaginase, 5-fluorouracil, carbamazepine, 5.1. Ammonium and hepatic encephalopathy and topiramate. Most of the conditions inducing hyperammonemia funnel to an imbalance between the amount of Advanced liver disease is usually accompanied by an ammonium produced and the body capacity to metabolize or elevation of blood ammonium levels, but the role of remove it, primarily by the liver. Congenital or acquired ammonium causing hepatic encephalopathy is controversial defective urea cycle function leading to deficient ammonium and conclusive evidence of a major causative role of metabolic removal is a major cause of hyperammonemia, but hyperammonemia on hepatic coma has not been provided increased ammonium generation may overcome the hepatic in human clinical studies. There is a marked variability in the clearance capacity and produce hyperammonemia as well. blood ammonium level in patients with liver disease with and Tolerance of healthy persons to elevated blood ammonium without encephalopathy and overlap of blood ammonium seems to be remarkable, as they may undergo a rise of arterial values between groups of patients at all levels of conscious- ammonium concentration up to 510.7 μmol/L without devel- ness is present. Furthermore, the correlation between the oping neurological alterations or electroencephalographic blood ammonium concentration and hepatic encephalopathy abnormalities [85,93]. Similarly, symptoms suggesting hyper- has not been consistent [94,104,115–121]. In a prospective ammonemia have not been observed in patients affected study aimed to evaluate the correlation between plasma with hyperinsulinism hyperammonemia syndrome, despite ammonium levels and chronic hepatic encephalopathy, a blood ammonium concentration that usually is 3- to 5-fold the moderate correlation between blood ammonium level and normal level [39]. Further, it has been recently shown that the the severity of encephalopathy was found, but there performance of psychological tasks is not affected by induced remains substantial overlap in blood ammonium concen- hyperammonemia (up to 225 μmol/L) in a double blind cross- tration between cirrhotic patients with and without hepatic over study with healthy volunteers [127]. encephalopathy [121]. As previously outlined, severe liver disease and develop- A number of clinical studies have analyzed the role of ment of portosystemic shunts usually result in hyperammo- ammonium as risk factor for brain edema in acute liver failure nemia due to defective hepatic ammonium clearance or (ALF). Univariate analyses suggest that the mean plasma diversion of the portal blood with high ammonium content ammonium concentration in patients with ALF is higher in into the systemic circulation. patients with cerebral edema or brain herniation than in those without these complications, but considerable overlap is 6.1. Urea cycle disorders observed between the groups of patients [122,123]. In a large cohort of patients with ALF, multivariate Cox regression Congenital or acquired deficiency of any of the enzymes analysis revealed that the adjusted hazard ratios for elevated involved in the urea cycle reactions (N-acetylglutamate blood ammonium concentration and risk for intracranial synthase, carbamoylphosphate synthetase-1, ornithine trans- hypertension and severe encephalopathy were 1.010 and carbamylase, argininosuccinate synthetase-1, argininosucci- 1.008, respectively, suggesting that other factors are addition- nate lyase, arginase-1, mitochondrial ornithine transporter, ally important in producing brain edema during fulminant and citrin) may produce hyperammonemia. Congenital defi- hepatic failure [124]. Patients with higher ammonium level at ciency of the mitochondrial isoenzyme of carbonic anhydrase admission develop more complications, including cerebral has not been reported as cause of hyperammonemia. edema, renal failure, and need for ventilation, suggesting Congenital urea cycle disorders (UCDs) are inherited as that they may suffer more severe disease [125]. The persis- autosomal recessive traits, except ornithine transcarbamylase tence of hyperammonemia instead of the ammonium level on deficiency, which is X-linked. The most frequent congenital admission may be associated with intracranial hypertension UCD in Japan, US, and Finland is ornithine transcarbamylase [124,126]. deficiency while the least frequent is arginase-1 deficiency. The overall frequency of congenital UCDs is estimated to vary from 1 in 30,000 live births to 1 in 50,000 live births 6. Hyperammonemia [64,74,128,129]. Patients with UCDs characteristically exhibit an encephalopathy commencing with hyperventilation and Elevation of blood ammonium concentration occurs in a early respiratory alkalosis followed by coma and hyperam- variety of situations, including hepatocellular dysfunction, monemia. Approximately half of the cases manifest during development of portal collateral circulation, urea cycle disor- the neonatal period and these patients have poorer outcome ders, lysinuric protein intolerance, carnitine deficiency, medi- than those who present later in life. Most long-term survivors um-chain acyl-CoA dehydrogenase deficiency, valproate with neonatal-onset UCD have intellectual disabilities which administration, organic acidemias, Reye's syndrome, infec- become more pronounced with increasing age [64,74,128–130]. tions with urea-splitting organisms, chemotherapy for hema- However, even patients with partial urea cycle enzyme tologic malignancies, lung transplantation, Barth syndrome, deficiencies who manifest with late-onset UCDs demonstrate and other conditions such as pyruvate carboxylase deficiency, evidence of neurocognitive and behavioral impairment, pyruvate dehydrogenase complex deficiency, hyperinsulinism including autism, learning disorders, and hyperactive and hyperammonemia syndrome, distal renal tubular acidosis, self-injurious behavior. Even asymptomatic ornithine trans- ureterosigmoidostomy procedures, use of glycine solution as carbamylase deficient heterozygotes have been reported to

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 10 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX have cognitive deficits and are at risk for learning disabilities to arginine and then ornithine, results in improved urea cycle and attention deficit hyperactivity disorder. Pregnancy, infec- performance and amelioration of the postprandial rise of tious illnesses or fasting with subsequent catabolism, or the plasma ammonium [132,134]. use of sodium valproate may unmask a latent case of CPSI or OTC deficiency [130,131]. For unknown reasons, arginase-1 6.3. Carnitine deficiency deficiency and ornithine transporter deficiency differ from other UCDs as the major neurological presentation is pyra- Carnitine is a molecule normally derived from dietary midal tract involvement and spasticity, with hyperammone- protein although it may be synthesized by the liver from mic coma being rare [131]. There is no direct correlation lysine and methionine as a methyl donor. It is required for between the disease-causing molecular change, the peak the transfer of medium- and long-chain fatty acids across ammonium level, and the phenotypic manifestation, so that the mitochondrial membrane to be oxidized. Carnitine prediction of neurological outcome is not straightforward. enters the cell across a plasma membrane carnitine trans- There is a significant negative linear correlation between porter, OCTN2, being conjugated with fatty acids on the duration of neonatal hyperammonemic coma and intelligence outer mitochondrial membrane by carnitine palmitoyltrans- quotient (IQ) at 12 months, suggesting that prolonged ferase-1 (CPT1). The acylcarnitine complex is transferred neonatal coma is associated with brain damage and impair- across the inner mitochondrial membrane by the enzyme ment of intellectual function. However, there is no significant carnitine-acylcarnitine translocase (CACT). The fatty acid is correlation between peak ammonium level and IQ score at 12 liberated inside the mitochondrial matrix by carnitine months and a multivariate analysis indicates that the peak palmitoyl transferase-2 (CPT2) for subsequent β-oxidation, ammonium level did not add to the significance of the while the carnitine molecule is returned to the outer association between duration of coma and IQ at 12 months mitochondrial membrane by the translocase, ready for [128]. Survivors who are severely mentally retarded have another cycle of fatty acid transfer. Congenital deficiency chronic neuropathological findings, including increased ven- of OCTN2, CPT1, CACT, or CPT2 may result in hyperammo- tricular size and areas of focal cortical necrosis that may nemia of unclear origin [136–139]. Deficient formation of reflect hypoxia and increased intracranial pressure [128,131]. acetyl-CoA as a result of impaired fatty acid oxidation inside In patients with ornithine transcarbamylase deficiency, themitochondriamaydisruptureacyclefunctionby carbamoylphosphate synthetase-1 deficiency, and arginino- reducing N-acetylglutamate production. Primary carnitine succinate lyase deficiency, the development of neurological deficiency due to dysfunction of OCTN2 has been associated symptoms seems to be inevitable despite aggressive therapy with partial deficiency of the enzyme N-acetylglutamate and avoidance of hyperammonemia and virtually all survi- synthase [51]. Hyperammonemia associated with acquired vors have developmental disabilities. In these patients, carnitine deficiency in a severely malnourished patient has therapy fails to prevent severe neurological sequelae during also been reported [140]. a hyperammonemic crisis [64,130]. 6.4. Medium-chain acyl-CoA dehydrogenase deficiency 6.2. Lysinuric protein intolerance The enzyme medium-chain acyl-CoA dehydrogenase Lysinuric protein intolerance is an autosomal recessive (MCAD) is involved in mitochondrial fatty acid oxidation, disorder caused by mutations in the SLC7A7 gene that induce being encoded by the human gene ACADM, located on defective cationic amino acids (lysine, arginine, ornithine) chromosome 1p31. Congenital MCAD deficiency is an transport at the basolateral membrane of epithelial cells in the autosomal recessive disorder ranking as one of the most intestine and kidney, resulting in hyperaminoaciduria, espe- frequent defects of fatty acid metabolism in the US [141]. cially lysinuria, and in low plasma levels of arginine, ornithine Clinical features of MCAD deficiency may be triggered by and lysine [132,133]. The highest incidence of lysinuric protein catabolic stress, such as infections and fasting, and include intolerance has been found in Finland with more than half of sudden infant death, hypoketotic hypoglycemia and hyper- the reported cases coming from this country [133,134]. There ammonemia [142]. Disruption of the fatty acid oxidation is high variability in the clinical presentation even within results in mitochondrial accumulation of unoxidized fatty individual families. Typically, the disorder presents in infancy acyl-CoA metabolites that are believed to inhibit the urea with feeding difficulties, vomiting, diarrhea, and poor growth, cycle [140,143]. but it may also present in adult life. Postprandial hyperammonemia produces episodes of lethargy, convulsions, or 6.5. Valproate therapy coma. Sometimes there is a variety of other symptoms, including aversion to protein-rich foods, hepatosplenome- Valproate administration has been consistently associated galy, lens opacities, hyperextensible joints, hyperelastic skin, with hyperammonemia, particularly when combined with osteoporosis, short stature, hemolytic anemia, pancytopenia, other anticonvulsant drugs, including phenytoin, phenobar- neurological involvement, mental retardation, focal glomer- bital, and topiramate. Serum levels of ammonium do not ulosclerosis, renal tubular disease, interstitial lung disease correlate with the severity of valproate-induced encephalop- and pulmonary alveolar proteinosis [132,133,135]. Recurring athy and both asymptomatic hyperammonemia and carnitine postprandial hyperammonemia is assumed to be due to a deficiency are common in patients receiving valproate. depletion of ornithine and arginine in liver mitochondria. Sometimes, valproate therapy has unmasked an underlining Citrulline administration, which is metabolized sequentially urea cycle disorder [144–146].

6.6. Organic acidemias following bone marrow transplantation [158] or stem cell autograft [159]. Necropsy findings observed in these patients Organic acidemias including propionic acidemia, methylma- include ischemic changes in cerebral cortex and cerebral lonic acidemia, and isovaleric acidemia may result in edema with astrocyte swelling. In the liver, sinusoidal hyperammonemia. Amino acids such as methionine, threo- dilatation, marked acute congestion, hemosiderin deposition, nine, valine, and isoleucine, the side chain of cholesterol, and and centrilobular fat infiltration are found. In the lungs, odd chain fatty acids produce propionate in their degradation hemorrhage and edema along with bilateral pleural effusion pathways. Propionyl-CoA is sequentially transformed into are present. Other findings include pericardial effusion, methylmalonyl-CoA and succinyl-CoA, which enters the peritoneal effusion, hemorrhagic cystitis, and ischemic colitis tricarboxylic acids cycle, serving as an anaplerotic com- [159,160]. pound. Vitamin B12 is necessary for the conversion of methylmalonate to succinate. Propionic acidemia and iso- 6.10. Lung transplantation valeric acidemia are autosomal recessive disorders caused by propionyl-CoA carboxylase deficiency and isovaleryl-CoA Hyperammonemia has been noted in 4% of patients following dehydrogenase deficiency, respectively [147–149]. Methylma- lung transplantation and its presence has been associated lonic aciduria is a heterogeneous disorder that recognizes with increased mortality [161]. Risk factors for hyperammo- both genotypic and phenotypic variability. It may be due to nemia in lung transplant recipients include development of defective cobalamine synthesis (responsive to vitamin B12)or major gastrointestinal complications, use of total parenteral to mutations in the MMACHC gene located in chromosome nutrition, and lung transplantation for primary pulmonary region 1p34-1, which produce combined methylmalonic hypertension [161]. aciduria and homocystinuria or cobalamin C disease. In a retrospective study of 30 patients with vitamin B12- 6.11. Barth syndrome unresponsive methylmalonic aciduria, half of them have a neonatal onset. Neurological deterioration and chronic kid- Barth syndrome is a rare X-linked disorder due to mutations on ney failure are frequent manifestations of the disease [150]. the TAZ gene, characterized by skeletal myopathy, cardiomy- Administration of N-carbamylglutamate, a carbamoylpho- opathy, left ventricular noncompaction, neutropenia, growth sphate synthetase-1 activator, improves hyperammonemia retardation, and 3-methylglutaconic aciduria. The TAZ gene is associated with organic acidemias, suggesting that a dis- located in Xq28 and its gene product, taffazin, is probably rupted urea cycle may be the cause of the hyperammonemia involved in cardiolipin synthesis, as the concentration of this in these conditions [151–154]. phospholipid is markedly decreased in skeletal and cardiac muscle and in platelets from affected patients. Barth syndrome 6.7. Reye's syndrome may be accompanied by acute metabolic decompensation, lactic acidosis and hyperammonemia of unclear cause [162,163]. Reye's syndrome occurs following an apparently uneventful recovery from a viral illness and may be precipitated by 6.12. Other causes of hyperammonemia salicylate administration, being characterized by cerebral edema, diffuse fatty infiltration of the viscera, and a liver Hyperammonemia has been reported to be associated with biopsy considered diagnostic of the syndrome. Reye's syn- pyruvate carboxylase deficiency (presumably due to shortage of drome also features hyperammonemia of unclear cause aspartate supply to the urea cycle) [164], pyruvate dehydrogenase and increased blood concentration of fatty acids and lactic complex deficiency (attributed to defective acetyl-CoA synthesis acid [155]. and secondary inhibition of ureagenesis) [165], hyperinsulinism hyperammonemia syndrome [39], distal kidney tubular acidosis 6.8. Infections with urease-producing organisms (assumed to be due to defective urinary ammonium excretion) [166], ureterosigmoidostomy [167], essential amino acid total Urinary tract infections with organisms that produce urease parenteral nutrition [168], and drugs including asparaginase [37], such as Proteus mirabilis, Klebsiella oxytoca, Klebsiella pneumo- 5-fluorouracil [169], carbamazepine [170], topiramate [145],and niae,andCorynebacterium urealyticum, may induce severe glycine solution used as an irrigant [171]. hyperammonemia and coma, particularly in association with urinary dilatation or dysfunctional neurogenic bladder. Urease breaks down urea generating ammonium which is 7. Hyperammonemia, cerebral edema, and released into the systemic circulation and may exceed liver respiratory alkalosis clearance capacity, resulting in hyperammonemia [6,156,157]. Pelvic abscesses have also been rarely associated with Hyperventilation and consequent respiratory alkalosis usually hyperammonemia [7]. precede or accompany the acute metabolic crisis with cerebral edema and coma typical of most diseases featuring hyper- 6.9. Chemotherapy for hematologic malignancy ammonemia. Indeed, any alteration in the level of conscious- ness associated with respiratory alkalosis should prompt the Severe hyperammonemia preceded by hyperventilation and determination of blood ammonium level [128,130]. Respiratory respiratory alkalosis has been repeatedly observed in patients alkalosis is the predominant acid–base anomaly in patients receiving chemotherapy for hematologic malignancy and with liver disease, particularly in those with cerebral edema

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 12 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX and hepatic coma [120,172–174] and it has been suggested that molecular basis of the different disorders causing hyperam- cirrhotic patients with simultaneous hyperammonemia and monemia has fundamental translational potential in order to reduction in the plasma partial pressure of carbon dioxide establish the pathogenic mechanisms and the therapy for

(pCO2) are more likely to be affected by cerebral edema and these diseases. coma [120,172]. Hyperventilation is also an early sign of the acute metabolic decompensations associated with urea cycle disorders and other diseases featuring hyperammonemia, Authors contributions being followed by an encephalopathy characterized by cerebral edema and coma. Tachypnea and respiratory alkalosis pro- M. Adeva and G. Souto carried out the literature search and gressing to cerebral edema and coma with hyperammonemia wrote the draft of the manuscript. N. Blanco reviewed urea have been reported in ornithine transcarbamylase deficiency cycle disorders. Ammonium metabolism in healthy humans [59], argininosuccinate synthetase deficiency, argininosucci- and liver disease was reviewed by C. Donapetry. M. Adeva and nate lyase deficiency [65], lysinuric protein intolerance [132], G. Souto reviewed glutamine synthetase, glutaminase and medium-chain acyl-CoA dehydrogenase deficiency [142],pro- glutamate dehydrogenase and hyperammonemia. All authors pionic acidemia [175], valproate administration [144],Reye's contributed to the final version of the manuscript. syndrome [176], urinary tract infections with urea-splitting organisms [6,156,157], chemotherapy for the treatment of hematologic malignancy [160], bone marrow transplantation [160], stem cell autograft for multiple myeloma [159], infusion of Funding essential amino acid total parenteral nutrition [168], and 5-fluorouracil administration [177]. There was no financial support for this work The cause of hyperventilation in these disorders is unknown. In humans, the central nervous system controls respiration via unclear mechanisms. The respiratory control Conflict of interest center in the medulla oblongata likely receives input from adjacent chemoreceptors which sense changes in blood There are no conflicts of interest. hydrogen ion concentration and pCO2. In patients with hepatic cirrhosis, it has been suggested that ammonium ions might induce ventilatory stimulation. Another possible mechanism REFERENCES that may explain hyperventilation in this condition is the intracellular acidosis that accompanies liver cirrhosis [174]. Hepatic failure is associated with elevated plasma concentra- [1] Graham TE, MacLean DA. Ammonia and amino acid tion of lactate, pyruvate, and other organic anions, resulting in metabolism in human skeletal muscle during exercise. Can J intracellular acidosis [116,120,173,174,178,179]. Furthermore, a Physiol Pharmacol 1992;70:132–41. rise in the erythrocyte concentration of 2,3-bisphosphoglyce- [2] Coomes MW. Amino acid metabolism. In: Devlin TM, editor. rate [180–182] and a displacement to the right of the Textbook of biochemistry with clinical correlations. Hobo- ken, NJ: Wiley-Liss; 2006. p. 743–82. oxyhemoglobin dissociation curve [174,181,183] have been [3] Kopstein J, Wrong OM. The origin and fate of salivary urea observed in patients with liver disease, supporting the notion and ammonia in man. Clin Sci Mol Med 1977;52:9–17. that tissue acidosis induces a reduction in the affinity of [4] Aoyagi T, Engstrom GW, Evans WB, et al. Gastrointestinal hemoglobin for oxygen aimed to improve carbon dioxide urease in man. I Activity of mucosal urease Gut 1966;7:631–5. extraction from tissue cells. Similarly to hepatic failure, most [5] Osaki T, Mabe K, Hanawa T, et al. Urease-positive bacteria in disorders leading to hyperammonemia and acute metabolic the stomach induce a false-positive reaction in a urea breath test for diagnosis of Helicobacter pylori infection. J Med decompensations with cerebral edema are also characterized Microbiol 2008;57:814–9. by intracellular acidosis due to accumulation of different [6] Sato S, Yokota C, Toyoda K, et al. Hyperammonemic organic anions. Hyperventilation may be an adaptative encephalopathy caused by urinary tract infection with physiological response to intracellular acidosis intended to urinary retention. Eur J Intern Med 2008;19:e78–9. enhance carbon dioxide removal, as intracellular pH reflects [7] Pimentel Jr JL, Brusilow SW, Mitch WE. Unexpected cellular metabolic status better than blood pH [184]. encephalopathy in chronic renal failure: hyperammonemia In this review, we have summarized relevant articles complicating acute peritonitis. J Am Soc Nephrol 1994;5: 1066–73. concerning the enzymes involved in human ammonium [8] Clancy KP, Berger R, Cox M, et al. Localization of the L- metabolism, the homeostasis of ammonium ions in healthy glutamine synthetase gene to chromosome 1q23. Genomics humans and patients with liver disease, and the main causes 1996;38:418–20. of hyperammonemia. We additionally discuss the occurrence [9] Mitchell S, Ellingson C, Coyne T, et al. Genetic variation in the of hyperventilation and respiratory alkalosis associated with urea cycle: a model resource for investigating key candidate – cerebral edema and hyperammonemia. Although a thorough genes for common diseases. Hum Mutat 2009;30:56 60. [10] Nose K, Mizuno T, Yamane N, et al. Identification of search for relevant articles in the medical literature has been ammonia in gas emanated from human skin and its performed, the broad scope of the topic makes it difficult to correlation with that in blood. Anal Sci 2005;21:1471–4. expand the content of the information in every section. The [11] Hunt JF, Erwin E, Palmer L, et al. Expression and activity knowledge regarding the human distribution and activity of of pH-regulatory glutaminase in the human airway the enzymes involved in ammonium metabolism and the epithelium. Am J Respir Crit Care Med 2002;165:101–7.

[12] Elgadi KM, Meguid RA, Qian M, et al. Cloning and analysis of [34] Suzuki S, Tanaka T, Poyurovsky MV, et al. Phosphate- unique human glutaminase isoforms generated by tissue- activated glutaminase (GLS2), a p53-inducible regulator of specific alternative splicing. Physiol Genomics 1999;1:51–62. glutamine metabolism and reactive oxygen species. Proc [13] Kravos M, Malesic I. Glutamate dehydrogenase activity in Natl Acad Sci U S A 2010;107:7461–6. leukocytes and ageing. Neurodegener Dis 2010;7:239–42. [35] Gao P, Tchernyshyov I, Chang TC, et al. c-Myc suppression of [14] Wang Y, Kudoh J, Kubota R, et al. Chromosomal mapping of miR-23a/b enhances mitochondrial glutaminase expression a family of human glutamine synthetase genes: functional and glutamine metabolism. Nature 2009;458:762–5. gene (GLUL) on 1q25, pseudogene (GLULP) on 9p13, and three [36] Earl M. Incidence and management of asparaginase-associated related genes (GLULL1, GLULL2, GLULL3) on 5q33, 11p15, and adverse events in patients with acute lymphoblastic leukemia. 11q24. Genomics 1996;37:195–9. Clin Adv Hematol Oncol 2009;7:600–6. [15] James LA, Lunn PG, Middleton S, et al. Distribution of [37] Leonard JV, Kay JD. Acute encephalopathy and glutaminase and glutamine synthetase activities in the hyperammonaemia complicating treatment of acute human gastrointestinal tract. Clin Sci (Lond) 1998;94:313–9. lymphoblastic leukaemia with asparaginase. Lance 1986;1: [16] Yamamoto H, Konno H, Yamamoto T, et al. Glutamine 162–3. synthetase of the human brain: purification and character- [38] Plaitakis A, Zaganas I. Regulation of human glutamate ization. J Neurochem 1987;49:603–9. dehydrogenases: implications for glutamate, ammonia [17] Danielyan L, Zellmer S, Sickinger S, et al. Keratinocytes as and energy metabolism in brain. J Neurosci Res 2001;66: depository of ammonium-inducible glutamine synthetase: 899–908. age- and anatomy-dependent distribution in human and rat [39] Stanley CA, Lieu YK, Hsu BY, et al. Hyperinsulinism and skin. PLoS One 2009;4:e4416. hyperammonemia in infants with regulatory mutations of [18] Olkku A, Mahonen A. Wnt and steroid pathways control the glutamate dehydrogenase gene. N Engl J Med 1998;338: glutamate signalling by regulating glutamine synthetase 1352–7. activity in osteoblastic cells. Bone 2008;43:483–93. [40] Spanaki C, Zaganas I, Kleopa KA, et al. Human GLUD2 [19] Vermeulen T, Gorg B, Vogl T, et al. Glutamine synthetase is glutamate dehydrogenase is expressed in neural and essential for proliferation of fetal skin fibroblasts. Arch testicular supporting cells. J Biol Chem 2010;285:16748–56. Biochem Biophys 2008;478:96–102. [41] Borompokas N, Papachatzaki MM, Kanavouras K, et al. [20] DeMarco V, McCain MD, Strauss D, et al. Characterization of Estrogen modification of human glutamate dehydrogenases glutamine synthetase transcript, protein, and enzyme is linked to enzyme activation state. J Biol Chem 2010;285: activity in the human placenta. Placenta 1997;18:541–5. 31380–7. [21] Elia M, Livesey G. Effects of ingested steak and infused [42] Jung K, Pergande M, Rej R, et al. Mitochondrial enzymes leucine on forelimb metabolism in man and the fate of the in human serum: comparative determinations of carbon skeletons and amino groups of branched-chain glutamate dehydrogenase and mitochondrial aspartate amino acids. Clin Sci (Lond) 1983;64:517–26. aminotransferase in healthy persons and patients with [22] Aoki TT, Brennan MF, Fitzpatrick GF, et al. Leucine meal chronic liver diseases. Clin Chem 1985;31:239–43. increases glutamine and total nitrogen release from forearm [43] Jenkins WJ, Rosalki SB, Foo Y, et al. Serum glutamate muscle. J Clin Invest 1981;68:1522–8. dehydrogenase is not a reliable marker of liver cell necrosis [23] Darmaun D, Dechelotte P. Role of leucine as a precursor of in alcoholics. J Clin Pathol 1982;35:207–10. glutamine alpha-amino nitrogen in vivo in humans. Am J [44] Kravos M, Malesic I. Kinetics and isoforms of serum Physiol 1991;260:E326–9. glutamate dehydrogenase in alcoholics. Alcohol Alcohol [24] Gelfand RA, Glickman MG, Jacob R, et al. Removal of infused 2008;43:281–6. amino acids by splanchnic and leg tissues in humans. Am J [45] Plaitakis A, Berl S, Yahr MD. Neurological disorders Physiol 1986;250:E407–13. associated with deficiency of glutamate dehydrogenase. [25] Haberle J, Gorg B, Rutsch F, et al. Congenital glutamine Ann Neurol 1984;15:144–53. deficiency with glutamine synthetase mutations. N Engl J [46] Kaiser S, Gerok W, Haussinger D. Ammonia and glutamine Med 2005;353:1926–33. metabolism in human liver slices: new aspects on the [26] Haberle J, Shahbeck N, Ibrahim K, et al. Natural course of pathogenesis of hyperammonaemia in chronic liver disease. glutamine synthetase deficiency in a 3 year old patient. Mol Eur J Clin Invest 1988;18:535–42. Genet Metab 2011;103:89–91. [47] Caldovic L, Morizono H, Gracia Panglao M, et al. Cloning and [27] Tuchman M, Lichtenstein GR, Rajagopal BS, et al. Hepatic expression of the human N-acetylglutamate synthase gene. glutamine synthetase deficiency in fatal hyperammonemia Biochem Biophys Res Commun 2002;299:581–6. after lung transplantation. Ann Intern Med 1997;127:446–9. [48] Elpeleg O, Shaag A, Ben-Shalom E, et al. N-acetylglutamate [28] Schmidt A, Braeuning A, Ruck P, et al. Differential synthase deficiency and the treatment of hyperammonemic expression of glutamine synthetase and cytochrome P450 encephalopathy. Ann Neurol 2002;52:845–9. isoforms in human hepatoblastoma. Toxicology 2011;281: [49] Caldovic L, Lopez GY, Haskins N, et al. Biochemical properties 7–14. of recombinant human and mouse N-acetylglutamate [29] Bode BP, Souba WW. Modulation of cellular proliferation synthase. Mol Genet Metab 2006;87:226–32. alters glutamine transport and metabolism in human [50] Nissim I, Horyn O, Daikhin Y, et al. Down-regulation of hepatoma cells. Ann Surg 1994;220:411–22. hepatic urea synthesis by oxypurines: xanthine and uric [30] DeLaBarre B, Gross S, Fang C, et al. Full-length human acid inhibit N-acetylglutamate synthase. J Biol Chem glutaminase in complex with an allosteric inhibitor. 2011;286:22055–68. Biochemistry 2011;50:10764–70. [51] Hwu WL, Chien YH, Tang NL, et al. Deficiency of the [31] Human glutaminase-2 gene. Assessed at http://www.ncbi. carnitine transporter (OCTN2) with partial nlm.nih.gov/gene?term=(gls2) on 8 December 2011. N-acetylglutamate synthase (NAGS) deficiency. J Inherit [32] Sarantos P, Abouhamze Z, Copeland EM, et al. Glucocorticoids Metab Dis 2007;30:816. regulate glutaminase gene expression in human intestinal [52] Caldovic L, Morizono H, Tuchman M. Mutations and epithelial cells. J Surg Res 1994;57:227–31. polymorphisms in the human N-acetylglutamate synthase [33] Hu W, Zhang C, Wu R, et al. Glutaminase 2, a novel p53 (NAGS) gene. Hum Mutat 2007;28:754–9. target gene regulating energy metabolism and antioxidant [53] Loscalzo ML, Galczynski RL, Hamosh A, et al. Interstitial function. Proc Natl Acad Sci U S A 2010;107:7455–60. deletion of chromosome 2q32-34 associated with multiple

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 14 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX

congenital anomalies and a urea cycle defect (CPS I [76] Komatsu M, Yazaki M, Tanaka N, et al. Citrin deficiency as a deficiency). Am J Med Genet 2004;128A:311–5. cause of chronic liver disorder mimicking non-alcoholic [54] Haberle J, Shchelochkov OA, Wang J, et al. Molecular defects fatty liver disease. J Hepatol 2008;49:810–20. in human carbamoy phosphate synthetase I: mutational [77] Tsai CW, Yang CC, Chen HL, et al. Homozygous SLC25A13 spectrum, diagnostic and protein structure considerations. mutation in a Taiwanese patient with adult-onset Hum Mutat 2011;32:579–89. citrullinemia complicated with steatosis and hepatocellular [55] Huo R, Zhu H, Lu L, et al. Molecular cloning, identification carcinoma. J Formos Med Assoc 2006;105:852–6. and characteristics of a novel isoform of carbamyl [78] Chang KW, Chen HL, Chien YH, et al. SLC25A13 gene phosphate synthetase I in human testis. J Biochem Mol Biol mutations in Taiwanese patients with non-viral 2005;38:28–33. hepatocellular carcinoma. Mol Genet Metab 2011;103:293–6. [56] Ju Y, Yang J, Liu R, et al. Antigenically dominant proteins [79] Tokuhara D, Iijima M, Tamamori A, et al. Novel diagnostic within the human liver mitochondrial proteome identified approach to citrin deficiency: analysis of citrin protein in by monoclonal antibodies. Sci China Life Sci 2011;54:16–24. lymphocytes. Mol Genet Metab 2007;90:30–6. [57] Cardona DM, Zhang X, Liu C. Loss of carbamoyl phosphate [80] Kuhara T, Ohse M, Inoue Y, et al. A GC/MS-based synthetase I in small-intestinal adenocarcinoma. Am J Clin metabolomic approach for diagnosing citrin deficiency. Anal Pathol 2009;132:877–82. Bioanal Chem 2011;400:1881–94. [58] Ihara K, Miyako K, Ishimura M, et al. A case of hyperinsulinism/ [81] van de Poll MC, Ligthart-Melis GC, Olde Damink SW, et al. hyperammonaemia syndrome with reduced carbamoyl- The gut does not contribute to systemic ammonia release in phosphate synthetase-1 activity in liver: a pitfall in enzymatic humans without portosystemic shunting. Am J Physiol diagnosis for hyperammonaemia. J Inherit Metab Dis 2005;28: Gastrointest Liver Physiol 2008;295:G760–5. 681–7. [82] van der Hulst RR, von Meyenfeldt MF, Deutz NE, et al. [59] Gordon N. Ornithine transcarbamylase deficiency: a urea Glutamine extraction by the gut is reduced in depleted cycle defect. Eur J Paediatr Neurol 2003;7:115–21. [corrected] patients with gastrointestinal cancer. Ann Surg [60] Lien J, Nyhan WL, Barshop BA. Fatal initial adult-onset 1997;225:112–21. presentation of urea cycle defect. Arch Neurol 2007;64:1777–9. [83] Albers CJ, Huizenga JR, Krom RA, et al. Composition of [61] Engel K, Hohne W, Haberle J. Mutations and polymorphisms human hepatic bile. Ann Clin Biochem 1985;22(Pt 2): in the human argininosuccinate synthetase (ASS1) gene. 129–32. Hum Mutat 2009;30:300–7. [84] van de Poll MC, Wigmore SJ, Redhead DN, et al. Effect of [62] Kobayashi K, Sinasac DS, Iijima M, et al. The gene mutated major liver resection on hepatic ureagenesis in humans. Am in adult-onset type II citrullinaemia encodes a putative J Physiol Gastrointest Liver Physiol 2007;293:G956–62. mitochondrial carrier protein. Nat Genet 1999;22:159–63. [85] Owen EE, Johnson JH, Tyor MP. The effect of induced [63] Abramson RD, Barbosa P, Kalumuck K, et al. Characterization hyperammonemia on renal ammonia metabolism. J Clin of the human argininosuccinate lyase gene and analysis of Invest 1961;40:215–21. exon skipping. Genomics 1991;10:126–32. [86] Owen EE, Tyor MP, Giordano D. The effect of acute alkalosis [64] Keskinen P, Siitonen A, Salo M. Hereditary urea cycle on renal metabolism of ammonia in cirrhotics. J Clin Invest diseases in Finland. Acta Paediatr 2008;97:1412–9. 1962;41:1139–44. [65] Erez A, Nagamani SC, Lee B. Argininosuccinate lyase [87] Owen EE, Robinson RR. Amino acid extraction and ammonia deficiency—argininosuccinic aciduria and beyond. Am J Med metabolism by the human kidney during the prolonged Genet C Semin Med Genet 2011;157:45–53. administration of ammonium chloride. J Clin Invest 1963;42: [66] Iyer R, Jenkinson CP, Vockley JG, et al. The human arginases 263–76. and arginase deficiency. J Inherit Metab Dis 1998;21(Suppl 1): [88] Tizianello A, De Ferrari G, Garibotto G, et al. Renal 86–100. metabolism of amino acids and ammonia in subjects with [67] Van Elsen AF, Leroy JG. Human hyperargininemia: a normal renal function and in patients with chronic renal mutation not expressed in skin fibroblasts? Am J Hum Genet insufficiency. J Clin Invest 1980;65:1162–73. 1977;29:350–5. [89] Tizianello A, Deferrari G, Garibotto G, et al. Renal [68] Spector EB, Rice SC, Cederbaum SD. Immunologic studies of ammoniagenesis in an early stage of metabolic acidosis in arginase in tissues of normal human adult and arginase- man. J Clin Invest 1982;69:240–50. deficient patients. Pediatr Res 1983;17:941–4. [90] Madison LL, Seldin DW. Ammonia excretion and renal [69] Munder M, Mollinedo F, Calafat J, et al. Arginase I is enzymatic adaptation in human subjects, as disclosed by constitutively expressed in human granulocytes and administration of precursor amino acids. J Clin Invest participates in fungicidal activity. Blood 2005;105:2549–56. 1958;37:1615–27. [70] Sparkes RS, Dizikes GJ, Klisak I, et al. The gene for human [91] Bessman SP, Bessman AN. The cerebral and peripheral liver arginase (ARG1) is assigned to chromosome band 6q23. uptake of ammonia in liver disease with an hypothesis for Am J Hum Genet 1986;39:186–93. the mechanism of hepatic coma. J Clin Invest 1955;34:622–8. [71] Grody WW, Kern RM, Klein D, et al. Arginase deficiency [92] Webster Jr LT, Gabuzda GJ. Ammonium uptake by the manifesting delayed clinical sequelae and induction of a extremities and brain in hepatic coma. J Clin Invest 1958;37: kidney arginase isozyme. Hum Genet 1993;91:1–5. 414–24. [72] Prasad AN, Breen JC, Ampola MG, et al. Argininemia: a [93] Tyor MP, Wilson WP. Peripheral biochemical changes treatable genetic cause of progressive spastic diplegia associated with the intravenous administration of simulating cerebral palsy: case reports and literature review. ammonium salts in normal subjects. J Lab Clin Med 1958;51: J Child Neurol 1997;12:301–9. 592–9. [73] Lee BH, Jin HY, Kim GH, et al. Argininemia presenting with [94] Ganda OP, Ruderman NB. Muscle nitrogen metabolism in progressive spastic diplegia. Pediatr Neurol 2011;44:218–20. chronic hepatic insufficiency. Metabolism 1976;25:427–35. [74] Tuchman M, Lee B, Lichter-Konecki U, et al. Cross-sectional [95] Allen SI, Conn HO. Observations on the effect of exercise on multicenter study of patients with urea cycle disorders in blood ammonia concentration in man. Yale J Biol Med the United States. Mol Genet Metab 2008;94:397–402. 1960;33:133–44. [75] Wang JF, Chou KC. Insights into the mutation-induced HHH [96] Sinniah D, Fulton TT, McCullough H. The effect of exercise syndrome from modeling human mitochondrial ornithine on the venous blood ammonium concentration in man. transporter-1. PLoS One 2012;7:e31048. J Clin Pathol 1970;23:715–9.

[97] Fukui H, Taniguchi S, Ueta Y, et al. Enhanced activity of the [116] Schwartz R, Phillips GB, Gabuzda Jr GJ, et al. Blood ammonia purine nucleotide cycle of the exercising muscle in patients and electrolytes in hepatic coma. J Lab Clin Med 1953;42: with hyperthyroidism. J Clin Endocrinol Metab 2001;86: 499–508. 2205–10. [117] Phear EA, Sherlock S, Summerskill WH. Blood-ammonium [98] Sabina RL, Swain JL, Olanow CW, et al. Myoadenylate levels in liver disease and hepatic coma. Lancet 1955;268: deaminase deficiency. Functional and metabolic abnormal- 836–40. ities associated with disruption of the purine nucleotide [118] Summerskill WH, Wolfe SJ, Davidson CS. Ammonia cycle. J Clin Invest 1984;73:720–30. intoxication and hepatic coma. AMA Arch Intern Med [99] Olde Damink SW, Jalan R, Dejong CH. Interorgan 1956;97:661–3. ammonia trafficking in liver disease. Metab Brain Dis [119] Sievers ML, Vander JB. Toxic effects of ammonium chloride 2009;24:169–81. in cardiac, renal and hepatic disease. J Am Med Assoc [100] Tyor MP, Owen EE, Berry JN, et al. The relative role of 1956;161:410–5. extremity, liver, and kidney as ammonia receivers and [120] Tyor MP, Sieker HO. Biochemical, blood gas and peripheral donors in patients with liver disease. Gastroenterology circulatory alterations in hepatic coma. Am J Med 1959;27: 1960;39:420–4. 50–9. [101] Ripoche P, Bertrand O, Gane P, et al. Human Rhesus- [121] Ong JP, Aggarwal A, Krieger D, et al. Correlation between associated glycoprotein mediates facilitated transport of ammonia levels and the severity of hepatic encephalopathy. NH(3) into red blood cells. Proc Natl Acad Sci U S A 2004;101: Am J Med 2003;114:188–93. 17222–7. [122] Clemmesen JO, Larsen FS, Kondrup J, et al. Cerebral [102] Plauth M, Roske AE, Romaniuk P, et al. Post-feeding herniation in patients with acute liver failure is correlated hyperammonaemia in patients with transjugular with arterial ammonia concentration. Hepatology 1999;29: intrahepatic portosystemic shunt and liver cirrhosis: role of 648–53. small intestinal ammonia release and route of nutrient [123] Kundra A, Jain A, Banga A, et al. Evaluation of plasma administration. Gut 2000;46:849–55. ammonia levels in patients with acute liver failure and [103] Olde Damink SW, Jalan R, Redhead DN, et al. Interorgan chronic liver disease and its correlation with the ammonia and amino acid metabolism in metabolically severity of hepatic encephalopathy and clinical features stable patients with cirrhosis and a TIPSS. Hepatology of raised intracranial tension. Clin Biochem 2005;38: 2002;36:1163–71. 696–9. [104] White LP, Phear EA, Summerskill WH, et al. Ammonium [124] Bernal W, Hall C, Karvellas CJ, et al. Arterial ammonia and tolerance in liver disease: observations based on clinical risk factors for encephalopathy and intracranial catheterization of the hepatic veins. J Clin Invest 1955;34: hypertension in acute liver failure. Hepatology 2007;46: 158–68. 1844–52. [105] Conn HO. Studies of the source and significance of blood [125] Bhatia V, Singh R, Acharya SK. Predictive value of arterial ammonia. IV. Early ammonia peaks after ingestion of ammonia for complications and outcome in acute liver ammonium salts. Yale J Biol Med 1972;45:543–9. failure. Gut 2006;55:98–104. [106] Rudman D, DiFulco TJ, Galambos JT, et al. Maximal rates of [126] Tofteng F, Hauerberg J, Hansen BA, et al. Persistent arterial excretion and synthesis of urea in normal and cirrhotic hyperammonemia increases the concentration of glutamine subjects. J Clin Invest 1973;52:2241–9. and alanine in the brain and correlates with intracranial [107] Rudman D, Galambos JT, Smith 3rd RB, et al. Comparison of pressure in patients with fulminant hepatic failure. J Cereb the effect of various amino acids upon the blood ammonia Blood Flow Metab 2006;26:21–7. concentration of patients with liver disease. Am J Clin Nutr [127] Wilkinson DJ, Smeeton NJ, Castle PC, et al. Absence of 1973;26:916–25. neuropsychological impairment in hyperammonaemia in [108] Owen EE, Tyor MP, Flanagan JF, et al. The kidney as a source healthy young adults; possible synergism in development of of blood ammonia in patients with liver disease: the effect of hepatic encephalopathy (HE) symptoms? Metab Brain Dis acetazolamide. J Clin Invest 1960;39:288–94. 2011;26:203–12. [109] Owen EE, Flanagan JF, Tyor MP. Kidney as a source of blood [128] Msall M, Batshaw ML, Suss R, et al. Neurologic outcome ammonia: effect of chlorothiazide. Proc Soc Exp Biol Med in children with inborn errors of urea synthesis. Outcome 1959;102:696–7. of urea-cycle enzymopathies. N Engl J Med 1984;310: [110] Baertl JM, Sancetta SM, Gabuzda GJ. Relation of acute 1500–5. potassium depletion to renal ammonium metabolism in [129] Uchino T, Endo F, Matsuda I. Neurodevelopmental outcome patients with cirrhosis. J Clin Invest 1963;42:696–706. of long-term therapy of urea cycle disorders in Japan. [111] Berry JN, Owen EE, Flanagan JF, et al. The effect of acute J Inherit Metab Dis 1998;21(Suppl 1):151–9. hyperventilation on the blood ammonia concenration of [130] Krivitzky L, Babikian T, Lee HS, et al. Intellectual, adaptive, patients with liver disease. The relative role of the and behavioral functioning in children with urea cycle kidney, liver, and extremity. J Lab Clin Med 1960;55: disorders. Pediatr Res 2009;66:96–101. 849–54. [131] Gropman AL, Summar M, Leonard JV. Neurological [112] Mpabanzi L, van den Broek MA, Visschers RG, et al. Urinary implications of urea cycle disorders. J Inherit Metab Dis ammonia excretion increases acutely during living donor 2007;30:865–79. liver transplantation. Liver Int 2011;31:1150–4. [132] Shaw PJ, Dale G, Bates D. Familial lysinuric protein [113] Bessman AN, Evans JM. The blood ammonia in congestive intolerance presenting as coma in two adult siblings. heart failure. Am Heart J 1955;50:715–9. J Neurol Neurosurg Psychiatry 1989;52:648–51. [114] Strauss GI, Knudsen GM, Kondrup J, et al. Cerebral [133] Sebastio G, Sperandeo MP, Andria G. Lysinuric protein metabolism of ammonia and amino acids in patients with intolerance: reviewing concepts on a multisystem disease. fulminant hepatic failure. Gastroenterology 2001;121: Am J Med Genet C Semin Med Genet 2011;157:54–62. 1109–19. [134] Gare M, Shalit M, Gutman A. Lysinuric protein intolerance [115] Phillips GB, Schwartz R, Gabuzda Jr GJ, et al. The syndrome of presenting as coma in a middle-aged man. West J Med impending hepatic coma in patients with cirrhosis of the 1996;165:231–3. liver given certain nitrogenous substances. N Engl J Med [135] DiRocco M, Garibotto G, Rossi GA, et al. Role of haematological, 1952;247:239–46. pulmonary and renal complications in the long-term

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007 16 METABOLISM CLINICAL AND EXPERIMENTAL XX (2012) XXX– XXX

prognosis of patients with lysinuric protein intolerance. Eur J [158] Davies SM, Szabo E, Wagner JE, et al. Idiopathic Pediatr 1993;152:437–40. hyperammonemia: a frequently lethal complication of bone [136] Rubio-Gozalbo ME, Vos P, Forget PP, et al. Carnitine– marrow transplantation. Bone Marrow Transplant 1996;17: acylcarnitine translocase deficiency: case report and review 1119–25. of the literature. Acta Paediatr 2003;92:501–4. [159] Frere P, Canivet JL, Gennigens C, et al. Hyperammonemia [137] Longo N, Amat di San Filippo C, Pasquali M. Disorders of after high-dose chemotherapy and stem cell transplantation. carnitine transport and the carnitine cycle. Am J Med Genet Bone Marrow Transplant 2000;26:343–5. C Semin Med Genet 2006;142C:77–85. [160] Mitchell RB, Wagner JE, Karp JE, et al. Syndrome of idiopathic [138] Al-Sannaa NA, Cheriyan GM. Carnitine–acylcarnitine hyperammonemia after high-dose chemotherapy: review of translocase deficiency. Clinical course of three Saudi nine cases. Am J Med 1988;85:662–7. children with a severe phenotype. Saudi Med J 2010;30:931–4. [161] Lichtenstein GR, Yang YX, Nunes FA, et al. Fatal [139] Rose EC, di San Filippo CA, Ndukwe Erlingsson UC, et al. hyperammonemia after orthotopic lung transplantation. Genotype–phenotype correlation in primary carnitine defi- Ann Intern Med 2000;132:283–7. ciency. Hum Mutat 2012;33:118–23. [162] Yen TY, Hwu WL, Chien YH, et al. Acute metabolic [140] Limketkai BN, Zucker SD. Hyperammonemic decompensation and sudden death in Barth syndrome: encephalopathy caused by carnitine deficiency. J Gen report of a family and a literature review. Eur J Pediatr Intern Med 2008;23:210–3. 2008;167:941–4. [141] Waddell L, Wiley V, Carpenter K, et al. Medium-chain [163] DiMauro S, Schon EA. Mitochondrial respiratory-chain acyl-CoA dehydrogenase deficiency: genotype–biochemical diseases. N Engl J Med 2003;348:2656–68. phenotype correlations. Mol Genet Metab 2006;87:32–9. [164] Greter J, Gustafsson J, Holme E. Pyruvate–carboxylase [142] Feillet F, Steinmann G, Vianey-Saban C, et al. Adult deficiency with urea cycle impairment. Acta Paediatr Scand presentation of MCAD deficiency revealed by coma and 1985;74:982–6. severe arrhythmias. Intensive Care Med 2003;29:1594–7. [165] Brown GK, Scholem RD, Hunt SM, et al. Hyperammonaemia [143] Corkey BE, Hale DE, Glennon MC, et al. Relationship between and lactic acidosis in a patient with pyruvate dehydrogenase unusual hepatic acyl coenzyme A profiles and the deficiency. J Inherit Metab Dis 1987;10:359–66. pathogenesis of Reye syndrome. J Clin Invest 1988;82:782–8. [166] Miller SG, Schwartz GJ. Hyperammonaemia with distal renal [144] Verbiest HB, Straver JS, Colombo JP, et al. Carbamyl tubular acidosis. Arch Dis Child 1997;77:441–4. phosphate synthetase-1 deficiency discovered after valproic [167] Kaufman JJ. Ammoniagenic coma following acid-induced coma. Acta Neurol Scand 1992;86:275–9. ureterosigmoidostomy. J Urol 1984;131:743–5. [145] Hamer HM, Knake S, Schomburg U, et al. Valproate-induced [168] Lamiell JJ, Ducey JP, Freese-Kepczyk BJ, et al. Essential hyperammonemic encephalopathy in the presence of amino acid-induced adult hyperammonemic topiramate. Neurology 2000;54:230–2. encephalopathy and hypophosphatemia. Crit Care Med [146] Mock CM, Schwetschenau KH. Levocarnitine for valproic- 1990;18:451–2. acid-induced hyperammonemic encephalopathy. Am J [169] Kim YA, Chung HC, Choi HJ, et al. Intermediate dose Health Syst Pharm 2012;69:35–9. 5-fluorouracil-induced encephalopathy. Jpn J Clin Oncol [147] Wei CC, Lin WD, Tsai FJ, et al. Isovaleric acidemia diagnosed 2006;36:55–9. promptly by tandem mass spectrometry: report of one case. [170] Ambrosetto G, Riva R, Baruzzi A. Hyperammonemia in Acta Paediatr Taiwan 2004;45:236–8. asterixis induced by carbamazepine: two case reports. Acta [148] Filipowicz HR, Ernst SL, Ashurst CL, et al. Metabolic changes Neurol Scand 1984;69:186–9. associated with hyperammonemia in patients with [171] Ryder KW, Olson JF, Kahnoski RJ, et al. Hyperammonemia propionic acidemia. Mol Genet Metab 2006;88:123–30. after transurethral resection of the prostate: a report of 2 [149] Sutton VR, Chapman KA, Gropman AL, et al. Chronic cases. J Urol 1984;132:995–7. management and health supervision of individuals with [172] Vanamee P, Poppell JW, Glicksman AS, et al. Respiratory propionic acidemia. Mol Genet Metab 2012;105:26–33. alkalosis in hepatic coma. AMA Arch Intern Med 1956;97: [150] Cosson MA, Benoist JF, Touati G, et al. Long-term outcome in 762–7. methylmalonic aciduria: a series of 30 French patients. Mol [173] Mulhausen R, Eichenholz A, Blumentals A. Acid–base Genet Metab 2009;97:172–8. disturbances in patients with cirrhosis of the liver. Medicine [151] Gebhardt B, Vlaho S, Fischer D, et al. N-carbamylglutamate (Baltimore) 1967;46:185–9. enhances ammonia detoxification in a patient with [174] Karetzky MS, Mithoefer JC. The cause of hyperventilation decompensated methylmalonic aciduria. Mol Genet Metab and arterial hypoxia in patients with cirrhosis of the liver. 2003;79:303–4. Am J Med Sci 1967;254:797–804. [152] Gebhardt B, Dittrich S, Parbel S, et al. N-carbamylglutamate [175] Ierardi-Curto L, Kaplan P, Saitta S, et al. The glutamine protects patients with decompensated propionic aciduria paradox in a neonate with propionic acidaemia and from hyperammonaemia. J Inherit Metab Dis 2005;28: severe hyperammonaemia. J Inherit Metab Dis 2000;23: 241–4. 85–6. [153] Kasapkara CS, Ezgu FS, Okur I, et al. N-carbamylglutamate [176] Shannon DC, De Long R, Bercu B, et al. Studies on the treatment for acute neonatal hyperammonemia in pathophysiology of encephalopathy in Reye's syndrome; isovaleric acidemia. Eur J Pediatr 2011;170:799–801. hyperammonemia in Reye's syndrome. Pediatrics 1975;56: [154] Soyucen E, Demirci E, Aydin A. Outpatient treatment of 999–1004. propionic acidemia-associated hyperammonemia with [177] Yeh KH, Cheng AL. High-dose 5-fluorouracil infusional N-carbamoyl-L-glutamate in an infant. Clin Ther 2010;32: therapy is associated with hyperammonaemia, lactic 710–3. acidosis and encephalopathy. Br J Cancer 1997;75:464–5. [155] DeLong GR, Glick TH. Ammonia metabolism in Reye syndrome [178] Amatuzio DS, Nesbitt S. A study of pyruvic acid in the and the effect of citrulline. Ann Neurol 1982;11:53–8. blood and spinal fluid of patients with liver disease [156] De Jonghe B, Janier V, Abderrahim N, et al. Urinary tract with and without hepatic coma. J Clin Invest 1950;29: infection and coma. Lancet 2002;360:996. 1486–90. [157] Samtoy B, DeBeukelaer MM. Ammonia encephalopathy [179] Bihari D, Gimson AE, Lindridge J, et al. Lactic acidosis in secondary to urinary tract infection with Proteus mirabilis. fulminant hepatic failure. Some aspects of pathogenesis and Pediatrics 1980;65:294–7. prognosis. J Hepatol 1985;1:405–16.

[180] Hurt GA, Chanutin A. Organic phosphate compounds of [183] Keys A, Snell AM. Respiratory properties of the arterial blood erythrocytes from individuals with cirrhosis of the liver. in normal man and in patients with disease of the liver: Proc Soc Exp Biol Med 1965;118:167–9. position of the oxygen dissociation curve. J Clin Invest [181] Astrup J, Rorth M. Oxygen affinity of hemoglobin and red cell 1938;17:59–67. 2,3-diphosphoglycerate in hepatic cirrhosis. Scand J Clin Lab [184] Tizianello A, De Ferrari G, Gurreri G, et al. Effects of Invest 1973;31:311–7. metabolic alkalosis, metabolic acidosis and uraemia on [182] Bihari D, Gimson AE, Waterson M, et al. Tissue hypoxia during whole-body intracellular pH in man. Clin Sci Mol Med fulminant hepatic failure. Crit Care Med 1985;13:1034–9. 1977;52:125–35.

Please cite this article as: Adeva MM, et al, Ammonium metabolism in humans, Metabolism (2012), http://dx.doi.org/10.1016/ j.metabol.2012.07.007